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WO2004018632A2 - Tamis a haut rendement pour modulateurs de l'activite de modification de la chromatine - Google Patents

Tamis a haut rendement pour modulateurs de l'activite de modification de la chromatine Download PDF

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WO2004018632A2
WO2004018632A2 PCT/US2003/026334 US0326334W WO2004018632A2 WO 2004018632 A2 WO2004018632 A2 WO 2004018632A2 US 0326334 W US0326334 W US 0326334W WO 2004018632 A2 WO2004018632 A2 WO 2004018632A2
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WO2004018632A3 (fr
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Andrew Snowden
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Sangamo Therapeutics Inc
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Sangamo Biosciences Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • TECHNICAL FIELD This disclosure resides in the fields of cellular engineering and drug discovery; in particular, methods and compositions for identifying modulators of chromatin modifying activity.
  • chromosomal DNA is packaged into nucleosomes.
  • a nucleosome comprises a core and a linker.
  • the nucleosome core comprises an octamer of core histones (two each of H2A, H2B, H3 and H4) around which is wrapped approximately 150 base pairs of chromosomal DNA.
  • a linker DNA segment of approximately 50 base pairs is associated with linker histone HI.
  • Nucleosomes are organized into a higher-order chromatin fiber and chromatin fibers are organized into chromosomes. See, for example, Wolffe “Chromatin: Structure and Function” 3 rd Ed., Academic Press, San Diego, 1998.
  • cellular chromatin, including nucleosome structure is organized into a higher order structure of regions or "domains.” In those tissues where a given gene or gene cluster is active, the domain is sensitive to DNase I, suggesting that the chromatin of an active domain is in a loose, decondensed configuration that is easily accessible to trans-acting factors (Lawson et al. (1982). J. Biol.
  • chromatin structure has been associated with the process of gene regulation in vivo and with specific epigenetic states associated with repressed, silenced and induced gene promoters and/or enhancers.
  • CMEs chromatin modifying enzymes
  • HATs histone acetyltransferases
  • SAGA histone acetyltransferases
  • CMEs include histone deacteylases, which remove acetyl modifications from histone tails.
  • histone deactelyase activity may play a role in transcriptional co-repression by molecules such as NCoR/SMRT (Laherty et al. (1997) Cell 89(3)349-356; Nagy et al. (1997) Cell 89(3):373-380; Huang et al. (2000) Genes Devel.
  • Histone methyltranferases an additional class of CME, including arginine methyltransferases in the PRMTl and CARMl families, target specific arginine residues of H3 and H4 and have been associated with transcriptional activation of nuclear hormone receptors.
  • CME CME
  • arginine methyltransferases in the PRMTl and CARMl families, target specific arginine residues of H3 and H4 and have been associated with transcriptional activation of nuclear hormone receptors.
  • H3K9 methyltransferases include Suv39Hl (Rea et al. (2000) Nature 406(6796):593-599); Suv39H2 (O'CarroU et al.
  • a further exemplary class of CME includes histone kinases, which may phosphorylate serine residues of histones, and corresponding histone phosphatases. Kinases involved in phosphorylation of serine 10 of histone H3 have been shown to be associated with transcriptional activation. Lo et al. (2001) Science 293: 1142-1146.
  • the structure of chromatin can also be altered through the activity of macromolecular assemblies known as chromatin remodeling complexes. See, for example, Cairns (1998) Trends Biochem. Sci.
  • CME modulatory compounds e.g., activator or inhibitor
  • broad spectrum inhibitors such as sodium butyrate and trichostatin A, in the case ofthe histone deacetylases, are used to modulate these activities.
  • Such compounds can have genome-wide effects and do not exhibit specificity for a particular CME.
  • Specific compounds have been difficult to identify due to the limitations ofthe activity assays available, which commonly entail a bulk immunoprecipitation-in vitro activity assay or related assay, precluding the development of any large-scale screening system.
  • a method to screen for a modulator of a modifying activity comprises (a) providing a cell, wherein the cell contains a fusion protein comprising a modifying activity and a DNA-binding activity, wherein the DNA-binding activity is targeted to a reporter gene, and wherein the fusion protein regulates the expression ofthe reporter gene; (b) contacting the cell with a substance; and (c) assaying expression ofthe reporter gene, wherein, if expression of the reporter gene is altered in the presence ofthe substance, compared to the absence ofthe substance, the substance is a modulator ofthe modifying activity.
  • the modifying activity can, for example, modify cliromosomal DNA or a chromosomal protein (e.g., a histone or a non-histone cliromosomal protein).
  • the modifying activity can be, for example, a DNA methyltransferase, a histone acetyl transferase, a histone methyl transferase, a histone deacetylase or a functional fragment of any ofthe aforementioned enzymes.
  • the reporter gene can be, for example, a cliromosomal gene and/or an endogenous gene (e.g., NEGF, H19 and IGF-2).
  • the substance can be, for example, an activator ofthe modifying activity or an inhibitor ofthe modifying activity.
  • the D ⁇ A-binding activity comprises a naturally-occurring D ⁇ A- binding domain.
  • one or more engineered zinc fingers provide D ⁇ A- binding activity.
  • the fusion protein can activate expression ofthe reporter gene or repress expression ofthe reporter gene. Further, expression can be monitored by assaying mR ⁇ A levels encoded by the reporter gene, by assaying expression of protein encoded by the reporter gene and/or by assaying for alteration of expression ofthe reporter gene that results in a change in phenotype.
  • the cell can be a plant cell, a mammalian cell, or a human cell. In certain embodiments, the cell comprises a polynucleotide encoding the fusion protein.
  • the method comprises (a) providing a cell, wherein the cell contains a fusion protein comprising an interaction domain and a D ⁇ A-binding domain, wherein the interaction domain recruits a modifying activity, wherein the D ⁇ A-binding domain is targeted to a reporter gene, and wherein the modifying activity regulates the expression ofthe reporter gene; (b) contacting the cell with a substance; and (c) assaying expression ofthe reporter gene, wherein, if expression ofthe reporter gene is altered in the presence ofthe substance, compared to the absence ofthe substance, the substance is a modulator ofthe modifying activity.
  • a fusion protein comprising a modifying activity (or functional fragment thereof) and a D ⁇ A-binding activity, wherein the D ⁇ A-binding activity is targeted to a reporter gene, and wherein the modifying activity regulates the expression ofthe reporter gene.
  • Polynucleotides encoding such fusion proteins are also provided.
  • a polynucleotide encoding the fusion protein is the product of an oncogenic cliromosomal translocation.
  • a fusion protein comprising an interaction domain (or functional fragment thereof) and a D ⁇ A-binding domain.
  • the interaction domain is capable of recruiting a chromatin modifying activity and the D ⁇ A-binding domain is targeted to a reporter gene, such that the recruited modifying activity regulates expression ofthe reporter gene.
  • Polynucleotides encoding such fusion proteins are also provided.
  • a polynucleotide encoding the fusion protein is the product of an oncogenic cliromosomal translocation.
  • the modifying activity can comprise, for example, a histone methyltransferase, a histone demethylase, a histone kinase (e.g., S ⁇ F1, pp90Rsk, IKK alpha), a histone phosphatase (e.g., PP2A), a histone ubiquitinating enzyme (e.g., RAD6), a histone de-ubiquitinating enzyme, a histone ADP-ribosylating enzyme (e.g., PARPl), a histone de-ribosylating enzyme (e.g., PARG), a histone aminotransferase, a histone deaminase, a histone iminase, a histone de-iminase (e.g., peptidylarginine deiminase, PAD), a DNA aminotransferase, a DNA deamina
  • the interaction domain can recruit a histone methyltransferase, a histone demethylase, a histone kinase, a histone phosphatase, a histone ubiquitinating enzyme, a histone de-ubiquitinating enzyme, a histone ADP-ribosylating enzyme, a histone de-ribosylating enzyme, a histone aminotransferase, a histone deaminase, a histone iminase, a histone de-iminase, a DNA aminotransferase, a DNA.
  • the disclosed fusion proteins can also be used for these purposes.
  • the enzyme peptidylarginine deiminase (PAD) converts arginine to citrulline in proteins.
  • PADs PAD I, PAD II, PAD III and PAD V, exist in humans.
  • PAD V converts arginine to citrulline in histones HI, H2, H3 and H4, and this modification can antagonize the transcription-stimulatory activities of nuclear hormone receptors.
  • PAD V converts arginine to citrulline in histones HI, H2, H3 and H4, and this modification can antagonize the transcription-stimulatory activities of nuclear hormone receptors.
  • transcription is activated by a nuclear hormone receptor, a fusion protein comprising a PAD (e.g., PAD V), or functional fragment thereof, and a zinc finger binding domain targeted to the gene, can be used to repress and/or block nuclear hormone receptor-stimulated transcription ofthe targeted gene.
  • a fusion protein comprising a PAD (e.g., PAD V), or functional fragment thereof, and a zinc finger binding domain targeted to the gene
  • PAD e.g., PAD V
  • Methylation of lysine 9 on histones H3 and H4 can result in repression of gene expression, if the methylated histones are present in a nucleosome in the vicinity ofthe gene.
  • fusions of a ZFP binding domain (targeted to a gene of interest) and the catalytic domain of a histone methyltransferase when expressed in a cell, can be used for targeted repression of gene expression.
