WO1996029433A1 - Rest protein and dna - Google Patents

Abstract

The invention provides a substantially pure nucleic acid encoding a protein that inhibits the expression of neural proteins in non-neural tissues. The invention also provides a substantially pure nucleic acid encoding a protein that binds to a promoter sequence having at least about 90 % homology to nucleotides 6-28 of the RE1 sequence and acting to suppress the acitivity of a promoter having the promoter sequence. The invention further provides a substantially pure nucleic acid encoding a protein having at least about 85 % homology to at least one of the DNA binding domain or the suppressor domain of an animal RE1-Silencing Transcription factor. The invention also relates to the proteins so encoded.

Description

REST PROTEIN AND DNA

The present invention is directed to purified nucleic acids encoding REl -Silencing Transcription factors ("REST proteins") and to purified proteins with REST activity.

Part of the work performed during the development of this invention utilized United States Government Funds under National Institutes of Health Grant NS22518 and National Science Foundation Grant GER9023237. The government has certain rights in this invention.

It has been suggested that neural development is substantially a default pathway of development that is repressed in non-neural cell types. Consistent with this idea, Kraner et al., Neuron 9, 37-44, 1992, identified a DNA sequence, the 28 base pair ("bp") REl sequence, found in the 5 ' flanking sequence of the gene for the membrane protein that forms the CNS-type voltage dependent sodium channel (i.e., "type II" voltage dependent sodium channel), that appears to be responsible for negatively regulating the use of this gene in non-neural tissue. REl nucleic acid sequences also appear to interact with a nuclear protein found in non-neural calls but not in most neural cells. Similar sequences having cell-specific silencer activity have been identified in the promoters for SCG10 (Mori et al., Neuron 9, 45-54, 1992), synapsin (Li et al., Proc. Natl. Acad. Sci. USA 90, 1460-1464, 1993) and dopamine /3-hydroxylase (Ishigoro et al., J. Biol. Chem. 268, 17987-17994, 1993). Summary of the Invention

Until now, however, the protein responsible for silencing promoters containing REl elements has not been identified. That protein herein referred to as "REST," and the gene encoding it, is herein identified as having the amino acid sequence included in SEQ ID NO: 1. The portion of the nucleic acid sequence included in SEQ ID NO: 1 that is an open reading frame for REST is identified as SEQ ID NO: 10. The protein sequence for human REST and the nucleic acid sequence of the CDNA for human REST are shown in Figure 1.

One preferred embodiment of the present invention is a substantially pure nucleic acid comprising a nucleic acid encoding a protein having at least about 85 % homology to at least the DNA binding domain or the suppressor domain of an animal REST protein; the same substantially pure nucleic acid further comprising a nucleic acid encoding at least the DNA binding domain or the suppressor domain of an animal REST protein; the same substantially pure nucleic acid, wherein the REST protein is a mammalian REST protein; the same substantially pure nucleic acid, wherein the REST protein is a human REST protein; the same substantially pure nucleic acid, wherein the nucleic acid comprises SEQ ID NO:2; the same substantially pure nucleic acid, wherein the nucleic acid comprises SEQ ID NO: 10; the same substantially pure nucleic acid, further comprising a nucleic acid encoding both the DNA binding domain and the suppressor domain of an animal REST protein; the same substantially pure nucleic acid, wherein the REST protein is a mammalian REST protein; the same substantially pure nucleic acid, wherein the REST protein is a human REST protein; the same substantially pure nucleic acid, wherein the nucleic acid comprises SEQ ID NO: 2; the same substantially pure nucleic acid, wherein the nucleic acid comprises SEQ ID NO: 10; the same substantially pure nucleic acid, comprising a nucleic acid encoding a protein differing from an animal REST protein by no more than about 20 point mutations. Preferred substantially pure nucleic acids also encode analogs to the REST protein, which include either the DNA binding domain or the suppressor domain thereof.

Another preferred embodiment of the present invention is a substantially pure nucleic acid that hybridizes with an animal REST nucleic acid under stringent conditions; the same substantially pure nucleic acid, comprising the nucleic acid of SEQ ID NO:l. A further preferred embodiment is a substantially pure nucleic acid comprising a nucleic acid encoding a protein that binds to a promoter having at least about 90% homology to nucleotides 6-28 of SEQ ID NO: 29 and acting to suppress the activity of a promoter having said promoter.

Yet another preferred embodiment is a substantially pure protein having at least about 85 % homology with at least the DNA binding domain or the suppressor domain of an animal REST protein; the same substantially pure protein, comprising at least the DNA binding domain or the suppressor domain of an animal REST protein; the same substantially pure protein, further comprising the protein of SEQ ID NO:2; the same substantially pure protein, further comprising both the DNA binding domain and the suppressor domain of an animal REST protein; the same substantially pure protein, further comprising the protein of SEQ ID NO: 10. Yet another preferred embodiment is a transformed eukaryotic or prokaryotic cell comprising a nucleic acid encoding a protein having at least about 85 % homology to at least one of the DNA binding domain or the suppressor domain of an animal REST protein; the same transformed cell, further comprising a nucleic acid encoding at least the DNA binding domain or the suppressor domain of an animal REST protein; the same transformed cell, wherein the REST protein is a mammalian REST protein; the same transformed cell, wherein the REST protein is a human REST protein; the same transformed cell, wherein the nucleic acid comprises SEQ ID NO: 2. Preferably, the transformed cell expresses one of the inventive proteins described herein. - 3 -

Yet another preferred embodiment is a vector capable of reproducing in a eukaryotic or prokaryotic cell comprising a nucleic acid encoding a protein having at least about 85% homology to at least the DNA binding domain or the suppressor domain of an animal REST protein; the same vector capable of reproducing in a eukaryotic or prokaryotic cell, further comprising a nucleic acid encoding at least the DNA binding domain or the suppressor domain of an animal REST protein; the same vector capable of reproducing in a eukaryotic or prokaryotic cell, wherein the REST protein is a mammalian REST protein; the same vector capable of reproducing in a eukaryotic or prokaryotic cell, wherein the REST protein is a human REST protein; the same vector capable of reproducing in a eukaryotic or prokaryotic cell, wherein the nucleic acid comprises SEQ ID NO:2. Preferably, the inventive vector expresses, intracellularly or extracellularly, one of the inventive proteins described herein. 10 Yet another preferred embodiment is a method of preparing a protein having REST activity, wherein the protein has at least about 85% homology with at least the DNA binding domain or the suppressor domain of an animal REST protein, the method comprising:

(a) transforming an appropriate eukaryotic or prokaryotic cell with an expression vector for expressing intracellularly or extracellularly a nucleic acid encoding the protein; 15 (b) growing the transformed cell in culture; and

(c) isolating the protein from the transformed cell or the culture medium.

Yet another preferred embodiment is a pharmaceutical composition for treating an animal having de-differentiated neural cells or neural cells exhibiting diminished activity comprising an effective amount of a REST-interfering nucleic acid, wherein the REST-interfering nucleic acid cSfiiprises an antisense molecule directed against REST expression or an expression vector for expressing REST DNA binding activity but not REST silencer activity, and a pharmaceutically acceptable carrier; the same pharmaceutical composition, wherein the animal has brain cancer; the same pharmaceutical composition, wherein said animal has a demyelinating myasthenia gravis, muscular dystrophy, botulism, peripheral neuropathies, traumatic nerve injury, post stroke degeneration, post-traumatic spinal and neural degeneration, poliomyelitis or rabies.

Yet another preferred embodiment is a pharmaceutical composition for an animal having neural cells exhibiting excessive neural activity comprising an effective amount of an expression vector comprising a nucleic acid encoding a protein that inhibits the expression of neural proteins in non-neural tissues, and a pharmaceutically acceptable carrier; the same pharmaceutical composition, vθ krein the animal has epilepsy, Lennox-Gastaut syndrome, spasticity, trauma-induced pain, schizophrenia, stroke or a neurodegenerative disease; the same pharmaceutical composition, wherein the animal has Alzheimer's, Parkinson's or Huntington's disease; the same pharmaceutical composition, wherein the animal has epilepsy; the same pharmaceutical composition, wherein the animal has a neurodegenerative disease.

Yet another preferred embodiment is a method of determining the level of REST expression in Si tissue sample comprising

(a) contacting the tissue sample with (i) a nucleic acid that binds to REST mRNA under stringent conditions or (ii) an antibody specific for REST;

(b) washing the tissue sample to remove non-specific hybridizations of the nucleic acid or non-specific antibody binding; and

10 (c) determining the level of hybridized nucleic acid or bound antibody.

Yet another preferred embodiment is an antibody that reacts specifically with the substantially pure protein having at least about 85% homology with at least the DNA binding domain or the suppressor domain of an animal REST protein, as recited above.

Bkief Description of the Drawings

Figure 1 shows the protein encoded by the open reading frame of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO:l.

Detailed Description of the Invention

20 The DNA binding domain of REST is made up of eight zinc finger domains. The portion of SEQ ID NO:l that encompasses the eight zinc finger domains of REST is identified as SEQ ID NO:2. The underlined residues shown in Figure 1 are the zinc finger domains. A search of the GenBank database found that the closest homology for this DNA binding domain is found with the Kruppel family of repressor proteins, particularly the GLI-Kriippel repressor protein. (For a review oϊ±inc finger proteins, see Colman, Ann. Rev. Biochem. 61, 897-946, 1992.) The size of the REl sequence, 28 bp, and the number of zinc finder domains in REST is consistent with research (Pauletich and Pabo, Science 242, 809-817, 1991) that suggests that each such zinc finger domain interacts with a triplet of nucleotide base pairs. The sequences of the zinc finger domains are indicated in the table below (with a space inserted into 6 of the 8 sequences to facilitate alignment of homologous sequence):

SEQ E>NO. Zinc Finger Sequence

11 C K P C Q Y E A E S E E Q F V H H I R V H

12 C D R C G Y N T N R Y D H Y T A H L K H H

13 C I I C T Y T T V S E Y H W R K H L R N H

14 C G K C N Y F S D R K N N Y V Q H V R T H

1015 C E L C P Y S S S Q K T H L T R H M R T H

16 C D ^Q C S Y V A S N Q H E V T R H A R Q V H

17 C P H C D Y K T A D R S N F K K H V E L H

18 C P V C D Y A A S K K C N L QY H F K S K H

15 C-terminal to the DNA binding domain, REST has six repeat sequences having the following sequences:

These sequences are indicated in Figure 1 by the double underlined amino acid residues. The sequence encompassing these repeats is designed SEQ ID NO:20. The most highly conserved residues of the six repeats are highlighted in the table above.

30 By studying the activity of the REl promoter, it has been determined that REST is expressed in undifferentiated neural progenitors, which is consistent with the view that REST plays a role in maintaining the undifferentiated state of these cells. Antisense oligonucleotides directed against the REST transcript accordingly, would promote the differentiated state. Also consistent with this view is the hypothesis that certain neuroblastoma cells have de-differentiated into analogs of neural progenitors. Accordingly, REST antisense therapy aides in reversing this de- differentiation and reducing or reversing the malignancy of these cells.

5 As used herein, a "REST nucleic acid" means the REST-encoding nucleic acid, whether RNA or DNA, synthetic or natural, found in a REST-expressing animal, or the complementary strand thereof. "REST protein-encoding nucleic acid" or "nucleic acid encoding a REST protein" refers to any nucleic acid, whether native or synthetic, RNA, DNA, or cDNA, that encodes a REST protein. For recombinant expression purposes, codon usage preferences for the organism in v-Jflch such a nucleic acid is to be expressed are advantageously considered in designing a synthetic REST protein-encoding nucleic acid. A "REST protein" is a REST homologous protein with the ability to bind an REl sequence and to repress the activity of a promoter containing an REl sequence. An "animal REST protein" is a REST protein expressed by a member of the animal kingdom; a "human REST protein" is a REST protein expressed by a human.

15 Vectors encoding a protein with REl -binding activity but not suppressor activity are shown herein to reverse the transcriptional suppression caused by REST, apparently by competing for the REl promoter element through which REST functions. Accordingly, gene therapy with such vectors are used like the aforementioned and other antisense therapies known in the art to reduce REST's suppressor activity. The vectors described in this paragraph and the antisense molecules dMkussed above are termed herein "REST-interfering nucleic acids."

Probes for REST expression are used to measure the extent of a de-differentiation in biopsy tissue from tumors that are derived from neural tissue. Such probes are used to predict the extent of tissue transformation and the virulence of the tumor. Such probes include antibodies directed against REST or fragments thereof, nucleic acid probes that hybridize to REST mRNA under sflSngent conditions, and oligonucleotides that specifically prime a PCR amplification of REST mRNA.

For a number of years physicians have sought to treat neurodegenerative diseases by administering neural stem cells, for instance stem cells derived from embryos, to produce replacements for a patient's lost neural cells. Such diseases include Alzheimer's disease, PMkinson's disease, Huntington's disease, amyotrophic lateral sclerosis ("Lou Gehrig's disease") and demyelinating diseases such as multiple sclerosis. Stem cells used in these therapies are induced to initiate differentiation to provide the needed replacement cells by treating them with REST antisense constructs or with vectors expressing the DNA-binding domain of REST but not the suppressor function of REST.

In diseases where pathological states are associated with excesses in neural activity, such as epilepsy, Lennox-Gastaut syndrome, spasticity, trauma-induced pain, schizophrenia, stroke and nefirodegenerative diseases (including Alzheimer's, Parkinson's and Huntington's diseases), the level of neural expression of the voltage-dependent sodium channel is usefully reduced. Toward this end, neural cells are transformed to express sufficient REST to down-regulate expression of the sodium channel.

In diseases that exhibit insufficient neural activity, such as demyelinating diseases (including n±fltiple sclerosis), myasthenia gravis, muscular dystrophy, botulism, peripheral neuropathies, traumatic nerve injury, post-stroke degeneration, post-traumatic spinal cord neural degeneration, poliomyelitis and rabies, up regulation of the expression of the neural voltage-dependent sodium channel is useful. This up regulation is done by antisense therapy based on REST nucleic acids to inhibit neural expression of REST or with gene therapy using a vector that expresses a protein that competes with REST for REl promoter sequences without suppressing the activity of the promoter.

The REST protein is also a useful target for drug screening efforts to identify drugs that interfere with its suppressor activity, either by inhibiting DNA binding or the negative effect of REST on transcription. Such drug screening assays in one embodiment include cell-free transcription systems using the REST protein, cell-free transcription systems such as those described b|CDignam et al., Nucl. Acids. Res. 11, 1475-1489, 1983 or that described in the cell-free transcription protocol available from Promega (Madison, WI) in an appropriate REl -containing promoter. The screening methods also utilize in other embodiments expression studies conducted in cell culture, such as the chloramphenicol acetyl transferase (CAT) assay methods described herein below.

25 The suppression domain of REST is fused by recombinant methods to a DNA-binding domain of a positive transcription factor to create a protein that represses the activity of one or more promoters. For instance, in one embodiment the suppressor domain is linked to pit- 1 » a transcription factor for the prolactin and growth hormone promoters (see Ingraham et al., Cell 55, 519-529, 1988), thereby creating a vector for gene therapeutics aimed at down regulating hyperactive pituitary production of growth hormone and/or prolactin. Other examples of specific targets for this kind of therapy are the DNA-binding domains of steroid hormone or thyroid hormone receptors. Fusion vectors expressing a DNA binding domain from a steroid hormone receptor and the REST suppressor domain are used in yet other embodiments to down regulate responsiveness to the steroid hormones in patients that overproduce the steroid or that have steroid hormone receptors that are too active. The fusion protein in one embodiment includes the target DNA-binding element and substantially all of the REST protein.

The antibodies and nucleic acid probes of the present invention are also useful as histochemical reagents for marking the pathways of nerves that do not express the CNS-type sodium channel. Also, the staining of most non-neural tissue serves as a contrast agent to highlight neurons that do not express REST or express very low levels of REST. Thus, these histochemical agents are used to produce histochemical slides and preserved anatomy specimens useful for training students and physicians.

10 The first embodiment of the invention relates to a purified nucleic acid comprising a nucleic acid having at least 85% homology to at least the DNA binding domain or the suppressor domain of an animal REST protein. Such a nucleic acid is referred to herein as a REST protein that binds the REl promoter element and/or suppresses the activity of the promoter for the CNS-type voltage- dependent sodium channel. The encoded protein is preferably a REST protein of a mammalian aifinal, more preferably the human REST protein. Preferably, the encoded protein has the sequence of SEQ ID NO: l, SEQ ID NO:2, or SEQ ID NO: 10.

Another embodiment of the invention provides for one or more nucleic acids encoding a protein that binds to a promoter sequence having at least about 90% homology, preferably 95% homology, to nucleotides 6-28 the REl sequence (SEQ ID NO: 29) and acting to suppress the a20vity of a promoter containing that promoter sequence. Yet another embodiment provides for a nucleic acid encoding a protein that inhibits the expression of neural proteins in non-neural tissues.

The nucleic acid embodiments of the invention are preferably deoxyribonucleic acids, preferably double-stranded deoxyribonucleic acids, except that, for hybridization probes, single- stranded nucleic acids are preferred. However, nucleic acids of the present invention also include rQ&nucleic acids. The nucleic acids of the present invention are also referred to as polynucleotides or polynucleic acids.

Numerous methods are known to delete a segment of a nucleic acid from or mutate a nucleic acid that encodes a protein and to confirm the function of the proteins encoded by these deleted or mutated nucleic acids. Accordingly, the invention also relates to a mutated or deleted vSftion of a REST protein-encoding nucleic acid that encodes a protein that retains the ability to bind specifically to the REl promoter element and/or the ability to suppress an REl -responsive promoter when appropriately bound to the vicinity of the promoter. The invention also relates to a nucleic acid encoding, in the proper order, at least 4 of the zinc finger domains of a REST protein, preferably at least 6 of the zinc finger domains, more preferably all of the zinc finger domains. The zinc finger domains for human REST are identified in Figure 2. Preferably, the nucleic acid is SEQ ID NO:2.

5 Transcription suppressive proteins, such as Kruppel, Kid-1, and ZNF2 generally have distinct suppressor domains which function so long as they are appropriately linked to DNA binding domains that suitably bring the suppressor domains into the vicinity of the target promoters. See, for instance, Licht et al., Nature 346, 76-79, 1990; Witzgall et al., Proc. Natl. Acad. Sci. USA 91 , 4514-4518, 1994. Such a suppressor domain can readily be identified for the REST protein using deCfctional approaches and recombinant fusion protein approaches that are well known in the art. Accordingly, the invention also is directed to a nucleic acid encoding a segment of the protein of a REST protein that is effective to repress the use of a promoter when attached to a protein that binds the promoter. Preferably, the encoded protein will be effective to repress the use of the promoter for the CNS-type voltage-dependent sodium channel gene. Studies with the aforementioned REl nt^βleic acid suggest that it is ineffective as a transcription silencing element when inserted into some gene promoters. Accordingly, the promoters discussed in reference to this embodiment are REl -responsive promoters.

It is recognized that many deletional or mutational analogs of nucleic acid sequences for a REST protein are effective hybridization probes for REST nucleic acid. Accordingly, the invention rSBtes to nucleic acid sequences that hybridize with such REST-encoding sequences under stringent conditions. Preferably, the nucleic acid of the present invention hybridizes with SEQ ID NO: 1 under stringent conditions. The invention also relates to nucleic acids that hybridize with SEQ ID NO:2 under such stringent conditions.

"Stringent conditions" refers to conditions that allow for the hybridization of substantially rδrited nucleic acids, where relatedness is a function of the sequence of nucleotides in the respective nucleic acids. For instance, for a nucleic acid of 100 nucleotides, such conditions will generally allow hybridization thereto of a second nucleic acid having at least about 85% homology, preferably having at least about 90% homology. Such hybridization conditions are described by Sambrook et al.. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989.

The invention further relates to REST proteins and to proteins having sufficient zinc finger domains to confer the ability to bind the REl promoter element. Preferably, the protein has at least 4 of the zinc finger domains REST, more preferably at least 6, yet more preferably at least 7. Still more preferably, the REl binding protein has all of the zinc finger domains. Preferably, the protein has the sequence of a contiguous stretch of at least about 252 amino acids of SEQ ID NO: l, more preferably, of a contiguous stretch of at least about 504 amino acids.

As discussed above, deletional or mutational methods of producing recombinant proteins that retain a given activity are well known. Thus, the embodiments of the present invention that relate to proteins also encompass analogs of REST proteins that retain one or both of the ability to bind the REl promoter element and to suppress the activity of a promoter to which the protein is bound. These analogs preferably lack no more than about 360 amino acid residues of deleted sequence at the C-terminal or N-terminal ends, more preferably no more than about 180 amino acid rdfldues of deleted sequence. The remaining sequence of the REST protein will preferably have no more than about 20 point mutations, preferably no more than about 10 point mutations, more preferably no more than about 5 point mutations. The point mutations are preferably conservative point mutations. Preferably, the analogs will have at least about 85% homology, preferably at least about 90% homology, more preferably at least about 95% homology to a portion of an animal REST protein retaining one or both of REST's known activities, such as the proteins of SEQ ID NO: l or SEQ ID NO:2.

Antigens for eliciting the production of antibodies against the REST protein can be produced recombinantly by expressing all of or a part of the nucleic acid of a REST protein in a bacteria or a yeast or other eukaryotic cell line. In one embodiemnt, the recombinant protein is expressed as a fusion protein, with the non-REST portion of the protein serving either to facilitate purification or to enhance the immunogenicity of the fusion protein. For instance, the non-REST portion comprises a protein for which there is a readily-available binding partner that is utilized for affinity purification of the fusion protein. The antigen includes an "antigenic determinant," i.e., a minimum segment of amino acids sufficient to bind specifically with an anti-REST antibody. 25 Rules for designing PCR primers are well known in the art, as reviewed by PCR Protocols, Cold Spring Harbor Press, 1991. Degenerate primers, i.e., preparations of primers that are heterogeneous at given sequence locations, are designed to amplify nucleic acid sequences that are highly related to, but not identical to, a REST protein. For instance, such degenerate primers, in one embodiment, are designed from the human REST cDNA and used to amplify nucleic acid sequences for REST proteins from non-human species, as illustrated in the examples.

The method by which human REST cDNA was isolated, which is described in detail in the examples, illustrates how readily REl-binding domains from REST proteins are identified. In the isolation method, a library was made of cDNA from a REST-expressing cell and inserted into a yeast expression vector for the GAL4 activation domain so that the library would express fusion proteins having one part derived from cDNA and another part that is the GAL4 activation domain. Initial partial cDNA clones were identified by their ability to bind an REl element on the promoters for two reporter genes and activate expression of those genes by causing the fused GAL4 activation domain to act on the promoters. These initial clones were of portions of the REl binding domain of the human REST protein. The same methodology can be used to identify other sequences from other animal sources that are sufficient to bind the REl element.

Additionally, the mutational and deletional methodologies that are well known in the art are applied to nucleic acids having the sequence of SEQ ID NO:2, which encodes the zinc finger dtf ain of human REST. Nucleic acid constructs that express such mutated or deleted zinc finger domains are tested for the REl binding activity of the expressed protein. One facile method of doing this is to sub-clone the constructs into the GAM vector discussed above. Successful constructs activate the two REl -containing reporter genes that were used in the initial cloning of human REST cDNA. 15 For identifying the suppressor domain of REST, one approach is to take a REST cDNA and create deletional mutants lacking segments at either the 5' or the 3' end by, for instance, partial digestion with SI nuclease, Bal 31 or Mung Bean nuclease (the latter approach described in literature available from Stratagene, San Diego, CA, in connection with a commercial deletion cloning kit). Alternatively, the deletion mutants are constructed by subcloning restriction fragments ofta REST cDNA. The deletional constructs are cloned into expression vectors and tested for their ability to suppress the expression of a promoter that has a functional REl element. For instance, a reporter construct having the promoter for the CNS-type voltage-dependent sodium channel linked to the gene for chloramphenicol acetyl transferase ("CAT") is used. Such a vector is described below in the examples. Functional constructs diminish the level of expression of CAT, an enzyme tfifi is readily measurable by well established techniques. See, for example, Gorman et al., Mol. Cell. Biol. 2, 1044-1051, 1982 and Young et al., DNA 4, 469-475, 1985.

Mutational and deletional approaches are applied to all of the nucleic acid sequences of the invention that express REST-related proteins. As discussed above, conservative mutations are preferred. Such conservative mutations include mutations that switch one amino acid for another w3flιin one of the following groups:

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr. Pro and Gly;

2. Polar, negatively charged residues and their amides: Asp, Asn, Glu and Gin;

3. Polar, positively charged residues: His, Arg and Lys;

4. Large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and 5. Aromatic residues: Phe, Tyr and Tip. A preferred listing of conservative substitutions is the following:

Original Residue Substitution

Ala Gly, Ser

Arg Lys

Asn Gin, His

Asp Glu

Cys Ser

Gin Asn

10 Glu Asp

Gly Ala, Pro

His Asn, Gin

He Leu, Val

Leu He, Val

15 Lys Arg, Gin, Glu

Met Leu, Tyr, He

Phe Met, Leu, Tyr

Ser Thr

Thr Ser

20 Trp Tyr

Tyr Trp, Phe

Val He, Leu

The types of substitutions selected may be based on the analysis of the frequencies of amino 25 acid substitutions between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure, Springer- Verlag, 1978, pp. 14-16, on the analyses of structure- forming potentials developed by Chou and Fasman, Biochemistry 13, 211, 1974 or other such methods reviewed by Schulz et al. Principles in Protein Structure, Springer- Verlag, 1978, pp. 108-130, and on the analysis of hydrophobicity patterns in proteins developed by Kyte and 30 Doolittle, J. Mol. Biol. 157: 105-132, 1982. Numerous methods for determining percent homology are known in the art. One preferred method is to use version 6.0 of the GAP computer program for making sequence comparisons. The program is available from the University of Wisconsin Genetics Computer Group and utilizes the alignment method of Needleman and Wunsch, J. Mol. Biol. 48, 443, 1970, as revised by Smith and Waterman Adv. Appl. Math. 2, 482, 1981.

Nucleic acid molecules that bind to a REST-encoding nucleic acid under high stringency conditions are identified functionally, using methods outlined above, or by using the hybridization rules reviewed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989. Antisera to REST are made by creating a REST antigen by linking a portion of the cDNA for human REST to a cDNA for glutathione s-transferase ("GST") found on a commercial vector. The resulting vector expresses a fusion protein containing an antigenic portion of REST and GST that is readily purified from the expressing bacteria using a glutathione affinity column. The purified antigenic fusion protein is used to immunize rabbits. The same approach is used to make antigens based on other portions of the REST protein. Procedures for making antibodies and for identifying antigenic portions of proteins are well known. See, for instance, Harlow, Antibodies, Cold Spring Harbor Press, 1989.

The proteins of the invention are made, in one embodiment, using the identical approach as for generating REST antisera. The cDNA specific for a given REST protein or analog thereof is linked using standard means to a cDNA for GST, found on a commercial vector, for example. The fusion protein expressed by such a vector construct includes the REST protein or analog and GST, and can be treated as above for purification. Should the GST segment of the fusion protein interfere with function, it is removed by partial proteolytic digestion approaches that preferentially attack unstructured regions, such as the linkers between GST and the REST-derived protein. The linkers are designed to lack structure, for instance using the rules for secondary structure-forming potential developed by Chou and Fasman, Biochemistry 13, 211 , 1974. The linker is also designed to incorporate protease target amino acids, such as, for trypsin, arginine and lysine residues. To create the linkers, standard synthetic approaches for making oligonucleotides are employed together with standard subcloning methodologies. Other fusion partners other than GST can be used.

Also, of course, the REST proteins can be directly synthesized from nucleic acid (by the cellular machinery) without use of fusion partners. For instance, nucleic acids having the sequence of SEQ ID NO: 10 are subcloned into an appropriate expression vector having an appropriate promoter and expressed in an appropriate organism. (Note that REST lacks consensus glycosylation sites and, especially since it is not a membrane or exported protein, should lack glycosylations.) Antibodies against REST are employed to facilitate purification. Additional purifications techniques are applied as needed, including without limitation, preparative electrophoresis, FPLC (Pharmacia, Uppsala, Sweden), HPLC (e.g., using gel filtration, reverse-phase or mildly hydrophobic columns), gel filtration, differential precipitation (for instance, "salting out" precipitations), ion-exchange chromatography and affinity chromatography (including affinity chromatography using the REl duplex nucleotide sequence as the affinity ligand). A protein or nucleic acid is "isolated" in accordance with the invention in that the molecular cloning of the nucleic acid of interest, for example, involves taking a human REST nucleic acid from a human cell, and isolating it from other human-derived nucleic acids. This isolated nucleic acid may then be inserted into a host cell, which may be yeast or bacteria, for example, or another human cell. A protein or nucleic acid is "substantially pure" in accordance with the invention if it is predominantly free of other proteins or nucleic acids, respectively. A macromolecule, such as a nucleic acid or a protein, is predominantly free if it constitutes at least about 50% by weight of the given macromolecule in a composition. Preferably, the protein or nucleic acid of the present invention constitutes at least about 60% by weight of the total proteins or nucleic acids, respectively, that are present in a given composition thereof, more preferably about 80%, still more preferably about 90% , yet more preferably about 95% , and most preferably about 100%. Such compositions are referred to herein as being proteins or nucleic acids that are 60% pure, 80% pure, 90% pure, 95% pure, or 100% pure, any of which are substantially pure.

One aspect of the present invention is directed to the use of "antisense" polynucleic acid to treat neural diseases, including de-differentiated neural tumor cells and diseases characterized by diminished neural activity. Such an approach is also used to trigger the differentiation of neural stem cells. The approach involves the use of an antisense molecule designed to bind nascent mRNA (or "sense" strand) for a REST protein, thereby stopping or inhibiting the translation of the mRNA, or to bind to the REST gene to interfere with its transcription. Once the sequence of the mRNA sought to be bound is known, an antisense molecule is designed that binds the sense strand by the Watson-Crick base-pairing rules, forming a duplex structure analogous to the DNA double helix. Gene Regulation: Biology of Antisense RNA and DNA, Erikson and Ixzant, eds.. Raven Press, New York, 1991. A serious barrier to fully exploiting this technology is the problem of efficiently introducing into cells a sufficient number of antisense molecules to effectively interfere with the translation of the targeted mRNA or the function of DNA. One method that has been employed to overcome this problem is to covalently modify the 5' or the 3' end of the antisense polynucleic acid molecule with hydrophobic substituents. These modified nucleic acids generally gain access to the cells interior with greater efficiency. See, for example, Boutorin et al., FEBS Lett. 23,1382-1390, 1989; Shea et al, Nucleic Acids Res. 18, 3777-3783, 1990. Additionally, the phosphate backbone of die antisense molecules has been modified to remove the negative charge (see, for example, Agris et al., Biochemistry 25, 6268, 1986; Cazenave and Helene in Antisense Nucleic Acids and Proteins: Fundamentals and Applications, Mol and Van der Krol, eds., p. 47 et seq.. Marcel Dekker, New York, 1991) or the purine or pyrimidine bases have been modified (see, for example, Antisense Nucleic Acids and Proteins: Fundamentals and Applications, Mol and Van der Krol, eds., p. 47 et seq., Marcel Dekker, New York, 1991; Milligan et al. in Gene Therapy For Neoplastic Diseases, Huber and Laso, eds., p. 228 et seq.. New York Academy of Sciences, New York, 1994). Other attempts to overcome the cell penetration barrier include incorporating the antisense polynucleic acid sequence into an expression vector that is inserted into the cell in low copy number, but which, when in the cell, directs the cellular machinery to synthesize more substantial amounts of antisense polynucleic molecules. See, for example, Farhood et al., Ann. N Y. Acad. Sex. 716, 23, 1994. This strategy includes the use of recombinant viruses that have an expression site into which the antisense sequence has been incorporated. See, e.g., Boris-Lawrie and Temin, Ann. N Y. Acad. Sci., 716:59 (1994). Others have tried to increase membrane permeability by neutralizing the negative charges on antisense molecules or other nucleic acid molecules with polycations. See, e.g. Wu and Wu, Biochemistry, 27:887-892, 1988; Behr et al., Proc. Natl. Acad Sci U.S.A. 86:6982-6986, 1989.

The polynucleotide or nucleic acid compositions of the invention can be administered orally, topically, rectally, vaginally, by pulmonary route by use of an aerosol, or parenterally, i.e. intramuscularly, intraventricularly, subcutaneously, intraperitoneallly or intravenously. The polynucleotide compositions are administered alone, or they are combined with a pharmaceutical ly-acceptable carrier or excipient according to standard pharmaceutical practice. For the oral mode of administration, the polynucleotide compositions are used in the form of tablets, capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like. In the case of tablets, carriers that are used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the polynucleotide compositions are combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added. For parenteral administration, sterile solutions of the conjugate are usually prepared, and the pH of the solutions are suitably adjusted and buffered. For intravenous use, the total concentration of solutes is controlled to render the preparation isotonic. For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art, such as applicators or eye droppers. Such compositions include ucomimetics, such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives, such as sorbic acid or EDTA, and the usual quantities of diluents and/or carriers well known in the art. For pulmonary administration, diluents and/or carriers are selected so as to allow the formation of an aerosol.

Generally, the polynucleotide compositions are administered in an effective amount. An effective amount is an amount effective to either (1) reduce the symptoms of the disease sought to be treated or (2) induce a pharmacological change relevant to treating or preventing the disease sought to be treated.

For viral gene therapy vectors, dosages are generally from about 1 μg to about 1 mg of nucleic acid per kg of body mass. For non-infective gene therapy vectors, dosages are generally from about 1 μg to about 100 mg of nucleic acid per kg of body mass. Antisense oligonucleotide dosages are generally from about 1 μg to about 100 mg of nucleic acid per kg of body mass.

The invention also encompasses the use of gene therapy approaches to insert a gene expressing an REl binding domain but not a suppressor domain into de-differentiated tumor cells or neural cells with diminished neural activity. Gene therapy approaches for inserting a gene for a protein with REST activity into overactive neural cells are also within the invention. Also, gene therapy approaches for inserting a gene for a REST suppressor domain linked to a promoter binding element to suppress the activity of the promoter bound by the binding element are also within the invention. For gene therapy, medical workers prefer to incorporate, into one or more cell types of an organism, a DNA vector capable of directing the synthesis of a protein missing from the cell or useful to the cell or organism when expressed in greater amounts. The methods for introducing DNA to cause a cell to produce a new protein or a greater amount of a protein are called "transfection" methods. See, generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989.

A number of the above-discussed methods of enhancing cell penetration by antisense nucleic acid are generally applicable methods of incorporating a variety of nucleic acids into cells. Other general methods include calcium phosphate precipitation of nucleic acid and incubation with the target cells (Graham and Van der Eb, Virology, 52:456, 1983), co- incubation of nucleic acid, DEAE-dextran and cells (Sompayrac and Danna, Proc. Natl. Acad. Sci. , 12:7575, 1981), electroporation of cells in the presence of nucleic acid (Potter et al., Proc. Natl. Acad. Sci., 81:7161-7165, 1984), incorporating nucleic acid into virus coats to create transfection vehicles (Gitman et al., Proc. Natl. Acad. Sci. U.S.A., 82:7309-7313, 1985) and incubating cells with nucleic acid incorporated into liposomes (Wang and Huang, Proc. Natl. Acad. Sci., 84:7851-7855, 1987). An approach in employing gene therapy is to incorporate the gene sought to be introduced into the cell into a virus, such as an adeno virus. See, for instance, Akli et al.. Nature Genetics 3, 224, 1993.

The stem cells that are useful in neural stem cell replacement therapy include human mesencephalic fetal brain cells, porcine fetal brain cells, human subventricular zone cells and glial progenitor cells, including O2A cells (which are progenitors for all glial cell types, including astrocytes and oligodendrocytes).

The invention also relates to methods of measuring a REST protein or mRNA from a tissue or staining a tissue for a REST protein or mRNA. Useful methods of measuring mRNA include Southern blot analysis, dot blot analysis, nuclear transcription analysis, histochemical staining for mRNA and polymerase chain reaction amplification methods. See generally, Ausubel et al., Current Protocols in Molecular Biology, Wiley Press, 1993; PCR Protocols, Cold Spring Harbor Press, 1991; and Sambrook et al.. Molecular Cloning: A Laboratory Manual 2nd ed.. Cold Spring Harbor Press, 1989. For m situ nucleic acid hybridization techniques, see Baldino et al.. Methods in Enzymology 168, 761-777, 1989; Meson et al., Methods in Enzymology 168, 753-761, 1989; Harper et al., Methods in Enzymology 151, 539- 551, 1987; Angerer et al.. Methods in Enzymology 152, 649-661, 1987; Wilcox et al., Methods in Enzymology 124, 510-533, 1986. Methods of measuring protein in a tissue include enzyme- linked immunoassays ("ELISA"), immuno-diffusion assays, radio-immunoassays, immunoelectrophoresis, Western blot analyses and immunohistochemical staining techniques. See generally, Ausubel et al., Current Protocols in Molecular Biology, Wiley Press, 1993; Antibodies, a Laboratory Manual, Cold Spring Harbor Press, 1988; and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989.

PCR methods of amplifying nucleic acids utilize at least two primers. One of these primers is capable of hybridizing to a first strand of the nucleic acid to be amplified and of priming enzyme-driven nucleic acid synthesis in a first direction. The other is capable of hybridizing the reciprocal sequence of the first strand (if die sequence to be amplified is single stranded, this sequence is initially hypothetical, but is synthesized in the first amplification cycle) and of priming nucleic acid synthesis from that strand in the direction opposite the first direction and towards die site of hybridization for the first primer. Conditions for conducting such amplifications, particularly under preferred high stringency conditions, are well known. See, for example, PCR Protocols, Cold Spring Harbor Press, 1991.

The samples that are amenable to assaying or staining for REST protein or nucleic acid include, without limitation, cells or tissues (including nerve tissues), protein extracts, nucleic acid extracts and biological fluids such as cerebral fluid, serum and plasma. Preferred samples are nervous system-derived samples. In screening assays for antagonists of the activity of REST, the agents to be screened include a great variety of chemicals including, but not limited to, biologically active molecules such as peptides, carbohydrates, alkaloids, aromatic compounds, polynucleotides and analogs thereof (particularly analogs that have been rendered more membrane permeable), DNA intercolating compounds and other pharmaceutical agents. One cell-free assay comprises the steps of: providing a nuclear extract, providing a REST protein, providing the nucleotide triphosphates necessary for transcription, providing a promoter sequence that includes an element effective to bind to REST and thereby be inhibited, providing a candidate compound or a cocktail of candidate compounds, mixing the extract, protein, promoter, nucleotide triphosphates, and candidate compound(s), incubating the mixture to allow transcription to proceed, and determining the level of the resulting transcription from the promoter, relative increases in transcription reflecting an inhibition of either the binding of REST to the promoter element or the activity of the suppressor domain of REST. For nuclear extracts from REST-expressing cells, the extract itself will generally provide sufficient amounts of die REST protein. Sufficient amounts of the nucleotide triphosphates may also be found in the nuclear extract; however, generally, additional nucleotide triphosphates are added to reduce the variability of the assay. The level of transcription is determined by primer extension as described by Bodner and Karin, Cell 50, 267-275, 1987. One embodiment of the cellular assay comprises the steps of: providing a eukaryotic cell line that expresses the REST protein (either natively or through a stable or transient transfection), providing a suitable medium for maintaining the cell line, adding to the medium a candidate compound or a cocktail of candidate compounds, incubating the cells to allow transcription to proceed, and determining the level of transcription from a REST-responsive promoter. One way of determining the level of transcription is to have provided the cells with a REST- responsive promoter coupled to a gene for a readily measurable gene product. This method is, of course, indirect, since it requires the transcript, which one would prefer to directly measure, to be translated into a protein that is then measured. Nonetheless, the method is widely recognized as a surrogate measure of transcription. The appropriate RNA transcript is also measured by methods well known in the art, such as dot-blot hybridization or by Northern Blot analysis.

The REST protein has a negative influence on the activity of many promoters having an REl or an REl -like sequence (such as that of the promoter for SCG10). Direct cloning strategies for such negative factors are difficult since they require time consuming measurements of the loss of a property. To create a positive signal that can more facilely be used to screen a cDNA library for REST-related cDNAs, a HeLa cell cDNA library was created to express fusion proteins between cDNA-encoded polypeptides and the activation domain of the yeast GAL4 regulatory protein. The library was designed to identify a clone encoding a fusion protein having an REl -binding domain and a GAM activation domain. Such a fusion protein acts as a positive transcription factor on appropriate REl -containing promoter. A HeLa cell library was selected because HeLa cells do not express the type II voltage dependent sodium channel and express an REl -binding activity.

The invention is described in more detail, but without limitation, by reference to the examples set forth below.

Example 1 - "One-Hvbrid" Cloning of Three Partial Sequences a. Yeast Strains

The cloning strategy employed yeast containing two reporter genes having REl regulatory sequences in or adjacent to their promoters. One reporter gene was HIS3, which confers to yeast the ability to grow in media that lacks the amino acid histidine, functionally attached to the yeast GALl promoter. The GALl promoter is normally inactive in the absence of a yeast activator protein such as GAM. The other reporter gene was the bacterial lac z gene functionally coupled to the yeast CYC1 promoter. The CYC1 promoter is normally inactive in the absence of a yeast activator protein such as GAM.

i. The HIS3 Construct Four copies of the 28 bp REl nucleic acid, SEQ ID NO:29, which had been synthesized by standard oligonucleotide synthesis methods, were cloned into a unique EcoRI site on yeast expression shuttle vector pTHl (described by Flick and Johnson, Mol. Cell. Biol. 10(9), 4757-4769, 1990). The EcoRI site is adjacent (and 5') to a yeast GALl promoter that is functionally linked to a HIS3 gene. The shuttle vector also contained a marker gene that directed the expression of a gene that confers to yeast the ability to grow in the absence of the pyrimidine base uracil. A derivative plasmid containing four properly oriented copies of the REl sequence, as confirmed by sequence analysis, was isolated and designated pJAC12.

ii. The Lac z Construct

Four copies of the 28 bp REl nucleic acid, SEQ ID NO:29, were cloned between the Pst and BamHI sites upstream of the CYC1 promoter found on expression vector pCZi3gal (described by Lue and Kornberg, Proc. Natl. Acad. Sci. USA 84, 8839-8843, 1993), which promoter is functionally linked to a bacterial lac z gene. The vector also contained a marker gene that directed the expression of a gene that confers to yeast the ability to grow in the absence of the amino acid tryptophan. A derivative plasmid containing four properly oriented copies of the REl nucleic acid, as confirmed by sequence analysis, was isolated and designated pJAC13.

KΪ. Yeast Transformation To Incorporate Reporter Genes The reporter plasmids were linearized and introduced sequentially into a standard yeast strain (strain W303) by the LiAc method (Schiestl and Geitz, Curr. Gen. 16, 339-346, 1989). Transformants were selected by growth on plates lacking uracil (indicating the integration of pJAC12) and tryptophan (indicating the integration of pJAC13). Small scale preparations of total yeast genomic DNA were prepared from four colonies according to the method of Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Press, 1986, to confirm integration of me pJAC12 and pJAC13 reporter vectors into the yeast genome by Southern blot analysis using the REl, CYC1 promoter, HIS3 gene, and TRP1 gene as probes. One of these four transformants was then utilized for the subsequent cDNA library transformation. This reporter strain was assessed for growth on his- plates and screened for /J-galactosidase activity and, as expected, was negative for both markers.

iv. Control Reporter Strain

By the same methods described above, a control strain derived from W303 was created that incorporated analogs of pJAC12 and pJAC13, wherein the REl nucleic acids were substituted with four copies of the inactive mutant REl nucleic acid, SEQ ID No. 30, described by Kraner et al., Neuron 9, 37-44, 1992.

b. cDNA Cloning

A HeLa cell cDNA library was constructed using the pGADGH plasmid containing the GAM activation domain (see Li and Herskowitz, Science 262: 1870-1874, 1993) functionally linked to a GAM promoter and having a polylinker site (including EcoRI and Xhol sites), located downstream of the activator domain sequence for inserting the cDNA. The library plasmid contains a marker for the ability to grow in the absence of the amino acid leucine. The library was linearized and introduced into the yeast reporter strain by the LiAc method. The cells were plated in leucine minus and histidine minus agar plates to select colonies that are putatively transformed with a cDNA to express an fusion protein having an REl binding domain (derived from cDNA) and a GAM activation domain. One hundred his⁺ colonies were impressed onto filter paper and permeabilized by freeze-thawing. The filter paper was layered onto another filter paper containing the β- galactosidase substrate 5-bromo-4-chloro-3-indoyl-b-D-galactoside (X-gal, available from Sigma Chemical Co., St. Louis). The filter paper was incubated at room temperature and monitored for blue spots, which indicate /3-galactosidase positive colonies. Four colonies that were positive for the lac z marker were isolated. Plasmids containing the cDNA from these four colonies was isolated as described by Bartel et al., in Cellular Interactions in Development: A Practical Approach, D.A. Hartley, ed.. New York: Oxford University Press, 1994, pp 53-179, and amplified in bacteria. The plasmids were introduced into the control yeast strain (wherein the reporter gene promoters contained mutant REl sequences). Three of the four plasmids failed to transform the control strain, indicating that the fusion proteins they encoded interacted specifically with the REl nucleic acid. These plasmids were designated p73, p90 and p613. The three insert cDNAs were sequenced by the chain termination method (Sanger et al., Proc. Natl. Acad. Sci. USA 74, 998-1002, 1977) and found to include the sequences of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, all of which encode overlapping portions of an apparent zinc-finger DNA-binding domain (nucleotides 216-1622, 636-1725 and 695-1622 of Fig. 1, respectively).