  • a histone methyltransferase e.g., Suv39Hl, G9A
  • Polynucleotides encoding the fusion proteins disclosed herein, as well as cells comprising said polynucleotides and fusion proteins are also provided.
  • Also disclosed herein is a method of screening for a compound that modulates the activity of a chromatin modifying enzyme, the method comprising the steps of: (a) contacting a cell with the compound, wherein the cell comprises a fusion protein comprising a zinc finger protein (ZFP) and a functional chromatin modifying enzyme or fragment thereof and wherein the ZFP binds to a reporter gene; and (b) determining the level of expression ofthe reporter gene.
  • the fusion protein can be provided as a polypeptide and/or as a polynucleotide encoding the fusion protein.
  • the cell can be stably or transiently transfected with the polynucleotide encoding the fusion protein.
  • the polynucleotide further comprises an inducible promoter (e.g., a tetracycline-inducible promoter) operably linked to the polynucleotide encoding the fusion protein.
  • exemplary chromatin modifying enzymes can be selected from the group consisting of a histone methyltransferase (HMT) (e.g., lysine or arginine HMTases such as those which methylate H3 lysine 4 (H3K4), H3 lysine 9 (H3K9), H3 lysine 27 (H3K27), H3 lysine 36 (H3K36) and H4 lysine 20 (H4K20), a histone deacetylase (HDAC) (e.g., Sir2 or any of HDACs 1-11), a DNA methyltransferase (DNMT) (e.g., DNMT1, DNMT3
  • HMT histone methyltransferas
  • exemplary interaction domains include, but are not limited to, v-erbA and protein component ofthe NCoR, Sin3A, or Rb complexes, as well as functional fragments thereof.
  • polynucleotides encoding any of the fusion proteins disclosed herein are provided.
  • cells comprising any ofthe fusion proteins or any polynucleotides described herein are disclosed.
  • Figure 1A shows schematic representations ofthe histone methyltransferases G9A and SUN39H1, indicating the regions of each protein used as a ZFP fusions in relation to known structural features.
  • Figure IB shows that ZFP-HMT fusions repress NEGF-A protein expression.
  • HEK293 cells transfected with the indicated plasmids were assayed for NEGF-A protein production using a human NEGF-A ELISA assay kit as described in Example 5.
  • Figure 1C shows that ZFP-HMT fusions repress NEGF-A mR ⁇ A levels.
  • VEGF-A mR ⁇ A levels were determined by real time PCR (TaqMan) after expression ofthe indicated ZFPs for 72hrs by transient transfection.
  • the VEGF-A mR ⁇ A levels were normalized relative to an internal control of GAPDH mR ⁇ A, and are expressed as this ratio.
  • Figure ID shows that ZFP-HMT fusions do not repress a NEGF-reporter plasmid.
  • Figure IE shows a Western blot of ZFP-HMT constructs.
  • HEK293 cells were transfected with the indicated plasmids and whole cell lysates prepared 72hrs post transfection. Extracts were resolved by SDS-PAGE and immunoblotted using an anti HA-epitope tag antibody. Equal protein levels were loaded in each lane. A band of roughly equivalent mobility to ZFP-Suv Del 76 cross- reacts in HEK293 extracts (see also Fig. 2A Panel II).
  • FIG. 2A Panel I shows that ZFP-HMTs fusions are catalytically active in vitro. Extracts from HEK293 cells transfected with the indicated plasmids were immunoprecipitated with either an anti-HA epitope tag antibody (Anti-HA IP) or a IgG control antibody (IgG control IP), and assayed for histone methyltransferase activity. Panel II shows a Western blot of extracts used in the HMT assay (Panel I). See also Fig. IE.
  • Anti-HA IP anti-HA epitope tag antibody
  • IgG control IP IgG control antibody
  • Figure 2B provides schematics showing the locations ofthe amino acid substitution mutants generated within the HMT catalytic core motif of SUN39H1 (Suv Del 76 wild type: SEQ ID ⁇ O:l; Suv Del 76 mutant A: SEQ ID NO:2; Suv Del 76 mutant B: SEQ ID NO:3; Suv Del 76 mutant AB: SEQ ID NO:4).
  • Figure 2C shows that ZFP-HMT fusions are dependent upon their catalytic HMT activity for repression function in vivo.
  • HEK293 cells transfected with the plasmids indicated were assayed for NEGF-A mR ⁇ A by quantitative RT-PCR (TaqMan) as described in Fig. lC.
  • Figure 2D shows that expression levels ofthe ZFP-HMT mutants are comparable to that of the wild type fusion protein. Extracts from HEK293 cells transfected with the indicated plasmids were irnn unoblotted as in Fig. IE.
  • Figure 3A shows a schematic ofthe NEGF-A gene promoter indicating the ZFP binding sites relative to the transcriptional start site. Positions of the CHIP primer-pairs used in Figure 4 are also indicated (gray boxes).
  • Figure 3B shows that HMT and N-ErbA functional domains repress NEGF-A transcription when linked to either ZFP-A or ZFP-B.
  • the indicated combinations of ZFP-A, ZFP-B and functional domain were assayed for NEGF-A mR ⁇ A levels by quantitative RT-PCR (TaqMan) as described in Fig. lC.
  • FIG. 3C shows that simultaneous targeting of both an HMT and v-ErbA enhances repression of NEGF-A transcription.
  • HEK293 cells transfected with the plasmids indicated were assayed for NEGF-A mR ⁇ A by quantitative RT-PCR (TaqMan) as described in Fig. lC.
  • Figure 3D shows that dual targeting ofthe same functional domain with both ZFP-A and
  • ZFP-B does not enhance NEGF-A repression.
  • HEK293 cells transfected with the plasmids indicated were assayed for NEGF-A mR ⁇ A by quantitative RT-PCR (TaqMan) as described in
  • FIG. lC. Figure 4A shows that ZFP-HMT fusions methylate H3K9 promoter nucleosomes at the
  • NEGF-A locus and require HMT catalytic activity to methylate H3K9 at the VEGF-A promoter.
  • HEK293 cells transfected with the indicated plasmids were assayed for H3K9 methylation by ChlP with primers specific for the ZFP proximal +400 region. Enrichment was quantified by RT-PCR.
  • Results are expressed as the fold-increase ofthe ratio to the GAPDH control relative to the results for non-transfected cells, the value of which is arbitrarily set to 1.
  • the same samples were analyzed with primers specific the pl6 locus as a second internal control (light gray bars). No enrichment was observed with pre-immune serum.
  • Figure 4B shows that ZFP-G9A induces the spread of H3K9 methylation across the VEGF-
  • HEK293 cells transfected with the plasmids indicated were assayed for methylation of H3K9 by ChlP with primers specific for the regions centered on +400, +1 , and -
  • Figure 4C shows that ZFP-SUN Del 76 is dependent upon its catalytic HMT activity for spreading of H3K9 methylation across NEGF-A promoter in vivo.
  • HEK293 cells transfected with the plasmids indicated were assayed for H3K9 by C P with primers specific for the regions centered on +400, +1, and -500. Samples were treated as in Fig.4A.
  • Figure 5 is a graph depicting levels of secreted VEGF protein in foci of stably transformed cells inducibly expressing a ZFP-CME fusion.
  • Isolated clonal populations of TREx U20S cells which had been stably transformed with a TREx regulated ZFP-G9A expression vector were screened for Dox-dependent NEGF-A repression, as assayed by ELISA.
  • Figure 6 is a graph depicting levels of secreted VEGF protein in foci of stably transformed cells inducibly expressing a ZFP-CME fusion in foci of TREx U20S cells. Isolated clonal populations of TREx U20S cells which had been stably transformed with a TREx regulated ZFP-
  • Suv del 76 expression vector were screened for Dox-dependent VEGF-A repression, as assayed by
  • Figure 7 is a graph depicting repression of IGF2 transcription by NOP32-G9A in TREx- inducible U20S cells.
  • Figure 8 is a graph depicting repression of H19 transcription by NOP32-G9A in TREx- inducible U20S cells.
  • Figure 9 is a graph depicting repression of IGF2 transcription by NOP32-Suvdel76 in TREx-inducible U20S cells.
  • Figure 10 is a graph depicting repression of H19 transcription by NOP32-Suvdel76 in TREx-inducible U20S cells.
  • Figure 11 is a graph depicting levels of NEGF mR ⁇ A, normalized to levels of GAPDH rnR ⁇ A, in human HEK 293 cultured cells transfected with various plasmids, as indicated.
  • Lane 1 Cells were transfected with 1.5ug of an expression plasmid encoding the Vop32 zinc finger D ⁇ A binding protein fused with full length PAD N enzyme at its carboxy terminus.
  • Lane 2 Cells were transfected with 1.5ug of an expression plasmid encoding the Nop30 zinc finger D ⁇ A binding protein fused with full length PAD N enzyme at it's carboxy terminus.
  • Lane 3 Cells were transfected with 1.5ug of an expression plasmid encoding the 5499 zinc finger D ⁇ A binding protein fused with full length PAD N enzyme at its carboxy terminus. 5499 does not target or regulate the NEGF-A gene and acts as a control for the function of Nop30 PAD N.
  • Lane 4 Cells were transfected with 1.25ug of an expression plasmid control pCD ⁇ A4.1, which functions as an empty expression vector control, together with 250ng of an expression vector encoding the Nop32 zinc finger D ⁇ A binding protein fused with the ER alpha LBD at its carboxy terminus.
  • Lane 5 Cells were transfected with 1.25ug of an expression plasmid encoding the Nop32 zinc finger D ⁇ A binding protein fused with full length PAD N enzyme at its carboxy terminus, together with 250ng of an expression vector encoding the Nop32 zinc finger D ⁇ A binding protein fused with the ER alpha LBD at its carboxy terminus. Cells were stimulated with the ER alpha ligand 24hours post transfection.
  • Lane 6 Cells were transfected with 1.25ug of an expression plasmid encoding the 5499 zinc finger D ⁇ A binding protein fused with full length PAD N enzyme at its carboxy terminus, together with 250ng of an expression vector encoding the Nop32 zinc finger D ⁇ A binding protein fused with the ER alpha LBD at its carboxy terminus. 5499 does not target or regulate the NEGF-A gene and acts as a control for the function of Nop30 PAD N. Cells were stimulated with the ER alpha ligand 24hours post transfection. Lane 7: Cells were transfected with 1.5ug of an expression plasmid control pCD ⁇ A4.1, which functions as an expression vector control.
  • Lane 8 Cells were transfected with 1.5ug of an expression plasmid control encoding the Green Fluorescent protein (GFP) fused at its carboxy terminus to the PAD V enzyme, which functions as a PAD V expression control.
  • Lane 9 Cells were mock transfected with Lipofectamine 2000 reagent in the absence of DNA and stimulated 24 hours post transfection with the ER alpha ligand Beta Estradiol.
  • GFP Green Fluorescent protein
  • compositions and methods disclosed herein include novel assays (e.g., cell-based assays) for screening candidate compounds for their ability to modulate chromatin-modifying enzymes (CMEs). Identification of these CME modulators is useful in a variety of instances, for example, in diagnosing and/or treating a variety of conditions or disease states. However, since currently available assay systems are based on immunoprecipitation and/or in vitro enzymatic activity assays, they do not readily allow for identification of such modulators.
  • CMEs chromatin-modifying enzymes
  • the currently available assays are problematic for one or more ofthe following reasons: they generally involve the use of potentially hazardous radioactive substrates (e.g., tritium or 14 C); they are not scale-able and are difficult to utilize and standardize; and/or they give no indication of in vivo function or cellular toxicity.
  • radioactive substrates e.g., tritium or 14 C
  • known experimental approaches are not very amenable to high throughput screening for CMEs.
  • modulators of chromatin modifying activity is that most ofthe enzymes which catalyze covalent chromatin modifications do not bind directly to DNA, but are targeted to specific genomic regions through recruitment by other DNA- binding proteins.
  • the present disclosure addresses these problems by providing fusions of chromatin modifying activities to targeted DNA-binding domains, allowing a particular chromatin modifying activity to be directed to a specific reporter gene and thereby regulate the expression of that gene. This then provides an assay for compounds which modulate the targeted chromatin modifying activity, as such compounds will elicit changes in the regulation of expression ofthe reporter gene by the targeted chromatin modifying activity.
  • cells are transfected with a polynucleotide encoding a chimeric protein.
  • the chimeric protein comprises a fusion of a full-length or a fragment of a CME (e.g., the catalytic domain) and a DNA binding domain (e.g. a naturally-occurring DNA-binding domain or an engineered ZFP DNA-binding domain), which targets one or more endogenous gene(s) that can act as reporter(s).
  • a gene encoding a CME-ZFP fusion is optionally under the control of an inducible promoter.
  • the chimeric (fusion) proteins are capable of significantly up- or down-regulating the transcription of these endogenous 'reporter' genes, which in turn allows for the direct and rapid screening of molecules that affect the CME (e.g., by modulating its activity).
  • the cells can be stably or transiently transfected with a ZFP-CME- encoding construct.
  • the CME-ZFP fusion proteins or polynucleotides encoding these proteins are introduced into cells (e.g., via stable or transient transfection).
  • the effect of a candidate compound on the molecular target e.g. , the CME
  • the effect of a candidate compound on the molecular target can be determined by comparing expression ofthe reporter gene(s) in the fusion protein-containing cells in the presence and absence ofthe compound.
  • fusion protein-containing cells exposed to a compound can be compared to cells which do not contain the fusion protein but are exposed to the same compound.
  • the aforementioned two populations of cells can be provided by a single cell line containing a polynucleotide encoding the fusion protein under the control of an inducible promoter.
  • MOLECULAR CLONING A LABORATORY MANUAL, Third edition, Cold Spring Harbor Laboratory Press, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, "Chromatin” (P.M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, "Chromatin Protocols" (P.B., Becker, ed.) Humana Press, Totowa, 1999, all of which are incorporated by reference in their entireties.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form.
  • polynucleotide refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form.
  • these terms are not to be construed as limiting with respect to the length of a polymer.
  • the terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties.
  • an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.
  • polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • Chromatin is the nucleoprotein structure comprising the cellular genome. "Cellular chromatin" comprises nucleic acid, primarily DNA, and protein, including histones and non- histone cliromosomal proteins.
  • nucleosome core comprises approximately 150 base pairs of DNA associated with an octamer comprising two each of histones H2A, H2B, H3 and H4; and linker DNA (of variable length depending on the organism) extends between nucleosome cores.
  • linker DNA extends between nucleosome cores.
  • a molecule of histone HI is generally associated with the linker DNA.
  • chromatin is meant to encompass all types of cellular nucleoprotein, both prokaryotic and eukaryotic.
  • Cellular chromatin includes both cliromosomal and episomal chromatin.
  • a "chromosome” is a chromatin complex comprising all or a portion ofthe genome of a cell.
  • the genome of a cell is often characterized by its karyotype, which is the collection of all the chromosomes that comprise the genome ofthe cell.
  • the genome of a cell can comprise one or more chromosomes.
  • an “episome” is a replicating nucleic acid, nucleoprotein complex or other structure comprising a nucleic acid that is not part ofthe cliromosomal karyotype of a cell.
  • Examples of episomes include plasmids and certain viral genomes.
  • control elements include, but are not limited to, transcription promoters, transcription enhancer elements, silencers, locus control regions, insulators, boundary elements, matrix attachment regions, replication origins, cw-acting transcription regulating elements (transcription regulators, e.g., a c ⁇ -acting element that affects the transcription of a gene, for example, a region of a promoter with which a transcription factor interacts to modulate expression of a gene), transcription termination signals, as well as polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5' to the coding sequence), translation enhancing sequences, and translation termination sequences.
  • transcription regulators e.g., a c ⁇ -acting element that affects the transcription of a gene, for example, a region of a promoter with which a transcription factor interacts to modulate expression of a gene
  • transcription termination signals as well as polyadenylation sequences (located 3' to the translation stop codon
  • Transcription promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, small molecule, drug, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, small molecule, drug, etc.), and constitutive promoters, which are characterized by a constant level of activity in the absence of inducing or repressing substances.
  • sequence identity also are known in the art. Typically, such techniques include determining the nucleotide sequence ofthe mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this fashion. In general, “identity” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • Two or more sequences can be compared by determining their "percent identity.”
  • the percent identity of two sequences, whether nucleic acid or amino acid sequences is the number of exact matches between two aligned sequences divided by the length ofthe shorter sequences and multiplied by 100.
  • An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure. M.O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res.
  • the Smith- Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity.”
  • Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.
  • percent identities between the disclosed sequences and the claimed sequences are at least 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity to the reference sequence (i.e., the sequences disclosed herein).
  • the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that allow formation of stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination ofthe digested fragments.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 70%-75%, preferably 80%-82%, more preferably 85%-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity to each other, or to a reference sequence, over a defined length ofthe molecules, as determined using the methods above.
  • substantially homologous also refers to sequences showing complete identity to a specified DNA or polypeptide sequence.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill ofthe art. See, e.g., Sambrook et al, supra; DNA Cloning: A Practical Approach, editor, D.M. Glover (1985) Oxford; Washington, DC; IRL Press; Nucleic Acid Hybridization: A Practical Approach, editors B.D. Hames and S.J. Higgins (1985) Oxford; Washington, DC; IRL Press.
  • “Selective hybridization" of two nucleic acid fragments can be determined as described herein.
  • the degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules.
  • Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
  • a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence "selectively hybridize," or bind, to each other to form a duplex or "hybrid" molecule.
  • a nucleic acid molecule that is capable of hybridizing selectively to a target sequence under "moderately stringent” hybridization conditions typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence ofthe selected nucleic acid probe.
  • Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence ofthe selected nucleic acid probe.
  • Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B.D. Hames and S.J. Higgins, (1985) Oxford; Washington, DC; IRL Press).
  • Hybridization stringency refers to the degree to which hybridization conditions disfavor the fonnation of duplexes containing mismatched nucleotides, with higher stringency correlated with a lower tolerance for mismatches.
  • Factors that affect the stringency of hybridization include, but are not limited to, temperature, pH, ionic strength, and concentration of organic solvents such as, for example, formamide and dimethylsulfoxide. As is known to those of skill in the art, hybridization stringency is increased by higher temperatures, lower ionic strength and lower solvent concentrations.
  • stringency conditions for hybridization it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition ofthe various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as varying wash conditions.