Example 2 - Cloning of Two Overlapping Sequences Encoding REST SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 were used to probe another HeLa cell cDNA library that was cloned into the Lambda Zap II phage (Stratagene, Inc., San Diego, CA). Two phage isolates containing overlapping cDNAs of 3082 and 4408 bp were isolated (phages NH2 and NH7, respectively). These cDNAs are designated SEQ ID NO: 6 and SEQ ID NO:7 and encode nucleotides -175-1616 and 1472-5324 of Fig. 1, respectively. From the overlap of these two cDNAs, most of the full length REST cDNA can be deduced. The 5' segment, up to position -325, was determined by applying the 5' RACE PCR technique to HeLa cell cDN A. This segment is designated SEQ ID NO: 1. The deduced amino acid sequence of REST is shown is Figure 1. Note that Lambda Zap II is readily convertible to the Bluescript plasmid using EcoRI as outlined by the supplier.

Example 3 - Expression of REST Antigen and Polvclonal Antibody Production

For example 3, a 1.5 kilobase EcoRI-XhoI fragment of p73 comprising all of SEQ ID NO:3 was cloned in phase with the cDNA for glutathione s-transferase ("GST") in the commercial vector pGEX4T3 (Pharmacia, Uppsala, Sweden). The GST-REST fusion protein was produced in E.coli strain XL-1 blue (Stratagene, San Diego, CA) and purified on a glutathione-Sepharose column (Pharmacia, Uppsala, Sweden). The purified fusion protein was used to immunize two rabbits (Pocono Rabbit Farms, PA) to produce a polyclonal antibody preparation against REST.

Example 4 - RNA Hybridization (Northern Blots)

Total cellular RNA from HeLa cells, PC12 cells, L6 skeletal muscle cells and dorsal root ganglion was isolated as described by Toledo-Aral et al., Neuron, in press) and poly-A⁺ -selected using a commercially available kit (Pharmacia, Inc., Uppsala, Sweden). Messenger RNA (2-4 μg) was fractionated on denaturing gels and then electrophoretically transferred onto nylon paper for hybridization. A DNA probe of human REST was generated by random primer labeling of the EcoRI - Xhol fragment of p73, which includes the nucleic acid of SEQ ID NO:3, to incorporate ³²P. A rat REST cDNA (600 bp) was obtained by PCR (with an initial reverse-transcriptase step) of rat skeletal muscle mRNA using a degenerate primer modelled on the sequence of amino acids 146 to 153 (nucleotides 481 to 504) of the plus strand of SEQ ID NO: l and a degenerate primer modelled on the amino-acid-encoding sequence of amino acid residues 363 to 370 (nucleotides 1087 to 1110) of the minus strand of SEQ ID NO: l. The PCR-amplified cDNA was cloned into pGEM-7Z (Promega.Madison, WI), and workable amounts of the plasmid were grown in bacteria. A rat REST riboprobe was manufactured by linearizing the plasmid with AccI and transcribing it with T7 polymerase in the presence of ³²P-UTP (Dupont, Wilmington, DE). A riboprobe for the CNS-type sodium channel was made as described by D'Arcangelo et al., J. Cell Biol., 10(9), 4757-4769, 1993. Hybridization and washing conditions used with the rat REST and sodium channel riboprobes were as described by Toledo- Aral et al., Neuron, in press; for the human REST DNA probe, the hybridization and washing solutions were the same as those used for the riboprobes, except that the blots were hybridized at 37°C and washed at 32°C.

Northern blot analysis for mRNS for the CNS-type sodium channel and REST in a number of cell types and tissues produced the following results: CNS-type Sodium Channel

Cell or Tissue Type mRNA REST mRNA

HeLa cells none high levels rat L6 skeletal muscle cells none high levels rat PC 12 cells high level extremely low levels mouse dorsal root ganglia extremely low levels high levels

Example 5 - Western Blot Analysis Western immunoblots of proteins derived from nuclear extracts were performed according to standard procedures, as described by Sambrook et al.. Molecular Cloning: A Laboratory Manual, Cold Spring Harber Lab., Cold Spring Harbor, NY, 1989. Nuclear extracts were prepared by the single lysis method (Sambrook et al., 1989). Extracts were combined with an equal volume of 2X Laemmli sample buffer (Laemmli, Nature, 227, 680- 685, 1970) and boiled for 15 minutes. Samples were resolved by SDS-PAGE on 7.5% gels, transferred to nitrocellulose, and the nitrocellulose was blocked with 10% milk in TTBS (Sambrook et al., 1989). Immunoblotting was performed using the enhanced chemiluminescence method using a commercial kit (Amersham, Burlington, MA). The antibody to REST-GST was used at a 1:20 dilution after purification by FPLC on an alkyl Superose (a highly crosslinked agarose substituted with octyl groups) column (Pharmacia, Uppsala, Sweden).

Nuclear extracts were made from the PC 12 cell line derived from a neural pheochromocytoma, which expresses the CNS-type voltage-dependent sodium channel and does not express an REl binding activity, and from HeLa cells, which do not express the CNS-type voltage-dependent sodium channel and do express an REl binding activity. Western blots probed with the polyclonal antibodies to human REST indicated the presence of an immunoreactive protein of molecular weight 121 kDa in HeLa cell nuclear extracts, but no immunoreactive protein in PC 12 cell nuclear extracts.

Example 6 - In Situ Hybridization

The developmental pattern of expression of REST was analyzed by in situ hybridization in mouse embryos. A 600 bp fragment of mouse REST cDNA (encompassing most of the zinc finger domain) was prepared from 8.5 day mouse embryos by the PCR method described in Example 4 for the preparation of rat REST cDNA. The amplification product was cloned into a Bluescript vector (Stratagene, San Diego, CA) and partially sequenced using the Sequenase Kit (US Biochemicals, Cleveland, OH). In situ hybridization of intact embryos using digoxigenin (DIG-11-UTP, available from Boehringer Mannheim) labeled RNA probes for mouse Hox-Bl (Frohman at al., Development, 110, 589-608, 1990), and Gbx-2 (Frohman et al., Mouse Genome, 91, 323-325, 1993). Hybridization was performed using a published protocol (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989). In brief, embryos were fixed overnight in paraformaldehyde, incubated in hydrogen peroxide to inactivate endogenous phosphatases, lightly proteinase K digested, refixed, and hybridized at 70°C in 1 ml of 50% formamide, 5 x SSC pH 4.5, 50 μg/ml yeast RNA, 1 % SDS, 50 μg/ml heparin, 0.1 % CHAPS, and 5mM EDTA containing 1 μg of probe. The embryos were rinsed in a low wash solution (50% formamide, 5 x SSC, pH 4.5, 1 % SDS, 0.1 % CHAPS; 70°C), treated with RNAse A, rinsed with a high stringency wash solution (50% formamide, 2 x SSC, pH 4.5, 0.1 % CHAPS; 65°C), and incubated with an alkaline-phosphatase coupled rabbit anti-digoxin antisera (Boehringer Mannheim, Indianapolis, IN) The enzyme activity of the reporter was detected by a color reaction with 5-bromo-4- chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT), which resulted in the deposition of a water-insoluble purple precipitate. Embryos were rinsed, washed into 80% glycerol, and photographed intact and in slices. The in situ hybridization results for 9.5 day embryos indicated the presence of abundant

REST mRNA in all tissues except the developing brain and spinal cord. Robust expression of REST mRNA was found in neural crest-derived dorsal root ganglia, indicating the expression of REST in some non-CNS neural tissue.

Example 7 - Mobility Shift Assays for Proteins That Bind REl Sequences

The presence of REl binding activity in various cells and tissues was tested using a gel mobility shift assay. Nuclear extracts from HeLa, L6, and primary cultures of rat embryonic skeletal muscle cells were prepared as described by Dignam et al., Nucl. Acids Res. , 11 , 1475- 1489, 1983. The extracts were preincubated 15 minutes at room temperature with either buffer control, competitor DNA, REST-GST polyclonal antisera, or rabbit preimmune serum, and then incubated for two hours at room temperature with a 114 bp ³ P end-labeled DNA probe containing nucleotides -1051 to 837 of the 5' flanking sequence for the CNS-type sodium channel gene, which promotes sequence includes the REl sequence. The samples were resolved by electrophoresis on a 5% non-denaturing polyacrylamide gel, which was then autoradiographed. The presence of binding was indicated by the presence of a DNA complex that moved more slowly in the gel than does the free DNA probe.

The results were that HeLa, L6 and rat embryonic skeletal muscle all contained an REl binding activity that was competed away with excess unlabelled REl containing DNA but not by DNA containing the inactive REl mutant described by Kraner et al., Neuron, 9, 37-44, 1992. The polyclonal antisera to the REST-GST fusion further retarded mobility, while pre- immune serum had no effect. This result indicates that a REST-like protein is responsible for the binding indicated by the gel shift assay.

Example 8 - Expression Vector Encoding The Complete Human REST Protein

The NH2 vector containing the nucleic acid of SEQ ID NO: 6 was digested with Hind III and Hinc II; and the NH7 vector containing the nucleic acid of SEQ ID NO:7 was digested with Hinc II and Bgl II. The excised inserts were subcloned into a Hind III and Bam HI digested pCMV I-amp (Invitrogen, Inc., San Diego) vector. The Hinc II digestion cleaved the overlap region of NH2 and NH7 at nucleotide 1575, allowing for a contiguous insert of nucleotides -175 through 3656 to be isolated.

Example 9 - Transfection Studies of REST Function Transient transfection of PC 12 cells with a plasmid containing the chloramphenicol acetyl transferase (CAT) gene attached to the REl -containing promoter for the CNS-type sodium channel results in the expression of CAT (the plasmid designated herein as "type II- CAT"). This plasmid has been described by Kraner et al., Neuron, 9, 37-44, 1992. A control CAT vector driven by the strong rous sarcoma virus (RSV) promoter has been described by Kraner et al., 1992 and Gorman et al., Proc. Natl. Acad. Sci. USA 79, 6777-6781, 1982. To test whether this expression could be shut-down by the REST protein, cotransfection experiments using the type II-CAT plasmid and a plasmid containing the REST cDNA coupled to the cytomegalovirus ("CMV") promoter were undertaken. A fragment of the REST cDNA, encoding the entire REST protein, with HindlH and Bgll termini (including nucleotides -175 to 3656 of SEQ ID NO:l) was subcloned downstream of the CMV promoter in the commercial mammalian expression vector pCDNA 1-amp (InVitrogen, Inc., San Diego, CA) between the HindlH and BamHI sites to create the CMV-REST vector. The resulting expression vector was designated REST-Express. Rat PC12 cells were transfected with 30 μg of REST-Express and 30 μg of either type II-CAT or RSV-CAT by electroporation (Kraner et al., 1992). Forty-eight hours after transfection the cells were harvested, centrifuged and lysed by freeze-thaw cycles. The supernatant was analyzed for CAT activity as previously described in Maue et al.. Neuron, 4, 223-231, 1990. A cDNA encoding the Zn finger region of REST (including nucleotides 481 to 1236 of SEQ ID NO:l) was cloned independently into the pCDNAl-amp vector and was used as an interfering form of REST in transient transfection assays. L6 muscle cells and PC 12 cells were transfected with 30 μg of the interfering REST vector along with 30 μg of type II- CAT plasmid by electroporation and treated as above.

The results were that co-transfection into PC 12 cells of REST-Express along with the type II-CAT resulted in a ten-fold decrease in activity versus the activity seen with type II-CAT alone. REST-Express had not effect on the expression of CAT by RSV-CAT. The interfering REST vector, encoding just the DNA binding domain of REST, had no effect on the expression of type II-CAT in PC12 cells. However, in L6 muscle cells, which contain an endogenous REST activity, the interfering REST vector derepressed the expression of type II-CAT, which is otherwise inactive in L6 cells. This latter result is consistent with REST having a suppressor function that is held in the vicinity of the promoter for the CNS-type sodium channel by the DNA-binding domain. By competing the complete REST protein from the promoter, the interfering form of REST - containing only the DNA-binding domain - de-represses the promoter.

Example 10 - Localization of the Repressor Function

A number of restriction fragments were isolated from the full length expression clone described in Example 8 or from the NH2 clone and subcloned into the CMV-promoted expression vector also described in Example 8. Two other REST fragments were available from cDNA library screenings. These were clones NH10 and NH12, which contain nucleotides 121-1581 and 25-1308 of Figure 1, respectively (which sequences are designated SEQ ID NO:27 and 28). The inserts of these clones were excised with EcoRI and subcloned into the CMV-promoted vector. In total, the inserts subcloned into the expression vector had the following sequence from Figure 1 : 1. Nucleotides 31-3976

2. Nucleotides 31-2234

3. Nucleotides 31-1940

4. Nucleotides 121-1581

5. Nucleotides 25-1308 6. Nucleotides 31-2491 and 2683-3976

In the last of these clones, the sequence between two BstXI restriction sites is excised. These subclones are co-transfected with PC 12 cells along with the type II-CAT plasmid as described above to determine the silencing potential of the expressed fragment.

Example 11 - Designing PCR Amplification Primers

The PCR primers used to amplify sequences encoding amino acid residues 146 through 370 in Example 4 were designed as follows. First, the 146 to 153 sequence was translated into the following sequence-encoding nucleic acid sequence (SEQ ID NO:8): TGYAARCCNTGYCARTAYGARGCN, where Y = T/C, R =• A/G and N = A/G/T/C. Next, the sequence of amino acid residues 363 to 370 was translated as above. This translated sequence was used to define the following opposite strand sequence (SEQ ID NO: 9): NGTYTTRTARTCRCARTGNGGRCA.

While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations in the preferred compositions and methods may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, U is invention includes all modifications encompassed within the spirit and scope of the invention as defined by the claims that follow the Sequence Listing.

SEQUKNCB LISTING

(1) GENERAL INFORMATION (i) APPLICANT: Mandel, Gail, Chong, Jayhong A. (ii) TITLE OF INVENTION: REST Protein and DNA (iii) NUMBER OF SEQUENCES: 29 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Dechert Price & Rhoads

(B) STREET: P.O. Box 5218 (C) CITY: Princeton

(D) STATE: New Jersey

(E) COUNTRY: USA

(F) ZIP: 08543-5218

(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 3.50 inch, 1.44 Mb storage

(B) COMPUTER: IBM-compatible

(C) OPERATING SYSTEM: DOS 5.0

(D) SOFTWARE: WordPerfect (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER:

(B) FILING DATE: March 23, 1995

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Allen Bloom (B) REGISTRATION NUMBER: 29,135

(C) REFERENCE/DOCKET NUMBER: 317743-101 WO (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (609) 520-3214

(B) TELEFAX: (609) 520-3259 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 5648 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A. , Tapia-Ramirez Jose, Toledo-Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D., Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell (D) VOLUME: 80

(E) ISSUE:

(F) PAGES:

(G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:l:FROM -1 TO 5648 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

ATCTGGCGCG GCGTAGCCCT GTGTTGGAAT GTGCGGCTGC CGCGAGCTCG 50

CGGCGCAGCA GCGGAGCGAG CGCCGCCGAG GCCCGGGGCC CCAGACCCTG 100

GCGGCGGCTG CGGCAGCCGA GACGGCAGGG CGAGGCCCGG AGGCCTGAGC 150

ACCCTCTGCA GCCCCACTCC TGGGCCTTCT TGGTCCACGA CGGCCCCAGC 200

ACCCAACTTT ACCACCCTCC CCCACCTCTC CCCCGAAACT CCAGCAACAA 250

AGAAAAGTAG TCGGAGAAGG AGCGGCGACT CAGGGTCGCC CGCCCCTCCT 300

CACCGAGGAA GGCCGAATAC AGTT 324

ATG GCC ACC CAG GTA ATG GGG CAG TCT TCT GGA GGA GGA GGG CTG 369 Met Ala Thr Gin Val Met Gly Gin Ser Ser Gly Gly Gly Gly Leu 1 5 10 15

TTT ACC AGC AGT GGC AAC ATT GGA ATG GCC CTG CCT AAC GAC ATG 414 Phe Thr Ser Ser Gly Asn lie Gly Met Ala Leu Pro Asn Asp Met 20 25 30

TAT GAC TTG CAT GAC CTT TCC AAA GCT GAA CTG GCC GCA CCT CAG 459 Tyr Asp Leu His Asp Leu Ser Lys Ala Glu Leu Ala Ala Pro Gin

35 40 45 CTT ATT ATG CTG GCA AAT GTG GCC TTA ACT GGG GAA GTA AAT GGC 504 Leu lie Met Leu Ala Asn Val Ala Leu Thr Gly Glu Val Asn Gly 50 55 60

AGC TGC TGT GAT TAC CTG GTC GGT GAA GAA AGA CAG ATG GCA GAA 549 Ser Cys Cys Asp Tyr Leu Val Gly Glu Glu Arg Gin Met Ala Glu 65 70 75

CTG ATG CCG GTT GGG GAT AAC AAC TTT TCA GAT AGT GAA GAA GGA 594 Leu Met Pro Val Gly Asp Asn Asn Phe Ser Asp Ser Glu Glu Gly

80 85 90

GAA GGA CTT GAA GAG TCT GCT GAT ATA AAA GGT GAA CCT CAT GGA 639 Glu Gly Leu Glu Glu Ser Ala Asp lie Lys Gly Glu Pro His Gly 95 100 105

CTG GAA AAC ATG GAA CTG AGA AGT TTG GAA CTC AGC GTC GTA GAA 684 Leu Glu Asn Met Glu Leu Arg Ser Leu Glu Leu Ser Val Val Glu 110 115 120

CCT CAG CCT GTA TTT GAG GCA TCA GGT GCT CCA GAT ATT TAC AGT 729 Pro Gin Pro Val Phe Glu Ala Ser Gly Ala Pro Asp lie Tyr Ser 125 130 135

TCA AAT AAA GCT CTT GCC CCT GAA ACA CCT GGA GCG GAG GAC AAA 774 Ser Asn Lys Ala Leu Ala Pro Glu Thr Pro Gly Ala Glu Asp Lys 140 145 150

GGC AAG AGC TCG AAG ACC AAA CCC TTT CGC TGT AAG CCA TGC CAA 819 Gly Lys Ser Ser Lys Thr Lys Pro Phe Arg Cys Lys Pro Cys Gin

155 160 165

TAT GAA GCA GAA TCT GAA GAA CAG TTT GTG CAT CAC ATC AGA GTT 864 Tyr Glu Ala Glu Ser Glu Glu Gin Phe Val His His He Arg Val 170 175 180

CAC AGT GCT AAG AAA TTT TTT GTG GAA GAG AGT GCA GAG AAG CAG 909

His Ser Ala Lys Lys Phe Phe Val Glu Glu Ser Ala Glu Lys Gin

185 190 195 GCA AAA GCC AGG GAA TCT GGC TCT TCC ACT GCA GAA GAG GGA GAT 954 Ala Lys Ala Arg Glu Ser Gly Ser Ser Thr Ala Glu Glu Gly Asp 200 205 210

TTC TCC AAG GGC CCC ATT CGC TGT GAC CGC TGC GGC TAC AAT ACT 999 Phe Ser Lys Gly Pro He Arg Cys Asp Arg Cys Gly Tyr Asn Thr 215 220 225

AAT CGA TAT GAT CAC TAT ACA GCA CAC CTG AAA CAC CAC ACC AGA 1044 Asn Arg Tyr Asp His Tyr Thr Ala His Leu Lys His His Thr Arg

230 235 240

GCT GGG GAT AAT GAG CGA GTC TAC AAG TGT ATC ATT TGC ACA TAC 1089 Ala Gly Asp Asn Glu Arg Val Tyr Lys Cys He He Cys Thr Tyr 245 250 255

ACA ACA GTG AGC GAG TAT CAC TGG AGG AAA CAT TTA AGA AAC CAT 1134 Thr Thr Val Ser Glu Tyr His Trp Arg Lys His Leu Arg Asn His 260 265 270

TTT CCA AGG AAA GTA TAC ACA TGT GGA AAA TGC AAC TAT TTT TCA 1179 Phe Pro Arg Lys Val Tyr Thr Cys Gly Lys Cys Asn Tyr Phe Ser 275 280 285

GAC AGA AAA AAC AAT TAT GTT CAG CAT GTT AGA ACT CAT ACA GGA 1224 Asp Arg Lys Asn Asn Tyr Val Gin His Val Arg Thr His Thr Gly 290 295 300

GAA CGC CCA TAT AAA TGT GAA CTT TGT CCT TAC TCA AGT TCT CAG 1269 Glu Arg Pro Tyr Lys Cys Glu Leu Cys Pro Tyr Ser Ser Ser Gin

305 310 315

AAG ACT CAT CTA ACT AGA CAT ATG CGT ACT CAT TCA GGT GAG AAG 1314 Lys Thr His Leu Thr Arg His Met Arg Thr His Ser Gly Glu Lys 320 325 330

CCA TTT AAA TGT GAT CAG TGC AGT TAT GTG GCC TCT AAT CAA CAT 1359 Pro Phe Lys Cys Asp Gin Cys Ser Tyr Val Ala Ser Asn Gin His 335 340 345 GAA GTA ACC CGC CAT GCA AGA CAG GTT CAC AAT GGG CCT AAA CCT 1404 Glu Val Thr Arg His Ala Arg Gin Val His Asn Gly Pro Lys Pro 350 355 360

CTT AAT TGC CCA CAC TGT GAT TAC AAA ACA GCA GAT AGA AGC AAC 1449 Leu Asn Cys Pro His Cys Asp Tyr Lys Thr Ala Asp Arg Ser Asn 365 370 375

TTC AAA AAA CAT GTA GAG CTA CAT GTG AAC CCA CGG CAG TTC AAT 1494 Phe Lys Lys His Val Glu Leu His Val Asn Pro Arg Gin Phe Asn

380 385 390

TGC CCT GTA TGT GAC TAT GCA GCT TCC AAG AAG TGT AAT CTA CAG 1539 Cys Pro Val Cys Asp Tyr Ala Ala Ser Lys Lys Cys Asn Leu Gin 395 400 405