  • the selection of a particular set of hybridization conditions is conducted following standard methods in the art (see, for example, Sambrook, et al, supra).
  • binding protein is a protein that is able to bind non-covalently to another molecule.
  • a binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a protein-binding protein).
  • a protein-binding protein In the case of a protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more molecules of a different protein or proteins.
  • a binding protein can have more than one type of binding activity.
  • zinc finger proteins have DNA-binding, RNA-binding and protein-binding activity.
  • a "zinc finger DNA binding protein” is a protein or segment within a larger protein that binds DNA in a sequence-specific mamier as a result of stabilization of protein structure through coordination of a zinc ion.
  • the term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.
  • a "designed" zinc finger protein is a protein not occurring in nature whose design composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data.
  • a "selected" zinc finger protein is a protein not found in nature whose production results primarily from an empirical process such as phage display. See e.g., US 5,789,538; US 6,007,988; US 6,013,453; US 6,140,081; US 6,140,466; WO 95/19431; WO 96/06166 and WO 98/54311. Both designed and selected ZFPs are examples of "engineered" ZFPs.
  • naturally-occurring is used to describe an object that can be found in nature, as distinct from being artificially produced by humans.
  • examples include naturally-occurring zinc fingers (e.g., a zinc finger that is encoded by the genome of an organism, as opposed to having been designed or selected), and naturally-occurring zinc finger proteins (e.g., a protein comprising multiple zinc fingers wherein the sequence ofthe entire protein, including the sequence and location ofthe zinc fingers in the protein, is encoded by the genome of an organism).
  • a protein comprising a collection of naturally-occurring zinc fingers, which are not normally present together in a naturally-occurring ZFP and/or which are not present in the order in which they occur in a naturally-occurring ZFP is not a naturally-occurring protein, but is considered to be a type of engineered ZFP.
  • Nucleic acid or amino acid sequences are "operably linked” (or “operatively linked”) when placed into a functional relationship with one another.
  • a promoter or enhancer is operably linked to a coding sequence if it regulates, or contributes to the modulation of, the transcription ofthe coding sequence.
  • Operably linked DNA sequences are typically joined in cis and can be contiguous, and operably linked amino acid sequences are typically contiguous and in the same reading frame.
  • enhancers generally function when separated from the promoter by up to several kilobases or more and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.
  • certain amino acid sequences that are noncontiguous in a primary polypeptide sequence may nonetheless be operably linked due to, for example folding of a polypeptide chain.
  • the term "operatively linked" can refer to the fact that each of the components performs the same function in linkage to the other component as it would if it were not so linked.
  • the ZFP DNA-binding domain and the transcriptional activation domain (or functional fragment thereof) are in operative linkage if, in the fusion polypeptide, the ZFP DNA-binding domain portion is able to bind its target site and/or its binding site, while the transcriptional activation domain (or functional fragment thereof) is able to activate transcription.
  • a "functional fragment" of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains the same function as the full-length protein, polypeptide or nucleic acid.
  • a functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions.
  • the DNA-binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility-shift, or immunoprecipitation assays. See Ausubel et al, supra.
  • the ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, both genetic and biochemical. See, for example, Fields et al. (1989) Nature 340:245-246; U.S. Patent No. 5,585,245 and PCT WO 98/44350.
  • Specific binding between, for example, a ZFP and a specific target site means a binding affinity (i.e, K d ) of at least 1 x 10 ⁇ M "1 .
  • a "fusion molecule” is a molecule in which two or more subunit molecules are linked, preferably covalently.
  • the subunit molecules can be the same chemical type of molecule, or can be different chemical types of molecules.
  • the first type of fusion molecule include, but are not limited to, fusion polypeptides (for example, a fusion between a ZFP DNA-binding domain and a protein that exhibits chromatm modifying activity) and fusion nucleic acids (for example, a nucleic acid encoding a ZFP-CME fusion polypeptide).
  • the second type of fusion molecule include, but are not limited to, a fusion between a triplex-forming nucleic acid and a polypeptide, and a fusion between a minor groove binder and a nucleic acid.
  • exogenous molecule is a molecule that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods. Normal presence in the cell is determined with respect to the particular developmental stage and environmental conditions ofthe cell. Thus, for example, a molecule that is present only during embryonic development of muscle is an exogenous molecule with respect to an adult muscle cell. Similarly, a molecule induced by heat shock is an exogenous molecule with respect to a non-lieat-shocked cell.
  • An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally functioning endogenous molecule.
  • An exogenous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotien, polysaccharide, any modified derivative ofthe above molecules, or any complex comprising one or more ofthe above molecules.
  • Nucleic acids include DNA and RNA, can be single- or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example,.U.S. Patent Nos. 5,176,996 and 5,422,251.
  • Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases.
  • Ai exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., protein or nucleic acid (e.g., an exogenous gene).
  • an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a cliromosome that is not normally present in the cell.
  • exogenous molecules include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.
  • an "endogenous molecule” is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions.
  • an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally occurring episomal nucleic acid.
  • Additional endogenous molecules can include endogenous genes and endogenous proteins, for example, transcription factors and components of chromatin remodeling complexes.
  • Endogenous gene is a gene that is native to a cell, which is in its normal genomic and chromatin context and which is not heterologous to the cell.
  • Endogenous genes can be cellular, microbial or viral.
  • Endogenous microbial and viral genes refer to genes that are part of a naturally- occurring microbial or viral genome in a microbially- or virally-infected cell.
  • the microbial or viral genome can be extrachromosomal, or it can be integrated into the host chromosome(s).
  • Gene expression refers to the conversion ofthe information, contained in a gene, into a gene product.
  • a gene product can be the direct transcriptional product of a gene (e.g., mRNA, fRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA.
  • Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
  • Gene activation and “augmentation of gene expression” refer to any process which results in an increase in production of a gene product.
  • a gene product can be either RNA (including, but not limited to, mRNA, rRNA, tRNA, enzymatic RNA and structural RNA) or protein.
  • gene activation includes those processes that increase transcription of a gene and/or translation of a mRNA. Examples of gene activation processes'which increase transcription include, but are not limited to, those which facilitate formation of a transcription initiation complex, those which increase transcription initiation rate, those which increase transcription elongation rate, those which increase processivity of transcription and those which relieve transcriptional repression (by, for example, blocking the binding of a transcriptional repressor).
  • Gene activation can constitute, for example, inhibition of repression as well as stimulation of expression above an existing level.
  • Examples of gene activation processes that increase translation include those that increase translational initiation, those that increase translational elongation and those that increase mRNA stability.
  • gene activation comprises any detectable increase in the production of a gene product, preferably an increase in production of a gene product by about 2-fold, more preferably from about 2- to about 5-fold or any integral value therebetween, more preferably between about 5- and about 10-fold or any integral value therebetween, more preferably between about 10- and about 20-fold or any integral value therebetween, still more preferably between about 20- and about 50-fold or any integral value therebetween, more preferably between about 50- and about 100-fold or any integral value therebetween, more preferably 100-fold or more.
  • Gene repression and “inhibition of gene expression” refer to any process that results in a decrease in production of a gene product.
  • a gene product can be either RNA (including, but not limited to, mRNA, rRNA, tRNA, enzymatic RNA and structural RNA) or protein.
  • gene repression includes those processes that decrease transcription of a gene and/or translation of a mRNA.
  • Examples of gene repression processes which decrease transcription include, but are not limited to, those which inhibit formation of a transcription initiation complex, those which decrease transcription initiation rate, those which decrease transcription elongation rate, those which decrease processivity of transcription and those which antagonize transcriptional activation (by, for example, blocking the binding of a transcriptional activator).
  • Gene repression can constitute, for example, prevention of activation as well as inhibition of expression below an existing level.
  • Examples of gene repression processes that decrease translation include those that decrease translational initiation, those that decrease translational elongation and those that decrease mRNA stability.
  • Transcriptional repression includes both reversible and irreversible inactivation of gene transcription.
  • gene repression comprises any detectable decrease in the production of a gene product, preferably a decrease in production of a gene product by about 2-fold, more preferably from about 2- to about 5-fold or any integral value therebetween, more preferably between about 5- and about 10-fold or any integral value therebetween, more preferably between about 10- and about 20-fold or any integral value therebetween, still more preferably between about 20- and about 50-fold or any integral value therebetween, more preferably between about 50- and about 100-fold or any integral value therebetween, more preferably 100-fold or more.
  • Modulation of gene expression includes both gene activation and gene repression. Modulation can be assayed by determining any parameter that is indirectly or directly affected by the expression ofthe target gene. Such parameters include, e.g., changes in RNA or protein levels; changes in protein activity; changes in product levels; changes in downstream gene expression; changes in transcription or activity of reporter genes such as, for example, luciferase, CAT, beta-galactosidase, or GFP (see, e.g., Mistili & Spector, (1997) Nature Biotechnology 15:961-964); changes in signal transduction; changes in phosphorylation and dephosphorylation; changes in receptor-ligand interactions; changes in concentrations of second messengers such as, for example, cGMP, cAMP, IP 3 , and Ca2 + ; changes in cell growth, changes in neovascularization, and/or changes in any functional effect of gene expression.
  • reporter genes such as, for example, luciferase, CAT, beta-galactos
  • Measurements can be made in vitro, in vivo, and/or ex vivo. Such functional effects can be measured by conventional methods, e.g., measurement of RNA or protein levels, measurement of RNA stability, and/or identification of downstream or reporter gene expression. Readout can be by way of, for example, chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand binding assays; changes in intracellular second messengers such as cGMP and inositol triphosphate (IP3); changes in intracellular calcium levels; cytokine release, and the like.
  • Esucaryotic cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells.
  • a “regulatory domain” or “functional domain” refers to a protein or a polypeptide sequence that has transcriptional modulation activity.
  • a regulatory domain is covalently or non- covalently linked to a ZFP to modulate transcription of a gene of interest.
  • a ZFP can act alone, without a regulatory domain, to modulate transcription.
  • transcription of a gene of interest can be modulated by a ZFP linked to multiple regulatory domains.
  • a regulatory domain can be linked to any DNA-binding domain having the appropriate specificity to modulate the expression of a gene of interest.
  • a “target site” or “target sequence” is a sequence that is bound by a binding protein or binding domain such as, for example, a ZFP.
  • Target sequences can be nucleotide sequences (eitlier DNA or RNA) or amino acid sequences.
  • a DNA target sequence for a three-finger ZFP is generally either 9 or 10 nucleotides in length, depending upon the presence and/or nature of cross-strand interactions between the ZFP and the target sequence.
  • Recombinant cells when used with reference to a cell, indicates that the cell replicates an exogenous nucleic acid, or expresses a peptide or protein encoded by an exogenous nucleic acid.
  • Recombinant cells can contain genes that are not found within the native (non- recombinant) form ofthe cell.
  • Recombinant cells can also contain genes found in the native form ofthe cell wherein the genes are modified and re-introduced into the cell.
  • a recombinant cell can • comprise an unmodified cellular gene which has been introduced into the cell for the purpose, e.g., of overexpression. Expression of such an unmodified gene may be under the control of its normal cellular regulatory sequences or heterologous regulatory sequences.
  • the tenn also encompasses cells that contain a nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, site- specific mutation, and related techniques.
  • a "recombinant expression cassette,” “expression cassette” or “expression construct” is a nucleic acid construct, generated recombinantly or synthetically, that has control elements that are capable of effecting expression of a structural gene that is operatively linked to the control elements in hosts compatible with such sequences.
  • Expression cassettes include at least promoters and optionally, transcription termination signals.
  • the recombinant expression cassette includes at least a nucleic acid to be transcribed (e.g., a nucleic acid encoding a desired polypeptide) and a promoter. Additional factors necessary or helpful in effecting expression can also be used as described herein.
  • an expression cassette can also include nucleotide sequences that encode a signal sequence that directs secretion of an expressed protein from the host cell, nuclear localization signals and/or epitope tags. Transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression, can also be included in an expression cassette.
  • K ( j" refers to the dissociation constant for the compound, i.e., the concentration of a compound (e.g., a zinc finger protein) that gives half maximal binding ofthe compound to its target (i.e., half of the compound molecules are bound to the target) under given conditions (i.e., when [target] « K ( j), as measured using a given assay system (see, e.g., U.S. Patent No. 5,789,538).
  • the assay system used to measure the K ⁇ should be chosen so that it gives the most accurate measure ofthe actual K ⁇ ofthe ZFP. Any assay system can be used, as long is it gives an accurate measurement ofthe actual K ( j ofthe ZFP.
  • compositions and methods that allow for the identification of modulators (e.g., inliibitors or activators) of any type of chromatin modifying activity, acting on either chromosomal DNA and/or chromosomal proteins, including, for example, chromatin modifying enzymes such as histone acetyl transferases, histone deacetylases, histone methyltransferases, DNA methyltransferases, as well as proteins involved in phosphorylation, ubiquitination, glycosylation and ADP-ribosylation of chromatin.
  • modulators e.g., inliibitors or activators
  • chromatin modifying enzymes such as histone acetyl transferases, histone deacetylases, histone methyltransferases, DNA methyltransferases, as well as proteins involved in phosphorylation, ubiquitination, glycosylation and ADP-ribosylation of chromatin.
  • HMTs Histone methyltransferases
  • HMTs HMTs
  • lysine methyltransferases methylate specific lysine residues within histone H3 and H4. Methylation of H3 lysine 4 appears to code for gene activation, in certain cases, and silencing at telomeres (chromosome ends) in others. Most other lysine methylation tags - H3K9, H3K27 and H3K36 appear to be repressive signals associated with gene repression and transcriptional silencing.
  • HMT proteins ofthe HMT family contain the SET protein domain, in which catalytic (i.e., methyltransferase) activity appears to reside.
  • catalytic activity i.e., methyltransferase activity appears to reside.
  • Recent studies link liistone methylation with long-term epigenetic repression, achieved in part by subsequent DNA methylation in chromatin comprising methylated histones.
  • Aberrant versions or translocations of HMT proteins, that are linked to cancer are also being identified.
  • HMT activities are utilized and required for the function of known tumor suppressor genes. This link has provoked significant interest in specific inhibitors of these activities as anti-cancer agents.
  • arginine methyltransferases Approximately 5 arginine methyltransferases have been identified so far (e.g, PRMTl, CARMl) and all of these methylate specific arginine residues of the histone tails of histone H3 or H4. In addition, all are associated with transcriptional coactivators and with gene activation in vivo.
  • Histone deacetylases Histone deacteylases (HDACs)
  • HDACs Histone deacteylases
  • HATs histone deacteylases
  • these proteins are associated with transcriptional repression and gene silencing, with low acetylation levels occurring at repressed genes. Twelve HDACs have been identified and can be sub-divided into three distinct enzyme sub-families. These proteins are found in vivo as components of one or more co-repressor complexes, such as NCoR and Rb.
  • HDACs appear to be involved in various disease states. For example, the majority of leukemias characterized to date conatin chromosomal translocations that aberrantly fuse DNA sequences encoding two different proteins. A significant proportion of these translocations occur between two different transcription factors; such translocations have been identified in multiple distinct leukemias.
  • APL is the result of a PML-RAR ⁇ translocation event, which splices a transcription factor that drives normal terminal differentiation (RAR ⁇ ) onto a potent repressor (PML).
  • DNA methyltransferases methylate C residues at CG doublets and at CpNpG sites, which occur preferentially near to/at gene regulatory elements. See, for example, WO 01/83793. High numbers/clusters of these CG doublets are called CpG islands and commonly occur at gene promoters (perhaps as much as 50% of all genes have associated CpG islands). Unmetliylated CGs occur in the vicinity of active genes, methyl-CpGs appear to be associated with silenced genes. This is of vital importance during transcriptional silencing and in development and is associated with transcriptional repression and silencing.
  • DNA methylation patterns are considerably altered in cancer and include genome-wide losses of normal patterns of DNA methylation, together with specific increases in DNA methylation in the vicinity of tumor suppressor genes such as pl6 and Rb. This oncogenic promoter hypermethylation and consequent transcriptional silencing at such genes has been shown to play important roles in tumorigenesis. Because of this linkage, inhibitors of DNA methyltransferases are of considerable interest as therapeutic compounds in the treatment of cancer. Three functional human DNA methyltransferases have been identified, DNMTl, DNMT3a and DNMT3b, with the precise role of each at present undetermined. Complexes that recognize this methyl-C residues in DNA also been identified (MeCP2, NuRD/MeCPl).
  • the methods and compositions disclosed herein can be used to identify such inliibitors (as well as activators) by " using one ofthe aforementioned proteins as a modifying activity in the disclosed fusion proteins.
  • HATs Histone acetyltransferases
  • Histone acetyltransferases acetylate lysine residues within liistone tails as well as in other proteins such as p53.
  • HATs are associated with transcriptional coactivators; with histone acetylation comiected to active genes and gene activation in vivo, as well as in DNA damage repair.
  • Exemplary HATs include p300, CBP, GCN5 and pCAF; additional HAT activities and HAT-containing complexes are disclosed, for example, in WO 01/83793.
  • compositions and methods disclosed herein involve use of DNA binding proteins, particular zinc finger proteins.
  • a DNA-binding domain can comprise any molecular entity capable of sequence-specific binding to chromosomal DNA. Binding can be mediated by electrostatic interactions, hydrophobic interactions, or any other type of chemical interaction. Examples of moieties which can comprise part of a DNA-binding domain include, but are not limited to, minor groove binders, major groove binders, antibiotics, intercalating agents, peptides, polypeptides, oligonucleotides, and nucleic acids. An example of a DNA-binding nucleic acid is a triplex-forming oligonucleotide.
  • Minor groove binders include substances which, by virtue of their steric and/or electrostatic properties, interact preferentially with the minor groove of double-stranded nucleic acids. Certain minor groove binders exhibit a preference for particular sequence compositions. For instance, netropsin, distamycin and CC-1065 are examples of minor groove binders that bind specifically to AT-rich sequences, particularly runs of A or T. WO 96/32496. Many antibiotics are known to exert their effects by binding to DNA. Binding of antibiotics to DNA is often sequence-specific or exhibits sequence preferences. Actinomycin, for instance, is a relatively GC-specific DNA binding agent.
  • a DNA-binding domain is a polypeptide.
  • Certain peptide and polypeptide sequences bind to double-stranded DNA in a sequence-specific manner.