TAT CAC TTC AAA TCT AAG CAT CCT ACT TGT CCT AAT AAA ACA ATG 1584 Tyr His Phe Lys Ser Lys His Pro Thr Cys Pro Asn Lys Thr Met 410 415 420

GAT GTC TCA AAA GTG AAA CTA AAG AAA ACC AAA AAA CGA GAG GCT 1629 Asp Val Ser Lys Val Lys Leu Lys Lys Thr Lys Lys Arg Glu Ala 425 430 435

GAC TTG CCT GAT AAT ATT ACC AAT GAA AAA ACA GAA ATA GAA CAA 1674 Asp Leu Pro Asp Asn He Thr Asn Glu Lys Thr Glu He Glu Gin 440 445 450

ACA AAA ATA AAA GGG GAT GTG GCT GGA AAG AAA AAT GAA AAG TCC 1719 Thr Lys He Lys Gly Asp Val Ala Gly Lys Lys Asn Glu Lys Ser

455 460 465

GTC AAA GCA GAG AAA AGA GAT GTC TCA AAA GAG AAA AAG CCT TCT 1764 Val Lys Ala Glu Lys Arg Asp Val Ser Lys Glu Lys Lys Pro Ser 470 475 480

AAT AAT GTG TCA GTG ATC CAG GTG ACT ACC AGA ACT CGA AAA TCA 1809 Asn Asn Val Ser Val He Gin Val Thr Thr Arg Thr Arg Lys Ser 485 490 495 GTA ACA GAG GTG AAA GAG ATG GAT GTG CAT ACA GGA AGC AAT TCA 1854 Val Thr Glu Val Lys Glu Met Asp Val His Thr Gly Ser Asn Ser 500 505 510

GAA AAA TTC AGT AAA ACT AAG AAA AGC AAA AGG AAG CTG GAA GTT 1899 Glu Lys Phe Ser Lys Thr Lys Lys Ser Lys Arg Lys Leu Glu Val 515 520 525

GAC AGC CAT TCT TTA CAT GGT CCT GTG AAT GAT GAG GAA TCT TCA 1944 Asp Ser His Ser Leu His Gly Pro Val Asn Asp Glu Glu Ser Ser

530 535 540

ACA AAA AAG AAA AAG AAG GTA GAA AGC AAA TCC AAA AAT AAT AGT 1989 Thr Lys Lys Lys Lys Lys Val Glu Ser Lys Ser Lys Asn Asn Ser 545 550 555

CAG GAA GTG CCA AAG GGT GAC AGC AAA GTG GAG GAG AAT AAA AAG 2034 Gin Glu Val Pro Lys Gly Asp Ser Lys Val Glu Glu Asn Lys Lys 560 565 570

CAA AAT ACT TGC ATG AAA AAA AGT ACA AAG AAG AAA ACT CTG AAA 2079 Gin Asn Thr Cys Met Lys Lys Ser Thr Lys Lys Lys Thr Leu Lys 575 580 585

AAT AAA TCA AGT AAG AAA AGC AGT AAG CCT CCT CAG AAG GAA CCT 2124 Asn Lys Ser Ser Lys Lys Ser Ser Lys Pro Pro Gin Lys Glu Pro 590 595 600

GTT GAG AAG GGA TCT GCT CAG ATG GAC CCT CCT CAG ATG GGG CCT 2169 Val Glu Lys Gly Ser Ala Gin Met Asp Pro Pro Gin Met Gly Pro

605 610 615

GCT CCC ACA GAG GCG GTT CAG AAG GGG CCC GTT CAG GTG GAG CTG 2214 Ala Pro Thr Glu Ala Val Gin Lys Gly Pro Val Gin Val Glu Leu 620 625 630

CCA CCT CCC ATG GAG CAT GCT CAG ATG GAG GGT GCC CAG ATA CGG 2259 Pro Pro Pro Met Glu His Ala Gin Met Glu Gly Ala Gin He Arg 635 640 645 CCT GCT CCT GAC GAG CCT GTT CAG ATG GAG GTG GTT CAG GAG GGG 2304 Pro Ala Pro Asp Glu Pro Val Gin Met Glu Val Val Gin Glu Gly 650 655 660

CCT GCT CAG AAG GAG CTG CTG CCT CCC GTG GAG CCT GCT CAG ATG 2349 Pro Ala Gin Lys Glu Leu Leu Pro Pro Val Glu Pro Ala Gin Met 665 670 675

GTG GGT GCC CAA ATT GTA CTT GCT CAC ATG GAG CTG CCT CCT CCC 2394 Val Gly Ala Gin He Val Leu Ala His Met Glu Leu Pro Pro Pro

680 685 690

ATG GAG ACT GCT CAG ACG GAG GTT GCC CAA ATG GGG CCT GCT CCC 2439 Met Glu Thr Ala Gin Thr Glu Val Ala Gin Met Gly Pro Ala Pro 695 700 705

ATG GAA CCT GCT CAG ATG GAG GTT GCC CAG GTA GAA TCT GCT CCC 2484 Met Glu Pro Ala Gin Met Glu Val Ala Gin Val Glu Ser Ala Pro 710 715 720

ATG CAG GTG GTC CAG AAG GAG CCT GTT CAG ATG GAG CTG TCT CCT 2529 Met Gin Val Val Gin Lys Glu Pro Val Gin Met Glu Leu Ser Pro 725 730 735

CCC ATG GAG GTG GTC CAG AAG GAG CCT GTT CAG ATA GAG CTG TCT 2574 Pro Met Glu Val Val Gin Lys Glu Pro Val Gin He Glu Leu Ser 740 745 750

CCT CCC ATG GAG GTG GTC CAG AAG GAA CCT GTT AAG ATA GAG CTG 2619 Pro Pro Met Glu Val Val Gin Lys Glu Pro Val Lys He Glu Leu

755 760 765

TCT CCT CCC ATA GAG GTG GTC CAG AAG GAG CCT GTT CAG ATG GAG 2664 Ser Pro Pro He Glu Val Val Gin Lys Glu Pro Val Gin Met Glu 770 775 780

TTG TCT CCT CCC ATG GGG GTG GTT CAG AAG GAG CCT GCT CAG AGG 2709 Leu Ser Pro Pro Met Gly Val Val Gin Lys Glu Pro Ala Gin Arg 785 790 795 GAG CCA CCT CCT CCC AGA GAG CCT CCC CTT CAC ATG GAG CCA ATT 2754

Glu Pro Pro Pro Pro Arg Glu Pro Pro Leu His Met Glu Pro He 800 805 810

TCC AAA AAG CCT CCT CTC CGA AAA GAT AAA AAG GAA AAG TCT AAC 2799

Ser Lys Lys Pro Pro Leu Arg Lys Asp Lys Lys Glu Lys Ser Asn 815 820 825

ATG CAG AGT GAA AGG GCA CGG AAG GAG CAA GTC CTT ATT GAA GTT 2844 Met Gin Ser Glu Arg Ala Arg Lys Glu Gin Val Leu He Glu Val

830 835 840

GGC TTA GTG CCT GTT AAA GAT AGC TGG CTT CTA AAG GAA AGT GTA 2889

Gly Leu Val Pro Val Lys Asp Ser Trp Leu Leu Lys Glu Ser Val 845 850 855

AGC ACA GAG GAT CTC TCA CCA CCA TCA CCA CCA CTG CCA AAG GAA 2934

Ser Thr Glu Asp Leu Ser Pro Pro Ser Pro Pro Leu Pro Lys Glu

860 865 870

AAT TTA AGA GAA GAG GCA TCA GGA GAC CAA AAA TTA CTC AAC ACA 2979

Asn Leu Arg Glu Glu Ala Ser Gly Asp Gin Lys Leu Leu Asn Thr

875 880 885

GGT GAA GGA AAT AAA GAA GCC CCT CTT CAG AAA GTA GGA GCA GAA 3024

Gly Glu Gly Asn Lys Glu Ala Pro Leu Gin Lys Val Gly Ala Glu 890 895 900

GAG GCA GAT GAG AGC CTA CCT GGT CTT GCT GCT AAT ATC AAC GAA 3069 Glu Ala Asp Glu Ser Leu Pro Gly Leu Ala Ala Asn He Asn Glu

905 910 915

TCT ACC CAT ATT TCA TCC TCT GGA CAA AAC TTG AAT ACG CCA GAG 3114 Ser Thr His He Ser Ser Ser Gly Gin Asn Leu Asn Thr Pro Glu 920 925 930

GGT GAA ACT TTA AAT GGT AAA CAT CAG ACT GAC AGT ATA GTT TGT 3159

Gly Glu Thr Leu Asn Gly Lys His Gin Thr Asp Ser He Val Cys 935 940 945 GAA ATG AAA ATG GAC ACT GAT CAG AAC ACA AGA GAG AAT CTC ACT 3204 Glu Met Lys Met Asp Thr Asp Gin Asn Thr Arg Glu Asn Leu Thr 950 955 960

GGT ATA AAT TCA ACA GTT GAA GAA CCA GTT TCA CCA ATG CTT CCC 3249 Gly He Asn Ser Thr Val Glu Glu Pro Val Ser Pro Met Leu Pro 965 970 975

CCT TCA GCA GTA GAA GAA CGT GAA GCA GTG TCC AAA ACT GCA CTG 3294 Pro Ser Ala Val Glu Glu Arg Glu Ala Val Ser Lys Thr Ala Leu

980 985 990

GCA TCA CCT CCT GCT ACA ATG GCA GCA AAT GAG TCT CAG GAA ATT 3339 Ala Ser Pro Pro Ala Thr Met Ala Ala Asn Glu Ser Gin Glu He 995 1000 1005

GAT GAA GAT GAA GGC ATC CAC AGC CAT GAA GGA AGT GAC CTA AGT 3384 Asp Glu Asp Glu Gly He His Ser His Glu Gly Ser Asp Leu Ser 1010 1015 1020

GAC AAC ATG TCA GAG GGT AGT GAT GAT TCT GGA TTG CAT GGG GCT 3429 Asp Asn Met Ser Glu Gly Ser Asp Asp Ser Gly Leu His Gly Ala 1025 1030 1035

CGG CCA GTT CCA CAA GAA TCT AGC AGA AAA AAT GCA AAG GAA GCC 3474 Arg Pro Val Pro Gin Glu Ser Ser Arg Lys Asn Ala Lys Glu Ala 1040 1045 1050

TTG GCA GTC AAA GCG GCT AAG GGA GAT TTT GTT TGT ATC TTC TGT 3519 Leu Ala Val Lys Ala Ala Lys Gly Asp Phe Val Cys He Phe Cys

1055 1060 1065

GAT CGT TCT TTC AGA AAG GGA AAA GAT TAC AGC AAA CAC CTC AAT 3564 Asp Arg Ser Phe Arg Lys Gly Lys Asp Tyr Ser Lys His Leu Asn 1070 1075 1080

CGC CAT TTG GTT AAT GTG TAC TAT CTT GAA GAA GCA GCT CAA GGG 3609 Arg His Leu Val Asn Val Tyr Tyr Leu Glu Glu Ala Ala Gin Gly 1085 1090 1095

CAG GAG TAATG AAACTTTGAA CAAGGTTTCA GTTCTTAGTT 3650

Gin Glu TGTAAGGTAT ATTACATTTT ATATTCATTT ATGATAGCAG ACAACCTTTT 3700

AAGATTGCTT TAATTAGTAT CTGATGTTGA TTTTTAAGTG GCATTCTTTT 3750

CCTTAGGACT TTTTATGTAT ACCTGTTGAT TGTTGTGTAA ATTTTAGTAA 3800

ATCTAAGAGA GTGTACTAAA CCAGCAGGTA TCTGTTAGCT TATGTGTTTA 3850

ATTGAAATTA GAAGGCTAAG ATGGTATAAC AGCATTTTAT TGCTTTGTCC 3900

AGCTACAACA TGTCATTTTT TTCTCCATGT CTTATCTTCC TGTTTCACTT 3950

TAGTTTATTC TTCGTTTTTT ATTGAGATCT ATAAAAAATT GGCTTACTTA 4000

ATAGCAAATT ACTTGAAGAA TTTGCCTGCT TTATATAAAG TTAGCACTTT 4050

AAGATTTTTT TTTTAGAGAT GAGAAGACAT TTAAATTGAA GAAAAATTCC 4100

CCCAGCAATA GACAGTCTAT CAGTCCAAGT ATTTACTTCC TGAGTTTTGA 4150

TCAATATTTT TTATTTGTGT ATGTTAATCG TCATAAAAAC AGTGATTTTG 4200

GTGTGTTTTT TATTTTGGTG CTTTAATGGC TTAAGATGTT GCACATTTTT 4250

-pTTTTCTTTT GGTTTCTGTT TATGTTTTTT TGCCTATGCA GTTAAATTTT 4300

TCCTAGAAAT AGCATTTGTG TTGAACAGTA ACACTTTATA CATATATATA 4350

TGCATGTTTA TTTTGTTTGG CGTCTTTGGA GGGATGCTTT TAGACTTGTT 4400

TGCAAAAGGG CAGTTTTCTT TTTCTTTGCT GCAGTTGTCT ATTTTGCAGA 4450

ATAATAGTGT GTGCAAGTTT GTGAGCAAAT GAAATATGCA GGTTCAATCT 4500

ATTGATTTTG ATTTTTACAT CTTATATCTA TGCCAGAATC TGTATTTCAT 4550

ATAACTTATT TATTTCGAAT GGATGTAGTA AATTCACAGC TATCAGTTTT 4600

GATTTTGCAA TAAATAAACC ACTAGGTTGC ATGTCGAACA AATTTTTATC 4650

TCAAATACCA ACCATCAGTT TTTTTTTTCA TGTGTTTTGG TACAGCTAAT 4700 TCCTAATTGT AGAGTGTTAA ATGTTTGAGG AGAACCTTTT CTCATAGATG 4750

GTTGGTGTTC ATATGGCNAC TTTACAATAA AGAGAACTGT AAGTGATATT 4800

TGGAAACTAC AAACCTGGAA TTAGGAGATA TAATTATTCC TTCAAGTTTT 4850

ATAGATATCA CTTGGGAGAT TCCAAAGCCA TAGCTATTAC GCNGCAAACC 4900

TAGGATAAGA AAGGTAGTAT GAGTGCTGGT AGACCAGCTG CAACATTTCC 4950

TATATCAGAT GAAAAAGGCT GGTGAAACAA GTACAGTCCA GATTTTTTAA 5000

AATCATACTT TCTCAGGGAT CTCCACAAAC TGGTGGGTGT CCTGGCTGTC 5050

TGTGTGATAG CCTCTTTCTA TAGGTGAGGC CTCAAATGAA TTGCAGCTAT 5100

CCTGGTGTTC CTATGAGGGC ACTTGTATGA AAAAGGCAGT ACTCCAAAAC 5150

ATTTTTGATG GTTCTTTGGC CAGTTGCCAA AGAGTGTGAA AGAATCCAAT 5200

AGAGGATTTT TCTTACTGAT AGCAGTCATT CATTGCAGTA AAATAAAATA 5250

TGAATTCCCA TTAGGGAATC TTGAATTCTG ACCTCCCATA CTCCGTTTTG 5300

AAATAACCAC TTATATTTCA TTTTTTAAAA ATCTGATGAT CTCTTTGAGG 5350

CAGGTTTCAG ATTTGGCAGT ACAACATGAA AGATTAGGAA AAGCATTAAT 5400

AACGTGTGGG TGGAAAGCTT GTTAAAAATC TGAGAGTGAA GTTTGAGTTA 5450

AAAGTTGTTT GACATGGCAT TGACTGGGAG GCCAAAGATT TAAAGAAGCG 5500

GAAGATTCTT CTCTTAAGAC ATGAGGAGTA AGTTGTGTGA TAATGGTATG 5550

TGTTTTGTGT GCATGAATGG ACATTGTAAA TGTTGAATTC TAGGCTCCGA 5600

CAATCATTGT CAACAGAAGA TAAAGCTGCA AATATTTATG TTTTAAAA 5648 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 756 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: CDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C. , Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D. , Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80

(E) ISSUE:

(F) PAGES: (G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:2:FROM 1 TO 756 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

TGT AAG CCA TGC CAA 15 Cys Lys Pro Cys Gin

165

TAT GAA GCA GAA TCT GAA GAA CAG TTT GTG CAT CAC ATC AGA GTT 60 Tyr Glu Ala Glu Ser Glu Glu Gin Phe Val His His He Arg Val 170 175 180

CAC AGT GCT AAG AAA TTT TTT GTG GAA GAG AGT GCA GAG AAG CAG 105

His Ser Ala Lys Lys Phe Phe Val Glu Glu Ser Ala Glu Lys Gin

185 190 195 GCA AAA GCC AGG GAA TCT GGC TCT TCC ACT GCA GAA GAG GGA GAT 150 Ala Lys Ala Arg Glu Ser Gly Ser Ser Thr Ala Glu Glu Gly Asp

200 205 210

TTC TCC AAG GGC CCC ATT CGC TGT GAC CGC TGC GGC TAC AAT ACT 195 Phe Ser Lys Gly Pro He Arg Cys Asp Arg Cys Gly Tyr Asn Thr

215 220 225

AAT CGA TAT GAT CAC TAT ACA GCA CAC CTG AAA CAC CAC ACC AGA 240 Asn Arg Tyr Asp His Tyr Thr Ala His Leu Lys His His Thr Arg 230 235 240

GCT GGG GAT AAT GAG CGA GTC TAC AAG TGT ATC ATT TGC ACA TAC 285 Ala Gly Asp Asn Glu Arg Val Tyr Lys Cys He He Cys Thr Tyr

245 250 255

ACA ACA GTG AGC GAG TAT CAC TGG AGG AAA CAT TTA AGA AAC CAT 330 Thr Thr Val Ser Glu Tyr His Trp Arg Lys His Leu Arg Asn His

260 265 270

TTT CCA AGG AAA GTA TAC ACA TGT GGA AAA TGC AAC TAT TTT TCA 375 Phe Pro Arg Lys Val Tyr Thr Cys Gly Lys Cys Asn Tyr Phe Ser 275 280 285

GAC AGA AAA AAC AAT TAT GTT CAG CAT GTT AGA ACT CAT ACA GGA 420 Asp Arg Lys Asn Asn Tyr Val Gin His Val Arg Thr His Thr Gly

290 295 300

GAA CGC CCA TAT AAA TGT GAA CTT TGT CCT TAC TCA AGT TCT CAG 465 Glu Arg Pro Tyr Lys Cys Glu Leu Cys Pro Tyr Ser Ser Ser Gin 305 310 315

AAG ACT CAT CTA ACT AGA CAT ATG CGT ACT CAT TCA GGT GAG AAG 510 Lys Thr His Leu Thr Arg His Met Arg Thr His Ser Gly Glu Lys 320 325 330

CCA TTT AAA TGT GAT CAG TGC AGT TAT GTG GCC TCT AAT CAA CAT 555 Pro Phe Lys Cys Asp Gin Cys Ser Tyr Val Ala Ser Asn Gin His 335 340 345

GAA GTA ACC CGC CAT GCA AGA CAG GTT CAC AAT GGG CCT AAA CCT 600 Glu Val Thr Arg His Ala Arg Gin Val His Asn Gly Pro Lys Pro 350 355 360 CTT AAT TGC CCA CAC TGT GAT TAC AAA ACA GCA GAT AGA AGC AAC 645

Leu Asn Cys Pro His Cys Asp Tyr Lys Thr Ala Asp Arg Ser Asn

365 370 375

TTC AAA AAA CAT GTA GAG CTA CAT GTG AAC CCA CGG CAG TTC AAT 690

Phe Lys Lys His Val Glu Leu His Val Asn Pro Arg Gin Phe Asn

380 385 390

TGC CCT GTA TGT GAC TAT GCA GCT TCC AAG AAG TGT AAT CTA CAG 735 Cys Pro Val Cys Asp Tyr Ala Ala Ser Lys Lys Cys Asn Leu Gin

395 400 405

TAT CAC TTC AAA TCT AAG CAT 756 Tyr His Phe Lys Ser Lys His 410

(2) INFORMATION FOR SEQ ID NO: 3 : (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 1407 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C. , Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D. , Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80 (E) ISSUE:

(F) PAGES:

(G) DATE: March 24, 1995 (K) RELEVANT RESIDUES IN SEQ ID NO:3:FROM 1 TO 1407 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :

G ATG GCA GAA 10 Met Ala Glu 75

CTG ATG CCG GTT GGG GAT AAC AAC TTT TCA GAT AGT GAA GAA GGA 55

Leu Met Pro Val Gly Asp Asn Asn Phe Ser Asp Ser Glu Glu Gly 80 85 90

GAA GGA CTT GAA GAG TCT GCT GAT ATA AAA GGT GAA CCT CAT GGA 100

Glu Gly Leu Glu Glu Ser Ala Asp He Lys Gly Glu Pro His Gly 95 100 105

CTG GAA AAC ATG GAA CTG AGA AGT TTG GAA CTC AGC GTC GTA GAA 145

Leu Glu Asn Met Glu Leu Arg Ser Leu Glu Leu Ser Val Val Glu 110 115 120

CCT CAG CCT GTA TTT GAG GCA TCA GGT GCT CCA GAT ATT TAC AGT 190

Pro Gin Pro Val Phe Glu Ala Ser Gly Ala Pro Asp He Tyr Ser 125 130 135

TCA AAT AAA GCT CTT GCC CCT GAA ACA CCT GGA GCG GAG GAC AAA 235 Ser Asn Lys Ala Leu Ala Pro Glu Thr Pro Gly Ala Glu Asp Lys

140 145 150

GGC AAG AGC TCG AAG ACC AAA CCC TTT CGC TGT AAG CCA TGC CAA 280

Gly Lys Ser Ser Lys Thr Lys Pro Phe Arg Cys Lys Pro Cys Gin 155 160 165

TAT GAA GCA GAA TCT GAA GAA CAG TTT GTG CAT CAC ATC AGA GTT 325

Tyr Glu Ala Glu Ser Glu Glu Gin Phe Val His His He Arg Val 170 175 180

CAC AGT GCT AAG AAA TTT TTT GTG GAA GAG AGT GCA GAG AAG CAG 370

His Ser Ala Lys Lys Phe Phe Val Glu Glu Ser Ala Glu Lys Gin 185 190 195

GCA AAA GCC AGG GAA TCT GGC TCT TCC ACT GCA GAA GAG GGA GAT 415

Ala Lys Ala Arg Glu Ser Gly Ser Ser Thr Ala Glu Glu Gly Asp 200 205 210 TTC TCC AAG GGC CCC ATT CGC TGT GAC CGC TGC GGC TAC AAT ACT 460 Phe Ser Lys Gly Pro He Arg Cys Asp Arg Cys Gly Tyr Asn Thr 215 220 225