  • transcription factors participate in transcription initiation by RNA Polymerase II through sequence- specific interactions with DNA in the promoter and/or enhancer regions of genes. Defined regions within the polypeptide sequence of various transcription factors have been shown to be responsible for sequence-specific binding to DNA. See, for example, Pabo et al. (1992) Ann. Rev. Biochem. 61:1053-1095 and references cited therein.
  • regions include, but are not limited to, motifs known as leucine zippers, helix-loop-helix (HLH) domains, helix-turn-helix domains, zinc fingers, ⁇ -sheet motifs, steroid receptor motifs, bZIP domains homeodomains, AT-hooks and others.
  • the amino acid sequences of these motifs are known and, in some cases, amino acids that are critical for sequence specificity have been identified.
  • Polypeptides involved in other process involving DNA, such as replication, recombination and repair will also have regions involved in specific interactions with DNA.
  • Peptide sequences involved in specific DNA recognition such as those found in transcription factors, can be obtained through recombinant DNA cloning and expression techniques or by chemical synthesis, and can be attached to other components of a fusion molecule by methods known in the art.
  • a DNA-binding domain comprises a zinc finger DNA- binding domain.
  • a zinc finger DNA- binding domain See, for example, Miller et al. (1985) EMBO J. 4:1609-1614; Rhodes et al. (1993) Scientific American Feb.:56-65; and Klug (1999) /. Mol. Biol. 293:215-218.
  • the three- fingered Zif268 murine transcription factor has been particularly well studied. (Pavletich, N. P. & Pabo, C. O. (1991) Science 252:809-17).
  • the X-ray co-crystal structure of Zif268 ZFP and double-stranded DNA indicates that each finger interacts independently with DNA (Nolte et al. (1998) Proc Natl Acad Sci USA 95:2938-43; Pavletich, N. P. & Pabo, C. O. (1993) Science
  • the organization ofthe 3-fingered domain allows recognition of three contiguous base-pair triplets by each finger.
  • Each finger is approximately 30 amino acids long, adopting a ⁇ fold.
  • the two ⁇ -strands form a sheet, positioning the recognition ⁇ -helix in the major groove for DNA binding.
  • Specific contacts with the bases are mediated primarily by four amino acids immediately preceeding and within the recognition helix.
  • these recognition residues are numbered -1, 2, 3, and 6 based on their positions in the ⁇ -helix.
  • ZFP DNA-binding domains are engineered (e.g., designed and/or selected) to recognize a particular target site as described in co-owned WO 00/42219; WO 00/41566 and WO 02/42459; as well as U.S. Patents 5,789,538; 6,007,408; 6,013,453; 6,140,081; 6,140,466 and 6,242,568; and PCT publications WO 95/19431, WO 98/53057, WO 98/53058, WO 98/53059, WO 98/53060, WO 98/54311 , WO 00/23464, WO 00/27878 and WO 01/53480.
  • a target site for a zinc finger DNA-binding domain is identified according to site selection rules disclosed in co- owned WO 00/42219.
  • a ZFP is selected by iterative processes of selection and optimization as described in co-owned International Patent Application PCT/US01/43568.
  • the binding specificity ofthe DNA-binding domain can be determined by identifying accessible regions in the sequence in question (e.g., in cellular chromatin). Accessible regions can be determined as described in co-owned PCT publications WO 01/83732 and WO 01/83751, the disclosures of which are hereby incorporated by reference herein.
  • a DNA- binding domain is then designed and/or selected as described herein to bind to a target site within the accessible region.
  • Two alternative methods are typically used to create the coding sequences required to express newly designed DNA-binding peptides.
  • One protocol is a PCR-based assembly procedure that utilizes six overlapping oligonucleotides.
  • Three oligonucleotides correspond to "universal" sequences that encode portions ofthe DNA-binding domain between the recognition helices. These oligonucleotides remain constant for all zinc finger constructs.
  • the other three "specific" oligonucleotides are designed to encode the recognition helices. These oligonucleotides contain substitutions primarily at positions -1, 2, 3 and 6 on the recognition helices maldng them specific for each ofthe different DNA-binding domains.
  • PCR synthesis is carried out in two steps.
  • a double stranded DNA template is created by combining the six oligonucleotides (three universal, three specific) in a four cycle PCR reaction with a low temperature annealing step, thereby annealing the oligonucleotides to form a DNA "scaffold.”
  • the gaps in the scaffold are filled in by high-fidelity thermostable polymerase, the combination of Taq and Pfu polymerases also suffices.
  • the zinc finger template is amplified by external primers designed to incorporate restriction sites at either end for cloning into a shuttle vector or directly into an expression vector.
  • Ai alternative method of cloning the newly designed DNA-binding proteins relies on annealing complementary oligonucleotides encoding the specific regions ofthe desired zinc finger protein. This particular apphcation requires that the oligonucleotides be phosphorylated p ⁇ or to the final hgation step. Phosphorylation is usually performed before annealing, but can also be done post-anneahng.
  • the "universal" oligonucleotides encoding the constant regions ofthe proteins are annealed with their complementary oligonucleotides.
  • the "specific" oligonucleotides encoding the finger recognition helices are annealed with their respective complementary oligonucleotides.
  • complementary oligos are designed to fill in the region, which was previously filled in by polymerase m the protocol described above.
  • the complementary oligos to the common oligos 1 and finger 3 are engineered to leave overhanging sequences specific for the restriction sites used in cloning into the vector of choice.
  • the second assembly protocol differs from the initial protocol in the following aspects: the "scaffold" encoding the newly designed zinc finger protein is composed entirely of synthetic DNA thereby eliminating the polymerase fill-m step, additionally the fragment to be cloned into the vector does not require amplification Lastly, inclusion in the design of sequence-specific overhangs eliminates the need for restriction enzyme digestion ofthe ZFP-encoding fragment prior to its insertion into the vector.
  • the resulting fragment encoding the newly designed zinc finger protem is ligated into an expression vector.
  • Expression vectors that are commonly utilized include, but are not limited to, a modified pMAL-c2 bacterial expression vector (New England BioLabs, "NEB") or a eukaryotic expression vector, pcDNA (Promega). Conventional methods of purification can be used (see Ausubel, supra, Sambrook, supra).
  • any suitable host can be used, e.g., bacterial cells, insect cells, yeast cells, mammalian cells, and the like.
  • zmc finger protein fused to a maltose binding protein (MBP-ZFP) m bacterial strain JM109 allows for straightforward purification through an amylose column (NEB).
  • High expression levels ofthe zmc finger chimeric protein can be obtained by induction with IPTG since the MBP-ZFP fusion m the pMal-c2 expression plasmid is under the control ofthe IPTG mducible tac promoter (NEB).
  • Bacteria containing the MBP-ZFP fusion plasmids are inoculated in to 2x YT medium containing lO ⁇ M ZnCl., 0.02% glucose, plus 50 ⁇ g/ml ampicillm and shaken at
  • IPTG is added to 0.3 mM and the cultures are allowed to shake. After 3 hours the bacteria are harvested by centrifugation, disrupted by somcation, and then insoluble matenal is removed by centrifugation.
  • the MBP-ZFP proteins are captured on an amylose-bound resm, washed extensively with buffer containing 20 mM T ⁇ s-HCl (pH 7.5), 200 mM NaCl, 5 mM DTT and 50 ⁇ M ZnCl , then eluted with maltose in essentially the same buffer
  • K ⁇ The biochemical properties ofthe purified proteins, e.g., K ⁇ , can be characterized by any suitable assay.
  • K ⁇ can be characterized via electrophoretic mobility shift assays ("EMSA")
  • Affinity is measured by titrating purified protein against a low fixed amount of labeled double-stranded oligonucleotide target.
  • the target comprises the natural binding site sequence (e.g., 9 or 18 bp), optionally flanked by the 3 bp found in the natural sequence. External to the binding site plus flanking sequence is a constant sequence.
  • the annealed oligonucleotide targets possess a 1-nucleotide 5' overhang that allows for efficient labeling ofthe target with T4 phage polynucleotide kinase.
  • the target is added at a concentration of 40 nM or lower (the actual concentration is kept at least 10-fold lower than the lowest protein dilution) and the reaction is allowed to equilibrate for at least 45 min.
  • the reaction mixture also contains 10 mM Tris (pH 7.5), 100 mM KCl, 1 mM MgCl 2 , 0.1 mM ZnCl 2 , 5 mM DTT, 10% glycerol, 0.02% BSA
  • the equilibrated reactions are loaded onto a 10% polyacrylamide gel, which has been pre- run for 45 min in Tris/glycine buffer, then bound and unbound labeled target is resolved be electrophoresis at 150V (alternatively, 10-20% gradient Tris-HCl gels, containing a 4% polyacrylamide stacker, can be used).
  • the dried gels are visualized by autoradiography or phosphoroimaging and the apparent K ⁇ is determined by calculating the protein concentration that gives half-maximal binding.
  • Similar assays can also include detennining active fractions in the protein preparations. Active fractions are determined by stoichiometric gel shifts where proteins are titrated against a high concentration of target DNA. Titrations are done at 100, 50, and 25% of target (usually at micromolar levels).
  • compositions and methods described herein involve fusions between at least one ofthe zinc finger proteins described herein (or functional fragments thereof) and one or more CMEs (or functional fragments thereof), or a polynucleotide encoding such a fusion, hi such a fusion molecule, the CME is brought into proximity with a sequence in a gene that is bound by the zinc finger protein and can function, for example, by regulating expression ofthe gene. Changes in regulation of a target gene by a fusion protein provides an assay for molecules that modulate the activity of a CME.
  • the zinc finger protein can be covalently or non-covalently associated with one or more regulatory domains (e.g., CMEs), alternatively two or more regulatory domains, with the two or more domains being two copies ofthe same domain, or two different domains.
  • the regulatory domains can be covalently linked to the zinc finger protein, e.g., via an amino acid linker, as part of a fusion protein.
  • the zinc finger proteins can also be associated with a regulatory domain via a non-covalent dimerization domain, e.g., a leucine zipper, a STAT protein N terminal domain, or a protein which binds cyclosporine, tetracycline, a steroid, FK506, FK520, rapamycin, and analogues or derivatives thereof.
  • a non-covalent dimerization domain e.g., a leucine zipper, a STAT protein N terminal domain, or a protein which binds cyclosporine, tetracycline, a steroid, FK506, FK520, rapamycin, and analogues or derivatives thereof.
  • FKBPs FK506 binding proteins
  • cyclophilin receptors cyclophilin receptors
  • tetracycline receptors steroid receptors
  • FRAPs e.g., US Patent No. 6,165,787; O'Shea, Science 254: 5
  • the regulatory domain can be associated with the zinc finger protein at any suitable position, including the C- or N- terminus ofthe zinc finger protein.
  • Fusion molecules may be constructed by methods of cloning and biochemical conjugation that are well known to those of skill in the art.
  • fusion molecules comprise a zinc finger protein and one or more CMEs (or functional fragments thereof).
  • CMEs or functional fragments thereof.
  • fusion molecules also comprise nuclear localization signals (such as, for example, that from an SV40 T- antigen) and epitope tags (such as, for example, FLAG, myc and hemagglutinin). Fusion proteins (and nucleic acids encoding them) are designed such that the translational reading frame is preserved among the components of the fusion.
  • the fusion molecules disclosed herein comprise a zinc finger binding protein that binds to a target site (in a reporter gene) and a CME.
  • the target site is in an endogenous gene whose level of expression can be readily assayed. Modulation of gene expression can be in the form of increased expression or repression. The effect of a compound or substance on the regulation of the reporter gene by the fusion protein can then be assayed to determine if a compound or substance is a modulator ofthe CME.
  • the fusion molecule(s) can be formulated with a pharmaceutically acceptable carrier, as is lmown to those of skill in the art. See, for example, Remington's Pharmaceutical Sciences, 17 th ed., 1985; and co-owned WO 00/42219.
  • Linker domains between polypeptide domains e.g., between the zinc finger proteins and the CME, can be included.
  • Such linkers are typically polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids.
  • Preferred linkers are typically flexible amino acid subsequences that are synthesized as part of a recombinant fusion protein, for example, the linkers DGGGS (SEQ ID NO:5); TGEKP (SEQ ID NO:6) (see, e.g., Liu et al., Proc. Natl. Acad. Sci. U.S.A. 5525-5530 (1997)); LRQKDGERP (SEQ ID NO:7); GGRR (SEQ ID NO:8)
  • a chemical linker can be used to connect synthetically or recombinantly produced domain sequences.
  • poly(ethylene glycol) linkers are available from Shearwater Polyi ⁇ ers, Inc. Huntsville, Alabama. Some linkers have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
  • non-covalent methods can be used to produce molecules with zinc finger proteins associated with regulatory domains. See, for example, US Patent No. 6,165,787 and WO 01/30843.
  • the fusion molecules may be in the form of nucleic acid sequences that encode the fusion molecule ,or in the form of a fusion between one or more polypeptides and/or one or more polypeptides and one or more non-polypeptide molecules.
  • the fusion molecule may also include one or more additional regulatory (functional) domains including, e.g., effector domains from transcription factors (activators, repressors, co-activators, co-repressors), silencers, nuclear hormone receptors, oncogene transcription factors (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members etc.); DNA repair enzymes and their associated factors and modifiers; DNA rearrangement enzymes and their associated factors and modifiers; chromatin associated proteins and their modifiers (e.g., kinases, acetylases, deacetylases, phosphatases, methyltransferases, ubiquitinylases); and DNA modifying enzymes (e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, and/or end
  • Transcription factor polypeptides from which one can obtain a regulatory domain include those that are involved in regulated and basal transcription. Such polypeptides include transcription factors, their effector domains, coactivators, silencers, nuclear hormone receptors (see, e.g., Goodrich et al, Cell 84:825-30 (1996) for a review of proteins and nucleic acid elements involved in transcription; transcription factors in general are reviewed in Barnes & Adcock, Clin. Exp. Allergy 25 Suppl. 2:46-9 (1995) and Roeder, Methods Enzymol. 273:165-71 (1996)). Databases dedicated to transcription factors are known (see, e.g., Science 269:630 (1995)).
  • Nuclear hormone receptor transcription factors are described in, for example, Rosen et al, J. Med. Chem. 38:4855-74 (1995).
  • the C/EBP family of transcription factors are reviewed in Wedel et al, Immunobiology 193:171-85 (1995).
  • Coactivators and co-repressors that mediate transcription regulation by nuclear hormone receptors are reviewed in, for example, Meier, Eur. J. Endocrinol. 134(2):158-9 (1996); Kaiser et al, Trends Biochem. Sci. 21:342-5 (1996); and Utley et al, Nature 394:498-502 (1998)).
  • GATA transcription factors which are involved in regulation of hematopoiesis, are described in, for example, Simon, Nat.
  • TATA box binding protein TBP
  • TAF polypeptides which include TAF30, TAF55, TAF80, TAF110, TAF150, and TAF250
  • TAF30, TAF55, TAF80, TAF110, TAF150, and TAF250 TAF30, TAF55, TAF80, TAF110, TAF150, and TAF250
  • TAF30, TAF55, TAF80, TAF110, TAF150, and TAF250 TAF30, TAF55, TAF80, TAF110, TAF150, and TAF250
  • the STAT family of transcription factors are reviewed in, for example, Barahmand-Pour et al, Curr. Top. Microbiol Immunol. 211:121-8 (1996).
  • An exemplary functional domain for fusing with a ZFP is a KRAB repression domain from the human KOX-1 protein (see, e.g., Thiesen et al., New Biologist 2, 363-374 (1990); Margolin et al., Proc. Natl. Acad. Sci. USA 91, 4509-4513 (1994); Pengue et al., Nucl. Acids Res. 22:2908- 2914 (1994); Witzgall et al, Proc. Natl. Acad. Sci. USA 91, 4514-4518 (1994).
  • MBD-2B methyl binding domain protein 2B
  • Aiother useful repression domain is that associated with the v-ErbA protein. See, for example, Damm, et al. (1989) Nature 339:593-597; Evans (1989) Int. J. Cancer Suppl. 4:26-28; Pain et al. (1990) New Biol. 2:284-294; Sap et al. (1989) Nature 340:242-244; Zenke et al. (1988) Cell 52:107-119; and Zenke et al. (1990) Cell 61:1035-1049.
  • Additional exemplary repression domains include, but are not limited to, thyroid hormone receptor (TR), SID, MBD2, MBD3, members ofthe DNMT family (e.g.,
  • DNMTl DNMT3A, DNMT3B
  • Rb MeCP2.
  • MeCP2 MeCP2.
  • Additional exemplary repression domains include, but are not limited to, ROM2 and AtHD2A. See, for example, Chern et al. (1996) Plant Cell 8:305-321; and Wu et al. (2000) Plant! 22:19- 27.
  • Suitable domains for achieving activation include the HSV NP16 activation domain (see, e.g., Hagmann et al., J. Nirol. 71, 5952-5962 (1997)) nuclear hormone receptors (see, e.g., Torchia et al, Curr. Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factor kappa B (Bitko & Barik, J. Nirol. 72:5610-5618 (1998)and Doyle & Hunt, Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28 (1998)), or artificial chimeric functional domains such as VP64 (Seifpal et al, EMBO J. 11, 4961-4968 (1992)).
  • HSV NP16 activation domain see, e.g., Hagmann et al., J. Nirol. 71, 5952-5962 (1997)
  • Additional exemplary activation domains include, but are not limited to, VPl 6, VP64, p300, CBP, PCAF,SRC1 PvALF, AtHD2A and ERF-2. See, for example, Robyr et al. (2000) Mol. Endocrinol. 14:329-347; CoUingwood et al. (1999) /. Mol. Endocrinol 23:255-275; Leo et al. (2000) Gene 245:1-11; Manteuffel-Cymborowska (1999) -4ct ⁇ 5z ' ocAfm. Pol. 46:77-89; McKenna et al (1999) J. Steroid Biochem. Mol. Biol.
  • Additional exemplary activation domains include, but are not limited to, OsGAI, HALF-1, Cl, API, ARF-5, -6, -7, and- 8, CPRFl, CPRF4, MYC-RP/GP, and TRAB1. See, for example, Ogawa et al (2000) Gene 245:21-29; Okanami et al. (1996) Genes Cells 1:87-99; Goff et al. (1991) Genes Dev.