AAT CGA TAT GAT CAC TAT ACA GCA CAC CTG AAA CAC CAC ACC AGA 505 Asn Arg Tyr Asp His Tyr Thr Ala His Leu Lys His His Thr Arg 230 235 240

GCT GGG GAT AAT GAG CGA GTC TAC AAG TGT ATC ATT TGC ACA TAC 550 Ala Gly Asp Asn Glu Arg Val Tyr Lys Cys He He Cys Thr Tyr

245 250 255

ACA ACA GTG AGC GAG TAT CAC TGG AGG AAA CAT TTA AGA AAC CAT 595 Thr Thr Val Ser Glu Tyr His Trp Arg Lys His Leu Arg Asn His 260 265 270

TTT CCA AGG AAA GTA TAC ACA TGT GGA AAA TGC AAC TAT TTT TCA 640 Phe Pro Arg Lys Val Tyr Thr Cys Gly Lys Cys Asn Tyr Phe Ser 275 280 285

GAC AGA AAA AAC AAT TAT GTT CAG CAT GTT AGA ACT CAT ACA GGA 685 Asp Arg Lys Asn Asn Tyr Val Gin His Val Arg Thr His Thr Gly 290 295 300

GAA CGC CCA TAT AAA TGT GAA CTT TGT CCT TAC TCA AGT TCT CAG 730 Glu Arg Pro Tyr Lys Cys Glu Leu Cys Pro Tyr Ser Ser Ser Gin 305 310 315

AAG ACT CAT CTA ACT AGA CAT ATG CGT ACT CAT TCA GGT GAG AAG 775 Lys Thr His Leu Thr Arg His Met Arg Thr His Ser Gly Glu Lys

320 325 330

CCA TTT AAA TGT GAT CAG TGC AGT TAT GTG GCC TCT AAT CAA CAT 820 Pro Phe Lys Cys Asp Gin Cys Ser Tyr Val Ala Ser Asn Gin His 335 340 345

GAA GTA ACC CGC CAT GCA AGA CAG GTT CAC AAT GGG CCT AAA CCT 865

Glu Val Thr Arg His Ala Arg Gin Val His Asn Gly Pro Lys Pro

350 355 360 CTT AAT TGC CCA CAC TGT GAT TAC AAA ACA GCA GAT AGA AGC AAC 910 Leu Asn Cys Pro His Cys Asp Tyr Lys Thr Ala Asp Arg Ser Asn 365 370 375

TTC AAA AAA CAT GTA GAG CTA CAT GTG AAC CCA CGG CAG TTC AAT 955 Phe Lys Lys His Val Glu Leu His Val Asn Pro Arg Gin Phe Asn 380 385 390

TGC CCT GTA TGT GAC TAT GCA GCT TCC AAG AAG TGT AAT CTA CAG 1000 Cys Pro Val Cys Asp Tyr Ala Ala Ser Lys Lys Cys Asn Leu Gin

395 400 405

TAT CAC TTC AAA TCT AAG CAT CCT ACT TGT CCT AAT AAA ACA ATG 1045

Tyr His Phe Lys Ser Lys His Pro Thr Cys Pro Asn Lys Thr Met 410 415 420

GAT GTC TCA AAA GTG AAA CTA AAG AAA ACC AAA AAA CGA GAG GCT 1090 Asp Val Ser Lys Val Lys Leu Lys Lys Thr Lys Lys Arg Glu Ala 425 430 435

GAC TTG CCT GAT AAT ATT ACC AAT GAA AAA ACA GAA ATA GAA CAA 1135 Asp Leu Pro Asp Asn He Thr Asn Glu Lys Thr Glu He Glu Gin 440 445 450

ACA AAA ATA AAA GGG GAT GTG GCT GGA AAG AAA AAT GAA AAG TCC 1180 Thr Lys He Lys Gly Asp Val Ala Gly Lys Lys Asn Glu Lys Ser 455 460 465

GTC AAA GCA GAG AAA AGA GAT GTC TCA AAA GAG AAA AAG CCT TCT 1225 Val Lys Ala Glu Lys Arg Asp Val Ser Lys Glu Lys Lys Pro Ser

470 475 480

AAT AAT GTG TCA GTG ATC CAG GTG ACT ACC AGA ACT CGA AAA TCA 1270 Asn Asn Val Ser Val He Gin Val Thr Thr Arg Thr Arg Lys Ser 485 490 495

GTA ACA GAG GTG AAA GAG ATG GAT GTG CAT ACA GGA AGC AAT TCA 1315 Val Thr Glu Val Lys Glu Met Asp Val His Thr Gly Ser Asn Ser 500 505 510 GAA AAA TTC AGT AAA ACT AAG AAA AGC AAA AGG AAG CTG GAA GTT 1360 Glu Lys Phe Ser Lys Thr Lys Lys Ser Lys Arg Lys Leu Glu Val 515 520 525

GAC AGC CAT TCT TTA CAT GGT CCT GTG AAT GAT GAG GAA TCT TCA 1405 Asp Ser His Ser Leu His Gly Pro Val Asn Asp Glu Glu Ser Ser 530 535 540

AC 1407

(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1090 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS : double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D., Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80

(E) ISSUE:

(F) PAGES: (G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO: :FROM 1 TO 1090 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : C AAG GGC CCC ATT CGC TGT GAC CGC TGC GGC TAC AAT ACT 40

Lys Gly Pro He Arg Cys Asp Arg Cys Gly Tyr Asn Thr 215 220 225

AAT CGA TAT GAT CAC TAT ACA GCA CAC CTG AAA CAC CAC ACC AGA 85 Asn Arg Tyr Asp His Tyr Thr Ala His Leu Lys His His Thr Arg 230 235 240

GCT GGG GAT AAT GAG CGA GTC TAC AAG TGT ATC ATT TGC ACA TAC 130 Ala Gly Asp Asn Glu Arg Val Tyr Lys Cys He He Cys Thr Tyr

245 250 255

ACA ACA GTG AGC GAG TAT CAC TGG AGG AAA CAT TTA AGA AAC CAT 175 Thr Thr Val Ser Glu Tyr His Trp Arg Lys His Leu Arg Asn His 260 265 270

TTT CCA AGG AAA GTA TAC ACA TGT GGA AAA TGC AAC TAT TTT TCA 220 Phe Pro Arg Lys Val Tyr Thr Cys Gly Lys Cys Asn Tyr Phe Ser 275 280 285

GAC AGA AAA AAC AAT TAT GTT CAG CAT GTT AGA ACT CAT ACA GGA 265 Asp Arg Lys Asn Asn Tyr Val Gin His Val Arg Thr His Thr Gly 290 295 300

GAA CGC CCA TAT AAA TGT GAA CTT TGT CCT TAC TCA AGT TCT CAG 310 Glu Arg Pro Tyr Lys Cys Glu Leu Cys Pro Tyr Ser Ser Ser Gin 305 310 315

AAG ACT CAT CTA ACT AGA CAT ATG CGT ACT CAT TCA GGT GAG AAG 355 Lys Thr His Leu Thr Arg His Met Arg Thr His Ser Gly Glu Lys

320 325 330

CCA TTT AAA TGT GAT CAG TGC AGT TAT GTG GCC TCT AAT CAA CAT 400 Pro Phe Lys Cys Asp Gin Cys Ser Tyr Val Ala Ser Asn Gin His 335 340 345

GAA GTA ACC CGC CAT GCA AGA CAG GTT CAC AAT GGG CCT AAA CCT 445

Glu Val Thr Arg His Ala Arg Gin Val His Asn Gly Pro Lys Pro 350 355 360 CTT AAT TGC CCA CAC TGT GAT TAC AAA ACA GCA GAT AGA AGC AAC 490 Leu Asn Cys Pro His Cys Asp Tyr Lys Thr Ala Asp Arg Ser Asn 365 370 375

TTC AAA AAA CAT GTA GAG CTA CAT GTG AAC CCA CGG CAG TTC AAT 535 Phe Lys Lys His Val Glu Leu His Val Asn Pro Arg Gin Phe Asn 380 385 390

TGC CCT GTA TGT GAC TAT GCA GCT TCC AAG AAG TGT AAT CTA CAG 580 Cys Pro Val Cys Asp Tyr Ala Ala Ser Lys Lys Cys Asn Leu Gin

395 400 405

TAT CAC TTC AAA TCT AAG CAT CCT ACT TGT CCT AAT AAA ACA ATG 625 Tyr His Phe Lys Ser Lys His Pro Thr Cys Pro Asn Lys Thr Met 410 415 420

GAT GTC TCA AAA GTG AAA CTA AAG AAA ACC AAA AAA CGA GAG GCT 670 Asp Val Ser Lys Val Lys Leu Lys Lys Thr Lys Lys Arg Glu Ala 425 430 435

GAC TTG CCT GAT AAT ATT ACC AAT GAA AAA ACA GAA ATA GAA CAA 715 Asp Leu Pro Asp Asn He Thr Asn Glu Lys Thr Glu He Glu Gin 440 445 450

ACA AAA ATA AAA GGG GAT GTG GCT GGA AAG AAA AAT GAA AAG TCC 760 Thr Lys He Lys Gly Asp Val Ala Gly Lys Lys Asn Glu Lys Ser 455 460 465

GTC AAA GCA GAG AAA AGA GAT GTC TCA AAA GAG AAA AAG CCT TCT 805 Val Lys Ala Glu Lys Arg Asp Val Ser Lys Glu Lys Lys Pro Ser

470 475 480

AAT AAT GTG TCA GTG ATC CAG GTG ACT ACC AGA ACT CGA AAA TCA 850 Asn Asn Val Ser Val He Gin Val Thr Thr Arg Thr Arg Lys Ser 485 490 495

GTA ACA GAG GTG AAA GAG ATG GAT GTG CAT ACA GGA AGC AAT TCA 895

Val Thr Glu Val Lys Glu Met Asp Val His Thr Gly Ser Asn Ser 500 505 510 GAA AAA TTC AGT AAA ACT AAG AAA AGC AAA AGG AAG CTG GAA GTT 940 Glu Lys Phe Ser Lys Thr Lys Lys Ser Lys Arg Lys Leu Glu Val 515 520 525

GAC AGC CAT TCT TTA CAT GGT CCT GTG AAT GAT GAG GAA TCT TCA 985 Asp Ser His Ser Leu His Gly Pro Val Asn Asp Glu Glu Ser Ser 530 535 540

ACA AAA AAG AAA AAG AAG GTA GAA AGC AAA TCC AAA AAT AAT AGT 1030 Thr Lys Lys Lys Lys Lys Val Glu Ser Lys Ser Lys Asn Asn Ser

545 550 555

CAG GAA GTG CCA AAG GGT GAC AGC AAA GTG GAG GAG AAT AAA AAG 1075 Gin Glu Val Pro Lys Gly Asp Ser Lys Val Glu Glu Asn Lys Lys 560 565 570

CAA AAT ACT TGC ATG 1090

Gin Asn Thr Cys Met 575

(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 928 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE:

(A) LIBRARY: CDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M., Frohman, Michael A., Kraner, Susan D., Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80 (E) ISSUE:

(F) PAGES:

(G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:5:FROM 1 TO 928 ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

CA GCA CAC CTG AAA CAC CAC ACC AGA 26

Ala His Leu Lys His His Thr Arg 235 240

GCT GGG GAT AAT GAG CGA GTC TAC AAG TGT ATC ATT TGC ACA TAC 71 Ala Gly Asp Asn Glu Arg Val Tyr Lys Cys He He Cys Thr Tyr 245 250 255

ACA ACA GTG AGC GAG TAT CAC TGG AGG AAA CAT TTA AGA AAC CAT 116 Thr Thr Val Ser Glu Tyr His Trp Arg Lys His Leu Arg Asn His 260 265 270

TTT CCA AGG AAA GTA TAC ACA TGT GGA AAA TGC AAC TAT TTT TCA 161 Phe Pro Arg Lys Val Tyr Thr Cys Gly Lys Cys Asn Tyr Phe Ser

275 280 285

GAC AGA AAA AAC AAT TAT GTT CAG CAT GTT AGA ACT CAT ACA GGA 206 Asp Arg Lys Asn Asn Tyr Val Gin His Val Arg Thr His Thr Gly 290 295 300

GAA CGC CCA TAT AAA TGT GAA CTT TGT CCT TAC TCA AGT TCT CAG 251 Glu Arg Pro Tyr Lys Cys Glu Leu Cys Pro Tyr Ser Ser Ser Gin 305 310 315

AAG ACT CAT CTA ACT AGA CAT ATG CGT ACT CAT TCA GGT GAG AAG 296 Lys Thr His Leu Thr Arg His Met Arg Thr His Ser Gly Glu Lys 320 325 330

CCA TTT AAA TGT GAT CAG TGC AGT TAT GTG GCC TCT AAT CAA CAT 341 Pro Phe Lys Cys Asp Gin Cys Ser Tyr Val Ala Ser Asn Gin His 335 340 345

GAA GTA ACC CGC CAT GCA AGA CAG GTT CAC AAT GGG CCT AAA CCT 386 Glu Val Thr Arg His Ala Arg Gin Val His Asn Gly Pro Lys Pro

350 355 360 CTT AAT TGC CCA CAC TGT GAT TAC AAA ACA GCA GAT AGA AGC AAC 431 Leu Asn Cys Pro His Cys Asp Tyr Lys Thr Ala Asp Arg Ser Asn 365 370 375

TTC AAA AAA CAT GTA GAG CTA CAT GTG AAC CCA CGG CAG TTC AAT 476 Phe Lys Lys His Val Glu Leu His Val Asn Pro Arg Gin Phe Asn 380 385 390

TGC CCT GTA TGT GAC TAT GCA GCT TCC AAG AAG TGT AAT CTA CAG 521 Cys Pro Val Cys Asp Tyr Ala Ala Ser Lys Lys Cys Asn Leu Gin

395 400 405

TAT CAC TTC AAA TCT AAG CAT CCT ACT TGT CCT AAT AAA ACA ATG 566 Tyr His Phe Lys Ser Lys His Pro Thr Cys Pro Asn Lys Thr Met 410 415 420

GAT GTC TCA AAA GTG AAA CTA AAG AAA ACC AAA AAA CGA GAG GCT 611 Asp Val Ser Lys Val Lys Leu Lys Lys Thr Lys Lys Arg Glu Ala

425 430 435

GAC TTG CCT GAT AAT ATT ACC AAT GAA AAA ACA GAA ATA GAA CAA 656 Asp Leu Pro Asp Asn He Thr Asn Glu Lys Thr Glu He Glu Gin

440 445 450

ACA AAA ATA AAA GGG GAT GTG GCT GGA AAG AAA AAT GAA AAG TCC 701 Thr Lys He Lys Gly Asp Val Ala Gly Lys Lys Asn Glu Lys Ser 455 460 465

GTC AAA GCA GAG AAA AGA GAT GTC TCA AAA GAG AAA AAG CCT TCT 746 Val Lys Ala Glu Lys Arg Asp Val Ser Lys Glu Lys Lys Pro Ser

470 475 480

AAT AAT GTG TCA GTG ATC CAG GTG ACT ACC AGA ACT CGA AAA TCA 791 Asn Asn Val Ser Val He Gin Val Thr Thr Arg Thr Arg Lys Ser 485 490 495

GTA ACA GAG GTG AAA GAG ATG GAT GTG CAT ACA GGA AGC AAT TCA 836 Val Thr Glu Val Lys Glu Met Asp Val His Thr Gly Ser Asn Ser 500 505 510 GAA AAA TTC AGT AAA ACT AAG AAA AGC AAA AGG AAG CTG GAA GTT 881 Glu Lys Phe Ser Lys Thr Lys Lys Ser Lys Arg Lys Leu Glu Val 515 520 525

GAC AGC CAT TCT TTA CAT GGT CCT GTG AAT GAT GAG GAA TCT TCA 926 Asp Ser His Ser Leu His Gly Pro Val Asn Asp Glu Glu Ser Ser 530 535 540

AC 928

(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 1791 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no

(vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: CDNA

(x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C. , Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D. , Mandel, Gail (B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80

(E) ISSUE: (F) PAGES:

(G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:6 :FROM 1 TO 1791 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

CACCCTCTGC AGCCCCACTC CTGGGCCTTC TTGGTCCACG ACGGCCCCAG 50

CACCCAACTT TACCACCCTC CCCCACCTCT CCCCCGAAAC TCCAGCAACA 100 AAGAAAAGTA GTCGGAGAAG GAGCGGCGAC TCAGGGTCGC CCGCCCCTCC 150

TCACCGAGGA AGGCCGAATA CAGTT 175

ATG GCC ACC CAG GTA ATG GGG CAG TCT TCT GGA GGA GGA GGG CTG 220 Met Ala Thr Gin Val Met Gly Gin Ser Ser Gly Gly Gly Gly Leu

1 5 10 15

TTT ACC AGC AGT GGC AAC ATT GGA ATG GCC CTG CCT AAC GAC ATG 265 Phe Thr Ser Ser Gly Asn He Gly Met Ala Leu Pro Asn Asp Met

20 25 30

TAT GAC TTG CAT GAC CTT TCC AAA GCT GAA CTG GCC GCA CCT CAG 310 Tyr Asp Leu His Asp Leu Ser Lys Ala Glu Leu Ala Ala Pro Gin 35 40 45

CTT ATT ATG CTG GCA AAT GTG GCC TTA ACT GGG GAA GTA AAT GGC 355 Leu He Met Leu Ala Asn Val Ala Leu Thr Gly Glu Val Asn Gly

50 55 60

AGC TGC TGT GAT TAC CTG GTC GGT GAA GAA AGA CAG ATG GCA GAA 400 Ser Cys Cys Asp Tyr Leu Val Gly Glu Glu Arg Gin Met Ala Glu

65 70 75

CTG ATG CCG GTT GOG GAT AAC AAC TTT TCA GAT AGT GAA GAA GGA 445 Leu Met Pro Val Gly Asp Asn Asn Phe Ser Asp Ser Glu Glu Gly 80 85 90

GAA GGA CTT GAA GAG TCT GCT GAT ATA AAA GGT GAA CCT CAT GGA 490 Glu Gly Leu Glu Glu Ser Ala Asp He Lys Gly Glu Pro His Gly

95 100 105

CTG GAA AAC ATG GAA CTG AGA AGT TTG GAA CTC AGC GTC GTA GAA 535 Leu Glu Asn Met Glu Leu Arg Ser Leu Glu Leu Ser Val Val Glu 110 115 120

CCT CAG CCT GTA TTT GAG GCA TCA GGT GCT CCA GAT ATT TAC AGT 580 Pro Gin Pro Val Phe Glu Ala Ser Gly Ala Pro Asp He Tyr Ser 125 130 135 TCA AAT AAA GCT CTT GCC CCT GAA ACA CCT GGA GCG GAG GAC AAA 625 Ser Asn Lys Ala Leu Ala Pro Glu Thr Pro Gly Ala Glu Asp Lys 140 145 150

GGC AAG AGC TCG AAG ACC AAA CCC TTT CGC TGT AAG CCA TGC CAA 670 Gly Lys Ser Ser Lys Thr Lys Pro Phe Arg Cys Lys Pro Cys Gin 155 160 165

TAT GAA GCA GAA TCT GAA GAA CAG TTT GTG CAT CAC ATC AGA GTT 715 Tyr Glu Ala Glu Ser Glu Glu Gin Phe Val His His He Arg Val

170 175 180

CAC AGT GCT AAG AAA TTT TTT GTG GAA GAG AGT GCA GAG AAG CAG 760 His Ser Ala Lys Lys Phe Phe Val Glu Glu Ser Ala Glu Lys Gin 185 190 195

GCA AAA GCC AGG GAA TCT GGC TCT TCC ACT GCA GAA GAG GGA GAT 805 Ala Lys Ala Arg Glu Ser Gly Ser Ser Thr Ala Glu Glu Gly Asp 200 205 210

TTC TCC AAG GGC CCC ATT CGC TGT GAC CGC TGC GGC TAC AAT ACT 850 Phe Ser Lys Gly Pro He Arg Cys Asp Arg Cys Gly Tyr Asn Thr 215 220 225

AAT CGA TAT GAT CAC TAT ACA GCA CAC CTG AAA CAC CAC ACC AGA 895 Asn Arg Tyr Asp His Tyr Thr Ala His Leu Lys His His Thr Arg 230 235 240

GCT GGG GAT AAT GAG CGA GTC TAC AAG TGT ATC ATT TGC ACA TAC 940 Ala Gly Asp Asn Glu Arg Val Tyr Lys Cys He He Cys Thr Tyr

245 250 255

ACA ACA GTG AGC GAG TAT CAC TGG AGG AAA CAT TTA AGA AAC CAT 985 Thr Thr Val Ser Glu Tyr His Trp Arg Lys His Leu Arg Asn His 260 265 270

TTT CCA AGG AAA GTA TAC ACA TGT GGA AAA TGC AAC TAT TTT TCA 1030 Phe Pro Arg Lys Val Tyr Thr Cys Gly Lys Cys Asn Tyr Phe Ser 275 280 285 GAC AGA AAA AAC AAT TAT GTT CAG CAT GTT AGA ACT CAT ACA GGA 1075 Asp Arg Lys Asn Asn Tyr Val Gin His Val Arg Thr His Thr Gly 290 295 300

GAA CGC CCA TAT AAA TGT GAA CTT TGT CCT TAC TCA AGT TCT CAG 1120 Glu Arg Pro Tyr Lys Cys Glu Leu Cys Pro Tyr Ser Ser Ser Gin 305 310 315

AAG ACT CAT CTA ACT AGA CAT ATG CGT ACT CAT TCA GGT GAG AAG 1165 Lys Thr His Leu Thr Arg His Met Arg Thr His Ser Gly Glu Lys

320 325 330

CCA TTT AAA TGT GAT CAG TGC AGT TAT GTG GCC TCT AAT CAA CAT 1210 Pro Phe Lys Cys Asp Gin Cys Ser Tyr Val Ala Ser Asn Gin His 335 340 345

GAA GTA ACC CGC CAT GCA AGA CAG GTT CAC AAT GGG CCT AAA CCT 1255 Glu Val Thr Arg His Ala Arg Gin Val His Asn Gly Pro Lys Pro 350 355 360

CTT AAT TGC CCA CAC TGT GAT TAC AAA ACA GCA GAT AGA AGC AAC 1300 Leu Asn Cys Pro His Cys Asp Tyr Lys Thr Ala Asp Arg Ser Asn 365 370 375

TTC AAA AAA CAT GTA GAG CTA CAT GTG AAC CCA CGG CAG TTC AAT 13 5 Phe Lys Lys His Val Glu Leu His Val Asn Pro Arg Gin Phe Asn 380 385 390

TGC CCT GTA TGT GAC TAT GCA GCT TCC AAG AAG TGT AAT CTA CAG 1390 Cys Pro Val Cys Asp Tyr Ala Ala Ser Lys Lys Cys Asn Leu Gin

395 400 405

TAT CAC TTC AAA TCT AAG CAT CCT ACT TGT CCT AAT AAA ACA ATG 1 35 Tyr His Phe Lys Ser Lys His Pro Thr Cys Pro Asn Lys Thr Met 410 415 420

GAT GTC TCA AAA GTG AAA CTA AAG AAA ACC AAA AAA CGA GAG GCT 1480 Asp Val Ser Lys Val Lys Leu Lys Lys Thr Lys Lys Arg Glu Ala 425 430 435 GAC TTG CCT GAT AAT ATT ACC AAT GAA AAA ACA GAA ATA GAA CAA 1525 Asp Leu Pro Asp Asn He Thr Asn Glu Lys Thr Glu He Glu Gin 440 445 450

ACA AAA ATA AAA GGG GAT GTG GCT GGA AAG AAA AAT GAA AAG TCC 1570 Thr Lys He Lys Gly Asp Val Ala Gly Lys Lys Asn Glu Lys Ser 455 460 465

GTC AAA GCA GAG AAA AGA GAT GTC TCA AAA GAG AAA AAG CCT TCT 1615 Val Lys Ala Glu Lys Arg Asp Val Ser Lys Glu Lys Lys Pro Ser

470 475 480

AAT AAT GTG TCA GTG ATC CAG GTG ACT ACC AGA ACT CGA AAA TCA 1660 Asn Asn Val Ser Val He Gin Val Thr Thr Arg Thr Arg Lys Ser 485 490 495

GTA ACA GAG GTG AAA GAG ATG GAT GTG CAT ACA GGA AGC AAT TCA 1705 Val Thr Glu Val Lys Glu Met Asp Val His Thr Gly Ser Asn Ser 500 505 510

GAA AAA TTC AGT AAA ACT AAG AAA AGC AAA AGG AAG CTG GAA GTT 1750 Glu Lys Phe Ser Lys Thr Lys Lys Ser Lys Arg Lys Leu Glu Val 515 520 525

GAC AGC CAT TCT TTA CAT GGT CCT GTG AAT GAT GAG GAA TC 1791 Asp Ser His Ser Leu His Gly Pro Val Asn Asp Glu Glu 530 535

(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 3705 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: CDNA (X) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M., Frohman, Michael A., Kraner, Susan D., Mandel, Gail (B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80

(E) ISSUE: (F) PAGES:

(G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:7 :FROM 1 TO 3705 ( i) SEQUENCE DESCRIPTION: SEQ ID NO:7:

GA ACT CGA AAA TCA 14

Thr Arg Lys Ser 495

GTA ACA GAG GTG AAA GAG ATG GAT GTG CAT ACA GGA AGC AAT TCA 59 Val Thr Glu Val Lys Glu Met Asp Val His Thr Gly Ser Asn Ser

500 505 510

GAA AAA TTC AGT AAA ACT AAG AAA AGC AAA AGG AAG CTG GAA GTT 104 Glu Lys Phe Ser Lys Thr Lys Lys Ser Lys Arg Lys Leu Glu Val 515 520 525

GAC AGC CAT TCT TTA CAT GGT CCT GTG AAT GAT GAG GAA TCT TCA 149 Asp Ser His Ser Leu His Gly Pro Val Asn Asp Glu Glu Ser Ser 530 535 540

ACA AAA AAG AAA AAG AAG GTA GAA AGC AAA TCC AAA AAT AAT AGT 194 Thr Lys Lys Lys Lys Lys Val Glu Ser Lys Ser Lys Asn Asn Ser 545 550 555

CAG GAA GTG CCA AAG GGT GAC AGC AAA GTG GAG GAG AAT AAA AAG 239 Gin Glu Val Pro Lys Gly Asp Ser Lys Val Glu Glu Asn Lys Lys 560 565 570

CAA AAT ACT TGC ATG AAA AAA AGT ACA AAG AAG AAA ACT CTG AAA 284 Gin Asn Thr Cys Met Lys Lys Ser Thr Lys Lys Lys Thr Leu Lys

575 580 585 AAT AAA TCA AGT AAG AAA AGC AGT AAG CCT CCT CAG AAG GAA CCT 329

Asn Lys Ser Ser Lys Lys Ser Ser Lys Pro Pro Gin Lys Glu Pro

590 595 600

GTT GAG AAG GGA TCT GCT CAG ATG GAC CCT CCT CAG ATG GGG CCT 374

Val Glu Lys Gly Ser Ala Gin Met Asp Pro Pro Gin Met Gly Pro

605 610 615

GCT CCC ACA GAG GCG GTT CAG AAG GGG CCC GTT CAG GTG GAG CTG 419 Ala Pro Thr Glu Ala Val Gin Lys Gly Pro Val Gin Val Glu Leu

620 625 630

CCA CCT CCC ATG GAG CAT GCT CAG ATG GAG GGT GCC CAG ATA CGG 464

Pro Pro Pro Met Glu His Ala Gin Met Glu Gly Ala Gin He Arg 635 640 645

CCT GCT CCT GAC GAG CCT GTT CAG ATG GAG GTG GTT CAG GAG GGG 509

Pro Ala Pro Asp Glu Pro Val Gin Met Glu Val Val Gin Glu Gly

650 655 660

CCT GCT CAG AAG GAG CTG CTG CCT CCC GTG GAG CCT GCT CAG ATG 554

Pro Ala Gin Lys Glu Leu Leu Pro Pro Val Glu Pro Ala Gin Met

665 670 675

GTG GGT GCC CAA ATT GTA CTT GCT CAC ATG GAG CTG CCT CCT CCC 599

Val Gly Ala Gin He Val Leu Ala His Met Glu Leu Pro Pro Pro

680 685 690

ATG GAG ACT GCT CAG ACG GAG GTT GCC CAA ATG GGG CCT GCT CCC 644 Met Glu Thr Ala Gin Thr Glu Val Ala Gin Met Gly Pro Ala Pro

695 700 705

ATG GAA CCT GCT CAG ATG GAG GTT GCC CAG GTA GAA TCT GCT CCC 689 Met Glu Pro Ala Gin Met Glu Val Ala Gin Val Glu Ser Ala Pro 710 715 720

ATG CAG GTG GTC CAG AAG GAG CCT GTT CAG ATG GAG CTG TCT CCT 734

Met Gin Val Val Gin Lys Glu Pro Val Gin Met Glu Leu Ser Pro

725 730 735 CCC ATG GAG GTG GTC CAG AAG GAG CCT GTT CAG ATA GAG CTG TCT 779 Pro Met Glu Val Val Gin Lys Glu Pro Val Gin He Glu Leu Ser 740 745 750

CCT CCC ATG GAG GTG GTC CAG AAG GAA CCT GTT AAG ATA GAG CTG 824 Pro Pro Met Glu Val Val Gin Lys Glu Pro Val Lys He Glu Leu 755 760 765

TCT CCT CCC ATA GAG GTG GTC CAG AAG GAG CCT GTT CAG ATG GAG 869 Ser Pro Pro He Glu Val Val Gin Lys Glu Pro Val Gin Met Glu

770 775 780

TTG TCT CCT CCC ATG GGG GTG GTT CAG AAG GAG CCT GCT CAG AGG 914 Leu Ser Pro Pro Met Gly Val Val Gin Lys Glu Pro Ala Gin Arg 785 790 795

GAG CCA CCT CCT CCC AGA GAG CCT CCC CTT CAC ATG GAG CCA ATT 959 Glu Pro Pro Pro Pro Arg Glu Pro Pro Leu His Met Glu Pro He 800 805 810

TCC AAA AAG CCT CCT CTC CGA AAA GAT AAA AAG GAA AAG TCT AAC 1004 Ser Lys Lys Pro Pro Leu Arg Lys Asp Lys Lys Glu Lys Ser Asn 815 820 825

ATG CAG AGT GAA AGG GCA CGG AAG GAG CAA GTC CTT ATT GAA GTT 1049 Met Gin Ser Glu Arg Ala Arg Lys Glu Gin Val Leu He Glu Val 830 835 840

GGC TTA GTG CCT GTT AAA GAT AGC TGG CTT CTA AAG GAA AGT GTA 1094 Gly Leu Val Pro Val Lys Asp Ser Trp Leu Leu Lys Glu Ser Val

845 850 855

AGC ACA GAG GAT CTC TCA CCA CCA TCA CCA CCA CTG CCA AAG GAA 1139 Ser Thr Glu Asp Leu Ser Pro Pro Ser Pro Pro Leu Pro Lys Glu 860 865 870

AAT TTA AGA GAA GAG GCA TCA GGA GAC CAA AAA TTA CTC AAC ACA 1184

Asn Leu Arg Glu Glu Ala Ser Gly Asp Gin Lys Leu Leu Asn Thr

875 880 885 GGT GAA GGA AAT AAA GAA GCC CCT CTT CAG AAA GTA GGA GCA GAA 1229 Gly Glu Gly Asn Lys Glu Ala Pro Leu Gin Lys Val Gly Ala Glu 890 895 900

GAG GCA GAT GAG AGC CTA CCT GGT CTT GCT GCT AAT ATC AAC GAA 1274 Glu Ala Asp Glu Ser Leu Pro Gly Leu Ala Ala Asn He Asn Glu 905 910 915

TCT ACC CAT ATT TCA TCC TCT GGA CAA AAC TTG AAT ACG CCA GAG 1319 Ser Thr His He Ser Ser Ser Gly Gin Asn Leu Asn Thr Pro Glu

920 925 930

GGT GAA ACT TTA AAT GGT AAA CAT CAG ACT GAC AGT ATA GTT TGT 1364 Gly Glu Thr Leu Asn Gly Lys His Gin Thr Asp Ser He Val Cys 935 940 945

GAA ATG AAA ATG GAC ACT GAT CAG AAC ACA AGA GAG AAT CTC ACT 1409 Glu Met Lys Met Asp Thr Asp Gin Asn Thr Arg Glu Asn Leu Thr 950 955 960

GGT ATA AAT TCA ACA GTT GAA GAA CCA GTT TCA CCA ATG CTT CCC 1454 Gly He Asn Ser Thr Val Glu Glu Pro Val Ser Pro Met Leu Pro 965 970 975

CCT TCA GCA GTA GAA GAA CGT GAA GCA GTG TCC AAA ACT GCA CTG 1499 Pro Ser Ala Val Glu Glu Arg Glu Ala Val Ser Lys Thr Ala Leu 980 985 990

GCA TCA CCT CCT GCT ACA ATG GCA GCA AAT GAG TCT CAG GAA ATT 1544 Ala Ser Pro Pro Ala Thr Met Ala Ala Asn Glu Ser Gin Glu He

995 1000 1005

GAT GAA GAT GAA GGC ATC CAC AGC CAT GAA GGA AGT GAC CTA AGT 1589 Asp Glu Asp Glu Gly He His Ser His Glu Gly Ser Asp Leu Ser 1010 1015 1020

GAC AAC ATG TCA GAG GGT AGT GAT GAT TCT GGA TTG CAT GGG GCT 1634 Asp Asn Met Ser Glu Gly Ser Asp Asp Ser Gly Leu His Gly Ala 1025 1030 1035 CGG CCA GTT CCA CAA GAA TCT AGC AGA AAA AAT GCA AAG GAA GCC 1679 Arg Pro Val Pro Gin Glu Ser Ser Arg Lys Asn Ala Lys Glu Ala 1040 1045 1050

TTG GCA GTC AAA GCG GCT AAG GGA GAT TTT GTT TGT ATC TTC TGT 1724 Leu Ala Val Lys Ala Ala Lys Gly Asp Phe Val Cys He Phe Cys 1055 1060 1065

GAT CGT TCT TTC AGA AAG GGA AAA GAT TAC AGC AAA CAC CTC AAT 1769 Asp Arg Ser Phe Arg Lys Gly Lys Asp Tyr Ser Lys His Leu Asn

1070 1075 1080

CGC CAT TTG GTT AAT GTG TAC TAT CTT GAA GAA GCA GCT CAA GGG 1814 Arg His Leu Val Asn Val Tyr Tyr Leu Glu Glu Ala Ala Gin Gly 1085 1090 1095

CAG GAG TAATG AAACTTTGAA CAAGGTTTCA GTTCTTAGTT 1855

Gin Glu

TGTAAGGTAT ATTACATTTT ATATTCATTT ATGATAGCAG ACAACCTTTT 1905

AAGATTGCTT TAATTAGTAT CTGATGTTGA TTTTTAAGTG GCATTCTTTT 1955

CCTTAGGACT TTTTATGTAT ACCTGTTGAT TGTTGTGTAA ATTTTAGTAA 2005

ATCTAAGAGA GTGTACTAAA CCAGCAGGTA TCTGTTAGCT TATGTGTTTA 2055

ATTGAAATTA GAAGGCTAAG ATGGTATAAC AGCATTTTAT TGCTTTGTCC 2105

AGCTACAACA TGTCATTTTT TTCTCCATGT CTTATCTTCC TGTTTCACTT 2155

TAGTTTATTC TTCGTTTTTT ATTGAGATCT ATAAAAAATT GGCTTACTTA 2205

ATAGCAAATT ACTTGAAGAA TTTGCCTGCT TTATATAAAG TTAGCACTTT 2255

AAGATTTTTT TTTTAGAGAT GAGAAGACAT TTAAATTGAA GAAAAATTCC 2305

CCCAGCAATA GACAGTCTAT CAGTCCAAGT ATTTACTTCC TGAGTTTTGA 2355

TCAATATTTT TTATTTGTGT ATGTTAATCG TCATAAAAAC AGTGATTTTG 2405 GTGTGTTTTT TATTTTGGTG CTTTAATGGC TTAAGATGTT GCACATTTTT 2 55

TTTTTCTTTT GGTTTCTGTT TATGTTTTTT TGCCTATGCA GTTAAATTTT 2505

TCCTAGAAAT AGCATTTGTG TTGAACAGTA ACACTTTATA CATATATATA 2555

TGCATGTTTA TTTTGTTTGG CGTCTTTGGA GGGATGCTTT TAGACTTGTT 2605

TGCAAAAGGG CAGTTTTCTT TTTCTTTGCT GCAGTTGTCT ATTTTGCAGA 2655

ATAATAGTGT GTGCAAGTTT GTGAGCAAAT GAAATATGCA GGTTCAATCT 2705

ATTGATTTTG ATTTTTACAT CTTATATCTA TGCCAGAATC TGTATTTCAT 2755

ATAACTTATT TATTTCGAAT GGATGTAGTA AATTCACAGC TATCAGTTTT 2805

GATTTTGCAA TAAATAAACC ACTAGGTTGC ATGTCGAACA AATTTTTATC 2855

TCAAATACCA ACCATCAGTT TTTTTTTTCA TGTGTTTTGG TACAGCTAAT 2905

TCCTAATTGT AGAGTGTTAA ATGTTTGAGG AGAACCTTTT CTCATAGATG 2955

GTTGGTGTTC ATATGGCNAC TTTACAATAA AGAGAACTGT AAGTGATATT 3005

TGGAAACTAC AAACCTGGAA TTAGGAGATA TAATTATTCC TTCAAGTTTT 3055

ATAGATATCA CTTGGGAGAT TCCAAAGCCA TAGCTATTAC GCNGCAAACC 3105

TAGGATAAGA AAGGTAGTAT GAGTGCTGGT AGACCAGCTG CAACATTTCC 3155

TATATCAGAT GAAAAAGGCT GGTGAAACAA GTACAGTCCA GATTTTTTAA 3205

AATCATACTT TCTCAGGGAT CTCCACAAAC TGGTGGGTGT CCTGGCTGTC 3255

TGTGTGATAG CCTCTTTCTA TAGGTGAGGC CTCAAATGAA TTGCAGCTAT 3305

CCTGGTGTTC CTATGAGGGC ACTTGTATGA AAAAGGCAGT ACTCCAAAAC 3355

ATTTTTGATG GTTCTTTGGC CAGTTGCCAA AGAGTGTGAA AGAATCCAAT 3405

AGAGGATTTT TCTTACTGAT AGCAGTCATT CATTGCAGTA AAATAAAATA 3455 TGAATTCCCA TTAGGGAATC TTGAATTCTG ACCTCCCATA CTCCGTTTTG 3505

AAATAACCAC TTATATTTCA TTTTTTAAAA ATCTGATGAT CTCTTTGAGG 3555

CAGGTTTCAG ATTTGGCAGT ACAACATGAA AGATTAGGAA AAGCATTAAT 3605

AACGTGTGGG TGGAAAGCTT GTTAAAAATC TGAGAGTGAA GTTTGAGTTA 3655

AAAGTTGTTT GACATGGCAT TGACTGGGAG GCCAAAGATT TAAAGAAGCG 3705

(2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS : single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D., Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80

(E) ISSUE:

(F) PAGES: (G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:8:FROM 1 TO 24 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

TGYAARCCNT GYCARTAYGA RGCN 24

(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (iii) HYPOTHETICAL: no

(iv) ANTI-SENSE: no

(x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C. , Altschuler,

Yelena M. , Frohman, Michael A., Kraner, Susan D. , Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell (D) VOLUME: 80

(E) ISSUE:

(F) PAGES:

(G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO: 9:FROM 1 TO 24 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

NGTYTTRTAR TCRCARTGNG GRCA 24

(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 3291 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C. , Altschuler,

Yelena M. , Frohman, Michael A., Kraner, Susan D. , Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell (D) VOLUME: 80

(E) ISSUE:

(F) PAGES: (G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:10:FROM 1 TO 3291 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

ATG GCC ACC CAG GTA ATG GGG CAG TCT TCT GGA GGA GGA GGG CTG 45 Met Ala Thr Gin Val Met Gly Gin Ser Ser Gly Gly Gly Gly Leu 1 5 10 15

TTT ACC AGC AGT GGC AAC ATT GGA ATG GCC CTG CCT AAC GAC ATG 90 Phe Thr Ser Ser Gly Asn He Gly Met Ala Leu Pro Asn Asp Met

20 25 30

TAT GAC TTG CAT GAC CTT TCC AAA GCT GAA CTG GCC GCA CCT CAG 135 Tyr Asp Leu His Asp Leu Ser Lys Ala Glu Leu Ala Ala Pro Gin 35 40 45

CTT ATT ATG CTG GCA AAT GTG GCC TTA ACT GGG GAA GTA AAT GGC 180 Leu He Met Leu Ala Asn Val Ala Leu Thr Gly Glu Val Asn Gly 50 55 60

AGC TGC TGT GAT TAC CTG GTC GGT GAA GAA AGA CAG ATG GCA GAA 225 Ser Cys Cys Asp Tyr Leu Val Gly Glu Glu Arg Gin Met Ala Glu 65 70 75

CTG ATG CCG GTT GGG GAT AAC AAC TTT TCA GAT AGT GAA GAA GGA 270 Leu Met Pro Val Gly Asp Asn Asn Phe Ser Asp Ser Glu Glu Gly 80 85 90

GAA GGA CTT GAA GAG TCT GCT GAT ATA AAA GGT GAA CCT CAT GGA 315 Glu Gly Leu Glu Glu Ser Ala Asp He Lys Gly Glu Pro His Gly

95 100 105

CTG GAA AAC ATG GAA CTG AGA AGT TTG GAA CTC AGC GTC GTA GAA 360 Leu Glu Asn Met Glu Leu Arg Ser Leu Glu Leu Ser Val Val Glu 110 115 120

CCT CAG CCT GTA TTT GAG GCA TCA GGT GCT CCA GAT ATT TAC AGT 405 Pro Gin Pro Val Phe Glu Ala Ser Gly Ala Pro Asp He Tyr Ser 125 130 135 TCA AAT AAA GCT CTT GCC CCT GAA ACA CCT GGA GCG GAG GAC AAA 450 Ser Asn Lys Ala Leu Ala Pro Glu Thr Pro Gly Ala Glu Asp Lys 140 145 150

GGC AAG AGC TCG AAG ACC AAA CCC TTT CGC TGT AAG CCA TGC CAA 495 Gly Lys Ser Ser Lys Thr Lys Pro Phe Arg Cys Lys Pro Cys Gin

155 160 165

TAT GAA GCA GAA TCT GAA GAA CAG TTT GTG CAT CAC ATC AGA GTT 540 Tyr Glu Ala Glu Ser Glu Glu Gin Phe Val His His He Arg Val

170 175 180

CAC AGT GCT AAG AAA TTT TTT GTG GAA GAG AGT GCA GAG AAG CAG 585 His Ser Ala Lys Lys Phe Phe Val Glu Glu Ser Ala Glu Lys Gin 185 190 195

GCA AAA GCC AGG GAA TCT GGC TCT TCC ACT GCA GAA GAG GGA GAT 630 Ala Lys Ala Arg Glu Ser Gly Ser Ser Thr Ala Glu Glu Gly Asp 200 205 210

TTC TCC AAG GGC CCC ATT CGC TGT GAC CGC TGC GGC TAC AAT ACT 675 Phe Ser Lys Gly Pro He Arg Cys Asp Arg Cys Gly Tyr Asn Thr 215 220 225

AAT CGA TAT GAT CAC TAT ACA GCA CAC CTG AAA CAC CAC ACC AGA 720 Asn Arg Tyr Asp His Tyr Thr Ala His Leu Lys His His Thr Arg 230 235 240

GCT GGG GAT AAT GAG CGA GTC TAC AAG TGT ATC ATT TGC ACA TAC 765 Ala Gly Asp Asn Glu Arg Val Tyr Lys Cys He He Cys Thr Tyr

245 250 255

ACA ACA GTG AGC GAG TAT CAC TGG AGG AAA CAT TTA AGA AAC CAT 810 Thr Thr Val Ser Glu Tyr His Trp Arg Lys His Leu Arg Asn His 260 265 270

TTT CCA AGG AAA GTA TAC ACA TGT GGA AAA TGC AAC TAT TTT TCA 855 Phe Pro Arg Lys Val Tyr Thr Cys Gly Lys Cys Asn Tyr Phe Ser 275 280 285 - 67 -

GAC AGA AAA AAC AAT TAT GTT CAG CAT GTT AGA ACT CAT ACA GGA 900 Asp Arg Lys Asn Asn Tyr Val Gin His Val Arg Thr His Thr Gly 290 295 300

GAA CGC CCA TAT AAA TGT GAA CTT TGT CCT TAC TCA AGT TCT CAG 945 Glu Arg Pro Tyr Lys Cys Glu Leu Cys Pro Tyr Ser Ser Ser Gin 305 310 315

AAG ACT CAT CTA ACT AGA CAT ATG CGT ACT CAT TCA GGT GAG AAG 990 Lys Thr His Leu Thr Arg His Met Arg Thr His Ser Gly Glu Lys

320 325 330

CCA TTT AAA TGT GAT CAG TGC AGT TAT GTG GCC TCT AAT CAA CAT 1035 Pro Phe Lys Cys Asp Gin Cys Ser Tyr Val Ala Ser Asn Gin His 335 340 345

GAA GTA ACC CGC CAT GCA AGA CAG GTT CAC AAT GGG CCT AAA CCT 1080 Glu Val Thr Arg His Ala Arg Gin Val His Asn Gly Pro Lys Pro 350 355 360

CTT AAT TGC CCA CAC TGT GAT TAC AAA ACA GCA GAT AGA AGC AAC 1125 Leu Asn Cys Pro His Cys Asp Tyr Lys Thr Ala Asp Arg Ser Asn 365 370 375

TTC AAA AAA CAT GTA GAG CTA CAT GTG AAC CCA CGG CAG TTC AAT 1170 Phe Lys Lys His Val Glu Leu His Val Asn Pro Arg Gin Phe Asn 380 385 390

TGC CCT GTA TGT GAC TAT GCA GCT TCC AAG AAG TGT AAT CTA CAG 1215 Cys Pro Val Cys Asp Tyr Ala Ala Ser Lys Lys Cys Asn Leu Gin

395 400 405

TAT CAC TTC AAA TCT AAG CAT CCT ACT TGT CCT AAT AAA ACA ATG 1260 Tyr His Phe Lys Ser Lys His Pro Thr Cys Pro Asn Lys Thr Met 410 415 420

GAT GTC TCA AAA GTG AAA CTA AAG AAA ACC AAA AAA CGA GAG GCT 1305 Asp Val Ser Lys Val Lys Leu Lys Lys Thr Lys Lys Arg Glu Ala 425 430 435 GAC TTG CCT GAT AAT ATT ACC AAT GAA AAA ACA GAA ATA GAA CAA 1350 Asp Leu Pro Asp Asn He Thr Asn Glu Lys Thr Glu He Glu Gin 440 445 450

ACA AAA ATA AAA GGG GAT GTG GCT GGA AAG AAA AAT GAA AAG TCC 1395 Thr Lys He Lys Gly Asp Val Ala Gly Lys Lys Asn Glu Lys Ser 455 460 465

GTC AAA GCA GAG AAA AGA GAT GTC TCA AAA GAG AAA AAG CCT TCT 1440 Val Lys Ala Glu Lys Arg Asp Val Ser Lys Glu Lys Lys Pro Ser

470 475 480

AAT AAT GTG TCA GTG ATC CAG GTG ACT ACC AGA ACT CGA AAA TCA 1485 Asn Asn Val Ser Val He Gin Val Thr Thr Arg Thr Arg Lys Ser 485 490 495

GTA ACA GAG GTG AAA GAG ATG GAT GTG CAT ACA GGA AGC AAT TCA 1530 Val Thr Glu Val Lys Glu Met Asp Val His Thr Gly Ser Asn Ser 500 505 510

GAA AAA TTC AGT AAA ACT AAG AAA AGC AAA AGG AAG CTG GAA GTT 1575 Glu Lys Phe Ser Lys Thr Lys Lys Ser Lys Arg Lys Leu Glu Val 515 520 525

GAC AGC CAT TCT TTA CAT GGT CCT GTG AAT GAT GAG GAA TCT TCA 1620 Asp Ser His Ser Leu His Gly Pro Val Asn Asp Glu Glu Ser Ser 530 535 540

ACA AAA AAG AAA AAG AAG GTA GAA AGC AAA TCC AAA AAT AAT AGT 1665 Thr Lys Lys Lys Lys Lys Val Glu Ser Lys Ser Lys Asn Asn Ser

545 550 555

CAG GAA GTG CCA AAG GGT GAC AGC AAA GTG GAG GAG AAT AAA AAG 1710 Gin Glu Val Pro Lys Gly Asp Ser Lys Val Glu Glu Asn Lys Lys 560 565 570

CAA AAT ACT TGC ATG AAA AAA AGT ACA AAG AAG AAA ACT CTG AAA 1755

Gin Asn Thr Cys Met Lys Lys Ser Thr Lys Lys Lys Thr Leu Lys 575 580 585 AAT AAA TCA AGT AAG AAA AGC AGT AAG CCT CCT CAG AAG GAA CCT 1800 Asn Lys Ser Ser Lys Lys Ser Ser Lys Pro Pro Gin Lys Glu Pro 590 595 600

GTT GAG AAG GGA TCT GCT CAG ATG GAC CCT CCT CAG ATG GGG CCT 1845 Val Glu Lys Gly Ser Ala Gin Met Asp Pro Pro Gin Met Gly Pro 605 610 615

GCT CCC ACA GAG GCG GTT CAG AAG GGG CCC GTT CAG GTG GAG CTG 1890 Ala Pro Thr Glu Ala Val Gin Lys Gly Pro Val Gin Val Glu Leu

620 625 630

CCA CCT CCC ATG GAG CAT GCT CAG ATG GAG GGT GCC CAG ATA CGG 1935 Pro Pro Pro Met Glu His Ala Gin Met Glu Gly Ala Gin He Arg 635 640 645

CCT GCT CCT GAC GAG CCT GTT CAG ATG GAG GTG GTT CAG GAG GGG 1980 Pro Ala Pro Asp Glu Pro Val Gin Met Glu Val Val Gin Glu Gly 650 655 660

CCT GCT CAG AAG GAG CTG CTG CCT CCC GTG GAG CCT GCT CAG ATG 2025 Pro Ala Gin Lys Glu Leu Leu Pro Pro Val Glu Pro Ala Gin Met 665 670 675

GTG GGT GCC CAA ATT GTA CTT GCT CAC ATG GAG CTG CCT CCT CCC 2070 Val Gly Ala Gin He Val Leu Ala His Met Glu Leu Pro Pro Pro 680 685 690

ATG GAG ACT GCT CAG ACG GAG GTT GCC CAA ATG GGG CCT GCT CCC 2115 Met Glu Thr Ala Gin Thr Glu Val Ala Gin Met Gly Pro Ala Pro

695 700 705

ATG GAA CCT GCT CAG ATG GAG GTT GCC CAG GTA GAA TCT GCT CCC 2160 Met Glu Pro Ala Gin Met Glu Val Ala Gin Val Glu Ser Ala Pro 710 715 720

ATG CAG GTG GTC CAG AAG GAG CCT GTT CAG ATG GAG CTG TCT CCT 2205 Met Gin Val Val Gin Lys Glu Pro Val Gin Met Glu Leu Ser Pro 725 730 735 CCC ATG GAG GTG GTC CAG AAG GAG CCT GTT CAG ATA GAG CTG TCT 2250 Pro Met Glu Val Val Gin Lys Glu Pro Val Gin He Glu Leu Ser 740 745 750

CCT CCC ATG GAG GTG GTC CAG AAG GAA CCT GTT AAG ATA GAG CTG 2295 Pro Pro Met Glu Val Val Gin Lys Glu Pro Val Lys He Glu Leu 755 760 765

TCT CCT CCC ATA GAG GTG GTC CAG AAG GAG CCT GTT CAG ATG GAG 2340 Ser Pro Pro He Glu Val Val Gin Lys Glu Pro Val Gin Met Glu

770 775 780

TTG TCT CCT CCC ATG GGG GTG GTT CAG AAG GAG CCT GCT CAG AGG 2385 Leu Ser Pro Pro Met Gly Val Val Gin Lys Glu Pro Ala Gin Arg 785 790 795

GAG CCA CCT CCT CCC AGA GAG CCT CCC CTT CAC ATG GAG CCA ATT 2430 Glu Pro Pro Pro Pro Arg Glu Pro Pro Leu His Met Glu Pro He 800 805 810

TCC AAA AAG CCT CCT CTC CGA AAA GAT AAA AAG GAA AAG TCT AAC 2475 Ser Lys Lys Pro Pro Leu Arg Lys Asp Lys Lys Glu Lys Ser Asn 815 820 825

ATG CAG AGT GAA AGG GCA CGG AAG GAG CAA GTC CTT ATT GAA GTT 2520 Met Gin Ser Glu Arg Ala Arg Lys Glu Gin Val Leu He Glu Val 830 835 840

GGC TTA GTG CCT GTT AAA GAT AGC TGG CTT CTA AAG GAA AGT GTA 2565 Gly Leu Val Pro Val Lys Asp Ser Trp Leu Leu Lys Glu Ser Val

845 850 855

AGC ACA GAG GAT CTC TCA CCA CCA TCA CCA CCA CTG CCA AAG GAA 2610 Ser Thr Glu Asp Leu Ser Pro Pro Ser Pro Pro Leu Pro Lys Glu 860 865 870

AAT TTA AGA GAA GAG GCA TCA GGA GAC CAA AAA TTA CTC AAC ACA 2655 Asn Leu Arg Glu Glu Ala Ser Gly Asp Gin Lys Leu Leu Asn Thr 875 880 885 GGT GAA GGA AAT AAA GAA GCC CCT CTT CAG AAA GTA GGA GCA GAA 2700 Gly Glu Gly Asn Lys Glu Ala Pro Leu Gin Lys Val Gly Ala Glu 890 895 900

GAG GCA GAT GAG AGC CTA CCT GGT CTT GCT GCT AAT ATC AAC GAA 2745 Glu Ala Asp Glu Ser Leu Pro Gly Leu Ala Ala Asn He Asn Glu 905 910 915

TCT ACC CAT ATT TCA TCC TCT GGA CAA AAC TTG AAT ACG CCA GAG 2790 Ser Thr His He Ser Ser Ser Gly Gin Asn Leu Asn Thr Pro Glu

920 925 930

GGT GAA ACT TTA AAT GGT AAA CAT CAG ACT GAC AGT ATA GTT TGT 2835 Gly Glu Thr Leu Asn Gly Lys His Gin Thr Asp Ser He Val Cys 935 940 945

GAA ATG AAA ATG GAC ACT GAT CAG AAC ACA AGA GAG AAT CTC ACT 2880 Glu Met Lys Met Asp Thr Asp Gin Asn Thr Arg Glu Asn Leu Thr 950 955 960

GGT ATA AAT TCA ACA GTT GAA GAA CCA GTT TCA CCA ATG CTT CCC 2925 Gly He Asn Ser Thr Val Glu Glu Pro Val Ser Pro Met Leu Pro 965 970 975

CCT TCA GCA GTA GAA GAA CGT GAA GCA GTG TCC AAA ACT GCA CTG 2970 Pro Ser Ala Val Glu Glu Arg Glu Ala Val Ser Lys Thr Ala Leu 980 985 990

GCA TCA CCT CCT GCT ACA ATG GCA GCA AAT GAG TCT CAG GAA ATT 3015 Ala Ser Pro Pro Ala Thr Met Ala Ala Asn Glu Ser Gin Glu He

995 1000 1005

GAT GAA GAT GAA GGC ATC CAC AGC CAT GAA GGA AGT GAC CTA AGT 3060 Asp Glu Asp Glu Gly He His Ser His Glu Gly Ser Asp Leu Ser 1010 1015 1020

GAC AAC ATG TCA GAG GGT AGT GAT GAT TCT GGA TTG CAT GGG GCT 3105

Asp Asn Met Ser Glu Gly Ser Asp Asp Ser Gly Leu His Gly Ala 1025 1030 1035 CGG CCA GTT CCA CAA GAA TCT AGC AGA AAA AAT GCA AAG GAA GCC 3150 Arg Pro Val Pro Gin Glu Ser Ser Arg Lys Asn Ala Lys Glu Ala 1040 1045 1050

TTG GCA GTC AAA GCG GCT AAG GGA GAT TTT GTT TGT ATC TTC TGT 3195 Leu Ala Val Lys Ala Ala Lys Gly Asp Phe Val Cys He Phe Cys 1055 1060 1065

GAT CGT TCT TTC AGA AAG GGA AAA GAT TAC AGC AAA CAC CTC AAT 3240 Asp Arg Ser Phe Arg Lys Gly Lys Asp Tyr Ser Lys His Leu Asn

1070 1075 1080

CGC CAT TTG GTT AAT GTG TAC TAT CTT GAA GAA GCA GCT CAA GGG 3285 Arg His Leu Val Asn Val Tyr Tyr Leu Glu Glu Ala Ala Gin Gly 1085 1090 1095

CAG GAG 3291

Gin Glu 1097

(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 63 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA

(x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C. , Altschuler, Yelena M., Frohman, Michael A., Kraner, Susan D. , Mandel, Gail (B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell (D) VOLUME: 80

(E) ISSUE:

(F) PAGES:

(G) DATE: March 24, 1995 (K) RELEVANT RESIDUES IN SEQ ID NO:11:FROM 1 TO 63 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

TGT AAG CCA TGC CAA TAT 18

Cys Lys Pro Cys Gin Tyr 165

GAA GCA GAA TCT GAA GAA CAG TTT GTG CAT CAC ATC AGA GTT CAC 63 Glu Ala Glu Ser Glu Glu Gin Phe Val His His He Arg Val His 170 175 180

(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 63 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: CDNA (X) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M., Frohman, Michael A., Kraner, Susan D. , Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80

(E) ISSUE:

(F) PAGES: (G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:12:FROM 1 TO 63 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TGT GAC CGC TGC GGC TAC AAT ACT 24

Cys Asp Arg Cys Gly Tyr Asn Thr 220 225

AAT CGA TAT GAT CAC TAT ACA GCA CAC CTG AAA CAC CAC 63

Asn Arg Tyr Asp His Tyr Thr Ala His Leu Lys His His 230 235

(2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 63 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler,

Yelena M. , Frohman, Michael A., Kraner, Susan D., Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell (D) VOLUME: 80

(E) ISSUE:

(F) PAGES:

(G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:13:FROM 1 TO 63 (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:13:

TGT ATC ATT TGC ACA TAC 18

Cys He He Cys Thr Tyr 250 255 ACA ACA GTG AGC GAG TAT CAC TGG AGG AAA CAT TTA AGA AAC CAT 63 Thr Thr Val Ser Glu Tyr His Trp Arg Lys His Leu Arg Asn His 260 265 270

(2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 63 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE:

(A) LIBRARY: CDNA

(x) PUBLICATION INFORMATION: (A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D. , Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons (C) JOURNAL: Cell

(D) VOLUME: 80

(E) ISSUE:

(F) PAGES:

(G) DATE: March 24, 1995 (K) RELEVANT RESIDUES IN SEQ ID NO: 14 :FROM 1 TO 63 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

TGT GGA AAA TGC AAC TAT TTT TCA 24

Cys Gly Lys Cys Asn Tyr Phe Ser 280 285

GAC AGA AAA AAC AAT TAT GTT CAG CAT GTT AGA ACT CAT 63

Asp Arg Lys Asn Asn Tyr Val Gin His Val Arg Thr His 290 295

(2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 63 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A. , Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler,

Yelena M. , Frohman, Michael A., Kraner, Susan D., Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell (D) VOLUME: 80

(E) ISSUE:

(F) PAGES:

(G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:15:FROM 1 TO 63 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

TGT GAA CTT TGT CCT TAC TCA AGT TCT CAG 30

Cys Glu Leu Cys Pro Tyr Ser Ser Ser Gin 310 315

AAG ACT CAT CTA ACT AGA CAT ATG CGT ACT CAT 63

Lys Thr His Leu Thr Arg His Met Arg Thr His 320 325

(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 66 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A. , Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D. , Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80 (E) ISSUE:

(F) PAGES:

(G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:16:FROM 1 TO 66 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

TGT GAT CAG TGC AGT TAT GTG GCC TCT AAT CAA CAT 36

Cys Asp Gin Cys Ser Tyr Val Ala Ser Asn Gin His 335 340 345

GAA GTA ACC CGC CAT GCA AGA CAG GTT CAC 66

Glu Val Thr Arg His Ala Arg Gin Val His 350 355

(2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 63 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D., Mandel, Gail (B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80

(E) ISSUE: (F) PAGES:

(G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:17:FROM 1 TO 63 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

TGC CCA CAC TGT GAT TAC AAA ACA GCA GAT AGA AGC AAC 39

Cys Pro His Cys Asp Tyr Lys Thr Ala Asp Arg Ser Asn 365 370 375

TTC AAA AAA CAT GTA GAG CTA CAT 63 Phe Lys Lys His Val Glu Leu His

380

(2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 66 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: CDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D., Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons (C) JOURNAL: Cell

(D) VOLUME: 80

(E) ISSUE:

(F) PAGES: (G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:18:FROM 1 TO 66 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

TGC CCT GTA TGT GAC TAT GCA GCT TCC AAG AAG TGT AAT CTA CAG 45 Cys Pro Val Cys Asp Tyr Ala Ala Ser Lys Lys Cys Asn Leu Gin

395 400 405

TAT CAC TTC AAA TCT AAG CAT 66

Tyr His Phe Lys Ser Lys His 410

(2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 441 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no

(vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA

(x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A. , Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D. , Mandel, Gail (B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80

(E) ISSUE: (F) PAGES:

(G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:20:FROM 1 TO 441 - 80 -

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

ATG GAG GTG GTT CAG GAG GGG 21

Met Glu Val Val Gin Glu Gly 655 660

CCT GCT CAG AAG GAG CTG CTG CCT CCC GTG GAG CCT GCT CAG ATG 66 Pro Ala Gin Lys Glu Leu Leu Pro Pro Val Glu Pro Ala Gin Met 665 670 675

GTG GGT GCC CAA ATT GTA CTT GCT CAC ATG GAG CTG CCT CCT CCC 111 Val Gly Ala Gin He Val Leu Ala His Met Glu Leu Pro Pro Pro 680 685 690

ATG GAG ACT GCT CAG ACG GAG GTT GCC CAA ATG GGG CCT GCT CCC 156 Met Glu Thr Ala Gin Thr Glu Val Ala Gin Met Gly Pro Ala Pro 695 700 705

ATG GAA CCT GCT CAG ATG GAG GTT GCC CAG GTA GAA TCT GCT CCC 201 Met Glu Pro Ala Gin Met Glu Val Ala Gin Val Glu Ser Ala Pro

710 715 720

ATG CAG GTG GTC CAG AAG GAG CCT GTT CAG ATG GAG CTG TCT CCT 246 Met Gin Val Val Gin Lys Glu Pro Val Gin Met Glu Leu Ser Pro 725 730 735

CCC ATG GAG GTG GTC CAG AAG GAG CCT GTT CAG ATA GAG CTG TCT 291 Pro Met Glu Val Val Gin Lys Glu Pro Val Gin He Glu Leu Ser 740 745 750

CCT CCC ATG GAG GTG GTC CAG AAG GAA CCT GTT AAG ATA GAG CTG 336 Pro Pro Met Glu Val Val Gin Lys Glu Pro Val Lys He Glu Leu 755 760 765

TCT CCT CCC ATA GAG GTG GTC CAG AAG GAG CCT GTT CAG ATG GAG 381 Ser Pro Pro He Glu Val Val Gin Lys Glu Pro Val Gin Met Glu 770 775 780 TTG TCT CCT CCC ATG GGG GTG GTT CAG AAG GAG CCT GCT CAG AGG 426 Leu Ser Pro Pro Met Gly Val Val Gin Lys Glu Pro Ala Gin Arg 785 790 795

GAG CCA CCT CCT CCC 441

Glu Pro Pro Pro Pro 800

(2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler,

Yelena M. , Frohman, Michael A., Kraner, Susan D. , Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell (D) VOLUME: 80

(E) ISSUE:

(F) PAGES:

(G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO-.21:FROM 1 TO 48 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

ATG GAG GTG GTT CAG GAG GGG 21

Met Glu Val Val Gin Glu Gly 655 660 CCT GCT CAG AAG GAG CTG CTG CCT CCC 48

Pro Ala Gin Lys Glu Leu Leu Pro Pro 665

(2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA

(x) PUBLICATION INFORMATION: (A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D. , Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons (C) JOURNAL: Cell

(D) VOLUME: 80

(E) ISSUE:

(F) PAGES:

(G) DATE: March 24, 1995 (K) RELEVANT RESIDUES IN SEQ ID NO:22:FROM 1 TO 48 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

ATG CAG GTG GTC CAG AAG GAG CCT GTT CAG ATG GAG CTG TCT CCT 45 Met Gin Val Val Gin Lys Glu Pro Val Gin Met Glu Leu Ser Pro 725 730 735

CCC 48

Pro

(2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 48 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA (X) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D., Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80 (E) ISSUE:

(F) PAGES:

(G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:23:FROM 1 TO 48 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

ATG GAG GTG GTC CAG AAG GAG CCT GTT CAG ATA GAG CTG TCT 42 Met Glu Val Val Gin Lys Glu Pro Val Gin He Glu Leu Ser 740 745 750

CCT CCC 48

Pro Pro

(2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: CDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D. , Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80

(E) ISSUE:

(F) PAGES: (G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:24:FROM 1 TO 48 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

ATG GAG GTG GTC CAG AAG GAA CCT GTT AAG ATA GAG CTG 39 Met Glu Val Val Gin Lys Glu Pro Val Lys He Glu Leu 755 760 765

TCT CCT CCC 48

Ser Pro Pro

(2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA (X) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M., Frohman, Michael A., Kraner, Susan D., Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell (D) VOLUME: 80

(E) ISSUE:

(F) PAGES:

(G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:25:FROM 1 TO 48 (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:25:

ATA GAG GTG GTC CAG AAG GAG CCT GTT CAG ATG GAG 36

He Glu Val Val Gin Lys Glu Pro Val Gin Met Glu 770 775 780

TTG TCT CCT CCC 48 Leu Ser Pro Pro

(2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE:

(A) LIBRARY: cDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A. , Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler,

Yelena M. , Frohman, Michael A., Kraner, Susan D., Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell (D) VOLUME: 80

(E) ISSUE:

(F) PAGES: (G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:26:FROM 1 TO 48 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

ATG GGG GTG GTT CAG AAG GAG CCT GCT CAG AGG 33

Met Gly Val Val Gin Lys Glu Pro Ala Gin Arg 785 790 795

GAG CCA CCT CCT CCC 48 Glu Pro Pro Pro Pro

800

(2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 1461 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A., Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C. , Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D., Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80 (E) ISSUE:

(F) PAGES:

(G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:26:FROM 1 TO 1461 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: CTG GCC GCA CCT CAG 15

Leu Ala Ala Pro Gin 45

CTT ATT ATG CTG GCA AAT GTG GCC TTA ACT GGG GAA GTA AAT GGC 60 Leu He Met Leu Ala Asn Val Ala Leu Thr Gly Glu Val Asn Gly 50 55 60

AGC TGC TGT GAT TAC CTG GTC GGT GAA GAA AGA CAG ATG GCA GAA 105 Ser Cys Cys Asp Tyr Leu Val Gly Glu Glu Arg Gin Met Ala Glu

65 70 75

CTG ATG CCG GTT GGG GAT AAC AAC TTT TCA GAT AGT GAA GAA GGA 150 Leu Met Pro Val Gly Asp Asn Asn Phe Ser Asp Ser Glu Glu Gly 80 85 90

GAA GGA CTT GAA GAG TCT GCT GAT ATA AAA GGT GAA CCT CAT GGA 195 Glu Gly Leu Glu Glu Ser Ala Asp He Lys Gly Glu Pro His Gly 95 100 105

CTG GAA AAC ATG GAA CTG AGA AGT TTG GAA CTC AGC GTC GTA GAA 240 Leu Glu Asn Met Glu Leu Arg Ser Leu Glu Leu Ser Val Val Glu

110 115 120

CCT CAG CCT GTA TTT GAG GCA TCA GGT GCT CCA GAT ATT TAC AGT 285 Pro Gin Pro Val Phe Glu Ala Ser Gly Ala Pro Asp He Tyr Ser 125 130 135

TCA AAT AAA GCT CTT GCC CCT GAA ACA CCT GGA GCG GAG GAC AAA 330 Ser Asn Lys Ala Leu Ala Pro Glu Thr Pro Gly Ala Glu Asp Lys

140 145 150

GGC AAG AGC TCG AAG ACC AAA CCC TTT CGC TGT AAG CCA TGC CAA 375 Gly Lys Ser Ser Lys Thr Lys Pro Phe Arg Cys Lys Pro Cys Gin 155 160 165

TAT GAA GCA GAA TCT GAA GAA CAG TTT GTG CAT CAC ATC AGA GTT 420 Tyr Glu Ala Glu Ser Glu Glu Gin Phe Val His His He Arg Val 170 175 180 CAC AGT GCT AAG AAA TTT TTT GTG GAA GAG AGT GCA GAG AAG CAG 465 His Ser Ala Lys Lys Phe Phe Val Glu Glu Ser Ala Glu Lys Gin 185 190 195

GCA AAA GCC AGG GAA TCT GGC TCT TCC ACT GCA GAA GAG GGA GAT 510 Ala Lys Ala Arg Glu Ser Gly Ser Ser Thr Ala Glu Glu Gly Asp 200 205 210

TTC TCC AAG GGC CCC ATT CGC TGT GAC CGC TGC GGC TAC AAT ACT 555 Phe Ser Lys Gly Pro He Arg Cys Asp Arg Cys Gly Tyr Asn Thr

215 220 225

AAT CGA TAT GAT CAC TAT ACA GCA CAC CTG AAA CAC CAC ACC AGA 600 Asn Arg Tyr Asp His Tyr Thr Ala His Leu Lys His His Thr Arg 230 235 240

GCT GGG GAT AAT GAG CGA GTC TAC AAG TGT ATC ATT TGC ACA TAC 645 Ala Gly Asp Asn Glu Arg Val Tyr Lys Cys He He Cys Thr Tyr 245 250 255

ACA ACA GTG AGC GAG TAT CAC TGG AGG AAA CAT TTA AGA AAC CAT 690 Thr Thr Val Ser Glu Tyr His Trp Arg Lys His Leu Arg Asn His 260 265 270

TTT CCA AGG AAA GTA TAC ACA TGT GGA AAA TGC AAC TAT TTT TCA 735 Phe Pro Arg Lys Val Tyr Thr Cys Gly Lys Cys Asn Tyr Phe Ser 275 280 285

GAC AGA AAA AAC AAT TAT GTT CAG CAT GTT AGA ACT CAT ACA GGA 780 Asp Arg Lys Asn Asn Tyr Val Gin His Val Arg Thr His Thr Gly

290 295 300

GAA CGC CCA TAT AAA TGT GAA CTT TGT CCT TAC TCA AGT TCT CAG 825 Glu Arg Pro Tyr Lys Cys Glu Leu Cys Pro Tyr Ser Ser Ser Gin 305 310 315

AAG ACT CAT CTA ACT AGA CAT ATG CGT ACT CAT TCA GGT GAG AAG 870 Lys Thr His Leu Thr Arg His Met Arg Thr His Ser Gly Glu Lys 320 325 330 CCA TTT AAA TGT GAT CAG TGC AGT TAT GTG GCC TCT AAT CAA CAT 915 Pro Phe Lys Cys Asp Gin Cys Ser Tyr Val Ala Ser Asn Gin His 335 340 345

GAA GTA ACC CGC CAT GCA AGA CAG GTT CAC AAT GGG CCT AAA CCT 960 Glu Val Thr Arg His Ala Arg Gin Val His Asn Gly Pro Lys Pro 350 355 360

CTT AAT TGC CCA CAC TGT GAT TAC AAA ACA GCA GAT AGA AGC AAC 1005 Leu Asn Cys Pro His Cys Asp Tyr Lys Thr Ala Asp Arg Ser Asn

365 370 375

TTC AAA AAA CAT GTA GAG CTA CAT GTG AAC CCA CGG CAG TTC AAT 1050 Phe Lys Lys His Val Glu Leu His Val Asn Pro Arg Gin Phe Asn 380 385 390

TGC CCT GTA TGT GAC TAT GCA GCT TCC AAG AAG TGT AAT CTA CAG 1095 Cys Pro Val Cys Asp Tyr Ala Ala Ser Lys Lys Cys Asn Leu Gin 395 400 405

TAT CAC TTC AAA TCT AAG CAT CCT ACT TGT CCT AAT AAA ACA ATG 1140

Tyr His Phe Lys Ser Lys His Pro Thr Cys Pro Asn Lys Thr Met 410 415 420

GAT GTC TCA AAA GTG AAA CTA AAG AAA ACC AAA AAA CGA GAG GCT 1185 Asp Val Ser Lys Val Lys Leu Lys Lys Thr Lys Lys Arg Glu Ala 425 430 435

GAC TTG CCT GAT AAT ATT ACC AAT GAA AAA ACA GAA ATA GAA CAA 1230 Asp Leu Pro Asp Asn He Thr Asn Glu Lys Thr Glu He Glu Gin

440 445 450

ACA AAA ATA AAA GGG GAT GTG GCT GGA AAG AAA AAT GAA AAG TCC 1275 Thr Lys He Lys Gly Asp Val Ala Gly Lys Lys Asn Glu Lys Ser 455 460 465

GTC AAA GCA GAG AAA AGA GAT GTC TCA AAA GAG AAA AAG CCT TCT 1320 Val Lys Ala Glu Lys Arg Asp Val Ser Lys Glu Lys Lys Pro Ser 470 475 480 AAT AAT GTG TCA GTG ATC CAG GTG ACT ACC AGA ACT CGA AAA TCA 1365 Asn Asn Val Ser Val He Gin Val Thr Thr Arg Thr Arg Lys Ser 485 490 495

GTA ACA GAG GTG AAA GAG ATG GAT GTG CAT ACA GGA AGC AAT TCA 1410 Val Thr Glu Val Lys Glu Met Asp Val His Thr Gly Ser Asn Ser 500 505 510

GAA AAA TTC AGT AAA ACT AAG AAA AGC AAA AGG AAG CTG GAA GTT 1455 Glu Lys Phe Ser Lys Thr Lys Lys Ser Lys Arg Lys Leu Glu Val

515 520 525

GAC AGC 1461

Asp Ser

(2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1284 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE: (A) ORGANISM: Human (H) CELL LINE: HeLa (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA (x) PUBLICATION INFORMATION:

(A) AUTHORS: Chong, Jayhong A. , Tapia-Ramirez Jose, Toledo- Aral, Juan, Zheng, Yingcong, Boutros, Michael C, Altschuler, Yelena M. , Frohman, Michael A., Kraner, Susan D. , Mandel, Gail

(B) TITLE: REST: A Mammalian Silencer Protein that Restricts Sodium Channel Gene Expression to Neurons

(C) JOURNAL: Cell

(D) VOLUME: 80

(E) ISSUE:

(F) PAGES: (G) DATE: March 24, 1995

(K) RELEVANT RESIDUES IN SEQ ID NO:26:FROM 1 TO 1284 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: TCT TCT GGA GGA GGA GGG CTG 21

Ser Ser Gly Gly Gly Gly Leu 10 15

TTT ACC AGC AGT GGC AAC ATT GGA ATG GCC CTG CCT AAC GAC ATG 66 Phe Thr Ser Ser Gly Asn He Gly Met Ala Leu Pro Asn Asp Met 20 25 30

TAT GAC TTG CAT GAC CTT TCC AAA GCT GAA CTG GCC GCA CCT CAG 111 Tyr Asp Leu His Asp Leu Ser Lys Ala Glu Leu Ala Ala Pro Gin

35 40 45

CTT ATT ATG CTG GCA AAT GTG GCC TTA ACT GGG GAA GTA AAT GGC 156 Leu He Met Leu Ala Asn Val Ala Leu Thr Gly Glu Val Asn Gly 50 55 60

AGC TGC TGT GAT TAC CTG GTC GGT GAA GAA AGA CAG ATG GCA GAA 201 Ser Cys Cys Asp Tyr Leu Val Gly Glu Glu Arg Gin Met Ala Glu 65 70 75

CTG ATG CCG GTT GGG GAT AAC AAC TTT TCA GAT AGT GAA GAA GGA 246 Leu Met Pro Val Gly Asp Asn Asn Phe Ser Asp Ser Glu Glu Gly 80 85 90

GAA GGA CTT GAA GAG TCT GCT GAT ATA AAA GGT GAA CCT CAT GGA 291 Glu Gly Leu Glu Glu Ser Ala Asp He Lys Gly Glu Pro His Gly 95 100 105

CTG GAA AAC ATG GAA CTG AGA AGT TTG GAA CTC AGC GTC GTA GAA 336 Leu Glu Asn Met Glu Leu Arg Ser Leu Glu Leu Ser Val Val Glu

110 115 120

CCT CAG CCT GTA TTT GAG GCA TCA GGT GCT CCA GAT ATT TAC AGT 381 Pro Gin Pro Val Phe Glu Ala Ser Gly Ala Pro Asp He Tyr Ser 125 130 135

TCA AAT AAA GCT CTT GCC CCT GAA ACA CCT GGA GCG GAG GAC AAA 426 Ser Asn Lys Ala Leu Ala Pro Glu Thr Pro Gly Ala Glu Asp Lys 140 145 150 GGC AAG AGC TCG AAG ACC AAA CCC TTT CGC TGT AAG CCA TGC CAA 471 Gly Lys Ser Ser Lys Thr Lys Pro Phe Arg Cys Lys Pro Cys Gin 155 160 165

TAT GAA GCA GAA TCT GAA GAA CAG TTT GTG CAT CAC ATC AGA GTT 516 Tyr Glu Ala Glu Ser Glu Glu Gin Phe Val His His He Arg Val 170 175 180

CAC AGT GCT AAG AAA TTT TTT GTG GAA GAG AGT GCA GAG AAG CAG 561 His Ser Ala Lys Lys Phe Phe Val Glu Glu Ser Ala Glu Lys Gin

185 190 195

GCA AAA GCC AGG GAA TCT GGC TCT TCC ACT GCA GAA GAG GGA GAT 606 Ala Lys Ala Arg Glu Ser Gly Ser Ser Thr Ala Glu Glu Gly Asp 200 205 210

TTC TCC AAG GGC CCC ATT CGC TGT GAC CGC TGC GGC TAC AAT ACT 651 Phe Ser Lys Gly Pro He Arg Cys Asp Arg Cys Gly Tyr Asn Thr 215 220 225

AAT CGA TAT GAT CAC TAT ACA GCA CAC CTG AAA CAC CAC ACC AGA 696 Asn Arg Tyr Asp His Tyr Thr Ala His Leu Lys His His Thr Arg 230 235 240

GCT GGG GAT AAT GAG CGA GTC TAC AAG TGT ATC ATT TGC ACA TAC 741 Ala Gly Asp Asn Glu Arg Val Tyr Lys Cys He He Cys Thr Tyr 245 250 255

ACA ACA GTG AGC GAG TAT CAC TGG AGG AAA CAT TTA AGA AAC CAT 786 Thr Thr Val Ser Glu Tyr His Trp Arg Lys His Leu Arg Asn His

260 265 270

TTT CCA AGG AAA GTA TAC ACA TGT GGA AAA TGC AAC TAT TTT TCA 831 Phe Pro Arg Lys Val Tyr Thr Cys Gly Lys Cys Asn Tyr Phe Ser 275 280 285

GAC AGA AAA AAC AAT TAT GTT CAG CAT GTT AGA ACT CAT ACA GGA 876

Asp Arg Lys Asn Asn Tyr Val Gin His Val Arg Thr His Thr Gly 290 295 300 GAA CGC CCA TAT AAA TGT GAA CTT TGT CCT TAC TCA AGT TCT CAG 921 Glu Arg Pro Tyr Lys Cys Glu Leu Cys Pro Tyr Ser Ser Ser Gin 305 310 315

AAG ACT CAT CTA ACT AGA CAT ATG CGT ACT CAT TCA GGT GAG AAG 966 Lys Thr His Leu Thr Arg His Met Arg Thr His Ser Gly Glu Lys 320 325 330

CCA TTT AAA TGT GAT CAG TGC AGT TAT GTG GCC TCT AAT CAA CAT 1011 Pro Phe Lys Cys Asp Gin Cys Ser Tyr Val Ala Ser Asn Gin His

335 340 345

GAA GTA ACC CGC CAT GCA AGA CAG GTT CAC AAT GGG CCT AAA CCT 1056 Glu Val Thr Arg His Ala Arg Gin Val His Asn Gly Pro Lys Pro 350 355 360

CTT AAT TGC CCA CAC TGT GAT TAC AAA ACA GCA GAT AGA AGC AAC 1101 Leu Asn Cys Pro His Cys Asp Tyr Lys Thr Ala Asp Arg Ser Asn 365 370 375

TTC AAA AAA CAT GTA GAG CTA CAT GTG AAC CCA CGG CAG TTC AAT 1146 Phe Lys Lys His Val Glu Leu His Val Asn Pro Arg Gin Phe Asn 380 385 390

TGC CCT GTA TGT GAC TAT GCA GCT TCC AAG AAG TGT AAT CTA CAG 1191 Cys Pro Val Cys Asp Tyr Ala Ala Ser Lys Lys Cys Asn Leu Gin 395 400 405

TAT CAC TTC AAA TCT AAG CAT CCT ACT TGT CCT AAT AAA ACA ATG 1236 Tyr His Phe Lys Ser Lys His Pro Thr Cys Pro Asn Lys Thr Met

410 415 420

GAT GTC TCA AAA GTG AAA CTA AAG AAA ACC AAA AAA CGA GAG GCT 1281 Asp Val Ser Lys Val Lys Leu Lys Lys Thr Lys Lys Arg Glu Ala 425 430 435

GAC 1284

Asp

(2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: rat (vii) IMMEDIATE SOURCE: (A) LIBRARY: Genomic

(x) PUBLICATION INFORMATION:

(A) AUTHORS: Maue, R.A., Kraner, Goodman, R.H., Mandel, Gail

(B) TITLE: REST: Neuron-Specific Expression of the Rat Brain Type II Sodium Channel Gene Is Directed by Upstream Regulatory Elements

(C) JOURNAL: Neuron

(D) VOLUME: 4

(F) PAGES: 223-231 (G) DATE: February, 1990

(K) RELEVANT RESIDUES IN SEQ ID NO:29:FROM 1 TO 28 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

ATTGGGTTTC AGAACCACGG ACAGCACC 28

(2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:

(A) ORGANISM: Rat (vii) IMMEDIATE SOURCE:

(A) LIBRARY: Genomic

(x) PUBLICATION INFORMATION: (A) AUTHORS: Maue, R.A. , Kraner, Goodman, R.H., Mandel, Gail

(B) TITLE: REST: Neuron-Specific Expression of the Rat Brain Type II Sodium Channel Gene Is Directed by Upstream Regulatory Elements

(C) JOURNAL: Neuron

(D) VOLUME: 4

(F) PAGES: 223-231

(G) DATE: February, 1990

(K) RELEVANT RESIDUES IN SEQ ID NO:30:FROM 2353 TO 2400 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

ATTGGGGGGA CGAACCACGG ACAGCACC 28

Claims

What is claimed is: 1. A substantially pure nucleic acid comprising a nucleic acid encoding a protein having at least about 85 % homology to at least the DNA binding domain or the suppressor domain of an animal REST protein.

2. The substantially pure nucleic acid of claim 1, comprising a nucleic acid encoding at least the DNA binding domain or the suppressor domain of an animal REST protein. .

3. The substantially pure nucleic acid of claim 2, wherein the REST protein is a mammalian REST protein.

4. The substantially pure nucleic acid of claim 3, wherein the REST protein is a human REST protein.

5. The substantially pure nucleic acid of claim 4, wherein the nucleic acid comprises SEQ ID NO:2.

6. The substantially pure nucleic acid of claim 5, wherein the nucleic acid comprises SEQ ID NO: 10.

7. The substantially pure nucleic acid of claim 1, comprising a nucleic acid encoding both the DNA binding domain and the suppressor domain of an animal REST protein.

8. The substantially pure nucleic acid of claim 7, wherein the REST protein is a mammalian REST protein.

9. The substantially pure nucleic acid of claim 8, wherein the REST protein is a human REST protein.

10. The substantially pure nucleic acid of claim 9. wherein the nucleic acid comprises SEQ ID NO:2.

11. The substantially pure nucleic acid of claim 10, wherein the nucleic acid comprises SEQ ID NO: 10.

12. The substantially pure nucleic acid of claim 1, comprising a nucleic encoding a protein differing from an animal REST protein by no more than about 20 point mutations.

13. A substantially pure nucleic acid that hybridizes with an animal REST nucleic acid under stringent conditions.

14. The substantially pure nucleic acid of claim 13, comprising the nucleic acid of SEQ ID NO: 1.

15. A substantially pure nucleic acid comprising a nucleic acid encoding a protein that binds to a promoter having at least about 90% homology to nucleotides 6-28 of SEQ ID NO:29 and acting to suppress the activity of a promoter having said promoter.

16. A substantially pure protein having at least about 85% homology with at least the DNA binding domain or the suppressor domain of an animal REST protein.

17. The substantially pure protein of claim 16, comprising at least the DNA binding domain or the suppressor domain of an animal REST protein.

18. The substantially pure protein of claim 17, comprising the protein of SEQ ID NO:2.

19. The substantially pure protein of claim 18, comprising both the DNA binding domain and the suppressor domain of an animal REST protein.

20. The substantially pure protein of claim 19, comprising the protein of SEQ ID NO: 10.

21. A transformed eukaryotic or prokaryotic cell comprising a nucleic acid encoding a protein having at least about 85% homology to at least one of the DNA binding domain or the suppressor domain of an animal REST protein.

22. The transformed cell of claim 21 comprising a nucleic acid encoding at least the DNA binding domain or the suppressor domain of an animal REST protein.

23. The transformed cell of claim 22, wherein the REST protein is a mammalian REST protein.

24. The transformed cell of claim 23, wherein the REST protein is a human REST protein.

25. The transformed cell of claim 24, wherein the nucleic acid comprises SEQ ID NO:2.

26. A vector capable of reproducing in a eukaryotic or prokaryotic cell comprising a nucleic acid encoding a protein having at least about 85 % homology to at least the DNA binding domain or the suppressor domain of an animal REST protein.

27. The vector capable of reproducing in a eukaryotic or prokaryotic cell of claim 26, comprising a nucleic acid encoding at least the DNA binding domain or the suppressor domain of an animal REST protein.

28. The vector capable of reproducing in a eukaryotic or prokaryotic cell of claim 27, wherein the REST protein is a mammalian REST protein.

29. The vector capable of reproducing in a eukaryotic or prokaryotic cell of claim 28, wherein the REST protein is a human REST protein.

30. The vector capable of reproducing in a eukaryotic or prokaryotic cell of claim 29, wherein the nucleic acid comprises SEQ ID NO:2.

31. A method of preparing a protein having REST activity, wherein the protein has at least about 85 % homology with at least the DNA binding domain or the suppressor domain of an animal REST protein, the method comprising: (a) transforming an appropriate eukaryotic or prokaryotic cell with an expression vector for expressing intracellularly or extracellularly a nucleic acid encoding the protein; (b) growing the transformed cell in culture; and (c) isolating the protein from the transformed cell or the culture medium.

32. A pharmaceutical composition for treating an animal having de-differentiated neural cells or neural cells exhibiting diminished activity comprising an effective amount of a REST-interfering nucleic acid, wherein the REST-interfering nucleic acid comprises an antisense molecule directed against REST expression or an expression vector for expressing REST DNA binding activity but not REST silencer activity, and a pharmaceutically acceptable carrier.

33. The pharmaceutical composition of claim 32, wherein the animal has brain cancer.

34. The pharmaceutical composition of claim 32, wherein said animal has a demyelinating myasthenia gravis, muscular dystrophy, botulism, peripheral neuropathies, traumatic nerve injury, post stroke degeneration, post-traumatic spinal and neural degeneration, poliomyelitis or rabies.

35. A pharmaceutical composition for an animal having neural cells exhibiting excessive neural activity comprising an effective amount of an expression vector comprising a nucleic acid encoding a protein that inhibits the expression of neural proteins in non-neural tissues, and a pharmaceutically acceptable carrier.

36. The pharmaceutical composition of claim 35, wherein the animal has epilepsy, Lennox-Gastaut syndrome, spasticity, trauma-induced pain, schizophrenia, stroke or a neurodegenerative disease.

37. The pharmaceutical composition of claim 36, wherein the animal has Alzheimer's, Parkinson's or Huntington's disease.

38. The pharmaceutical composition of claim 36, wherein the animal has epilepsy.

39. The pharmaceutical composition of claim 36, wherein the animal has a neurodegenerative disease.

40. A method of determining the level of REST expression in a tissue sample comprising: (a) contacting the tissue sample with (i) a nucleic acid that binds to REST mRNA under stringent conditions or (ii) an antibody specific for REST; (b) washing the tissue sample to remove non-specific hybridizations of the nucleic acid or non-specific antibody binding; and (c) determining d e level of hybridized nucleic acid or bound antibody.

41. An antibody that reacts specifically with the substantially pure protein of claim 16.

42. A pair of PCR primers capable of directing the amplification of the substantially pure nucleic acid of claim 1.