  • Additional functional domains are disclosed, for example, in co-owned WO 00/41566. Further, insulator domains, chromatin remodeling proteins such as ISWI-containing domains and/or methyl binding domain proteins suitable for use in fusion molecules are described, for example, in co-owned International Publications WO 02/26959; WO 02/26960; and WO 02/44376.
  • Kinases, phosphatases, and other proteins that modify polypeptides involved in gene regulation are also useful as functional domains for zinc finger proteins. Such modifiers are often involved in switching on or off transcription mediated by, for example, hormones.
  • Kinases involved in transcription regulation are reviewed in Davis, Mol. Reprod. Dev. 42:459-67 (1995), Jackson et al, Adv. Second Messenger Phosphoprotein Res. 28:279-86 (1993), and Boulikas, Crit. Rev. Eulcaryot. Gene Expr. 5:1-77 (1995), while phosphatases are reviewed in, for example, Schonthal & Semin, Cancer Biol. 6:239-48 (1995).
  • Nuclear tyrosine kinases are described in Wang, Trends Biochem. Sci. 19:373-6 (1994).
  • Useful domains can also be obtained from the gene products of oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members) and their associated factors and modifiers.
  • oncogenes e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members
  • Oncogenes are described in, for example, Cooper, Oncogenes, The Jones andBartlett
  • the myb gene family is reviewed in Kanei-Ishii et al, Curr. Top. Microbiol. Immunol. 211:89-98 (1996).
  • the mos family is reviewed in Yew et al, Curr. Opin. Genet. Dev. 3:19-25 (1993).
  • Zinc finger proteins can include functional domains obtained from DNA repair enzymes and their associated factors and modifiers.
  • DNA repair systems are reviewed in, for example, Nos, Curr. Opin. Cell Biol. 4:385-95 (1992); Sancar, -4 ⁇ m. Rev. Genet. 29:69-105 (1995); Lehmann, Genet. Eng. 17:1-19 (1995); and Wood, Ann. Rev. Biochem. 65:135-67 (1996).
  • D ⁇ A rearrangement enzymes and their associated factors and modifiers can also be used as regulatory domains (see, e.g., Gangloff et al, Experientia 50:261-9 (1994); Sadowski, FASEB J. 7:760-7 (1993)).
  • the zinc finger protein is expressed as a fusion protein such as maltose binding protein ("MBP"), glutathione S transferase (GST), hexahistidine, c-myc, and the FLAG epitope, for ease of purification, monitoring expression, or monitoring cellular and subcellular localization.
  • MBP maltose binding protein
  • GST glutathione S transferase
  • hexahistidine hexahistidine
  • c-myc hexahistidine
  • FLAG epitope FLAG epitope
  • any component of a cell can serve as a molecular target (reporter) for the ZFP component of the fusion protein.
  • the product (mRNA or protein) of an endogenous cellular genes such as, e.g., VEGF, H19 or IGF-2, can serve as reporter.
  • a gene whose product is used as a reporter is denoted a "reporter gene.”
  • An exogenous gene can also serve as a reporter gene, for example, if it is integrated into the cliromosome so that it adopts a chromatin configuration.
  • Additional non-limiting examples of endogenous reporters include growth factor receptors (e.g., FGFR, PDGFR, EGFR, NGFR, and VEGFR).
  • G- protein receptors include substance K receptor, the angiotensin receptor, the ⁇ - and ⁇ - adrenergic receptors, the serotonin receptors, and PAF receptor. See, e.g., Gilman, Ann. Rev. Biochem. 56:625-649 (1987).
  • Other suitable reporters that may be employed include ion channels (e.g., calcium, sodium, potassium channels), muscarinic receptors, acetylcholine receptors, GABA receptors, glutamate receptors, and dopamine receptors (see Harpold, 5,401,629 and US
  • cytokines such as interleukins IL-1 through IL-13, tumor necrosis factors ⁇ & ⁇ , interferons ⁇ , ⁇ and ⁇ , tumor growth factor Beta (TGF- ⁇ ), colony stimulating factor (CSF) and granulocyte monocyte colony stimulating factor (GM-CSF).
  • TGF- ⁇ tumor growth factor Beta
  • CSF colony stimulating factor
  • GM-CSF granulocyte monocyte colony stimulating factor
  • Target molecules that serve as reporter molecules can be human, mammalian viral, plant, fungal or bacterial.
  • Other targets are antigens, such as proteins, glycoproteins and carbohydrates from microbial pathogens, both viral and bacterial, and tumors. Still other targets are described in U.S. Patent No. 4,366,241.
  • Reporter expression can be directly detected by detecting formation of transcript or of translation product.
  • transcription product can be detected using Northern blots, branched DNA signal amplification systems (e.g., US Patent Nos. 5,124,246; 5,624,802; 5,635,352; 5,681,697; 5,849,481), RNA tags (Aclara Biosciences, Mountain View, CA) or realtime PCR (Taqman ® , Roche) and the formation of certain proteins can be detected, e.g., by gel electrophoresis, immunoassay (e.g., ELISA), using a characteristic stain or by detecting an inherent characteristic (e.g., enzymatic activity) ofthe protein.
  • expression of reporter is determined by detecting a product formed as a consequence of an activity of the reporter.
  • Exemplary reporter genes encoding proteins having enzymatic activity include, but are not limited to, those encoding phosphatases, hydrolases, myeloperoxidases and proteases. Additional exemplary reporter genes include those encoding cell-surface proteins such as, for example, CD antigens, immunoglobulins, T-cell receptors, growth factor receptors and transmembrane proteins (e.g. , placental alkaline phosphatase).
  • cell based screening assays can be performed in which ZFP-CME fusions are introduced into a cell and used to identify compounds that inhibit or activate the CME(s). Readout is provided by the product of a reporter gene, the reporter gene being targeted by the ZFP portion ofthe chimeric protein.
  • the ZFP-CME is constitutively expressed and the effect of a compound on expression of the reporter gene is tested and compared to a baseline expression level prior to administration ofthe compound.
  • expression ofthe ZFP-CME is controlled by an inducible promoter and the ability of a compound to modulate CME activity is tested in the presence of inducer (and compared to values obtained in the presence of inducer and absence of compound). If the compound under test targets an intracellular component otlier than the CME of interest, effects of the compound in the absence of inducer will be detected.
  • the compound can be administered directly into a cell using methods known in the art and described herein.
  • any ofthe methods described herein can be used with any reporter and/or selectable marker.
  • Reporters that can be directly detected include GFP (green fluorescent protein). Fluorescence generated from this protein can be detected using a variety of commercially available fluorescent detection systems, including a FACS system for example.
  • Other reporters are enzymes that catalyze the formation of a detectable product. Suitable enzymes include proteases, nucleases, liposes, phosphatases, sugar hydrolases and esterases.
  • the substrate is substantially impermeable to eukaryotic plasma membranes, thus making it possible to tightly control signal formation.
  • reporter genes that encode enzymes include, for example, CAT (chloramphenicol acetyl transferase; Alton and Vapnek (1979) Nature 282:864-869), luciferase (lux), ⁇ -galactosidase, ⁇ -glucuronidase (GUS) and alkaline phosphatase (Toll, et al. (1980) Eur. J. Biochem. 182:231-238; and Hall et al. (1983) J. Mol. Appl. Gen. 2:101).
  • CAT chloramphenicol acetyl transferase
  • Alton and Vapnek (1979) Nature 282:864-869
  • lux luciferase
  • ⁇ -galactosidase ⁇ -glucuronidase
  • GUS alkaline phosphatase
  • Positive selection markers are those polynucleotides that encode a product that enables only cells that carry and express the gene to survive and/or grow under certain conditions. For example, cells that express neomycin resistance (Neo 1 ) gene are resistant to the compound G418, while cells that do not express Neo r are killed by G418. Other examples of positive selection markers including hygromycin resistance and the like will be known to those of skill in the art. Negative selection markers are those polynucleotides that encode a produce that enables only cells that carry and express the gene to be killed under certain conditions.
  • thymidine ldnase e.g., herpes simplex virus thymidine kinase, HSV-TK
  • HSV-TK herpes simplex virus thymidine kinase
  • Other negative selection markers are known to those skilled in the art.
  • the selectable marker need not be a transgene and, additionally, reporters and selectable markers can be used in various combinations.
  • High throughput screening involves several steps: creating an assay that is predictive of a particular physiological response; automating the assay so that it can be reproducibly performed a large number of times; and, sequentially testing samples from a chemical library to identify chemical structures able to "hit" the assay, suggesting that such structures might be capable of provoking the intended physiological response. Hits from the high throughput screen are followed up in a variety of secondary assays to eliminate artifactual results, particularly toxic compounds.
  • the assays used in high throughput screens are intended to detect the presence of chemical samples (e.g., compounds, substances, molecules) possessing specific biological or biochemical properties. These properties are chosen to identify compounds with the potential to elicit a specific biological response when applied in vivo.
  • High throughput screens identify both agents that can be used as drugs themselves and, in addition, drug candidates that will ultimately be used as drugs.
  • a compound of a certain chemical class that is found to have some level of desired biological property in a high-tliroughput assay can then be the basis for synthesis of derivative compounds.
  • Biochemical assays utilize pure or semi-pure components outside of a cellular environment. Enzyme assays and receptor binding assays are typical examples of biochemical assays.
  • Cell- based assays utilize intact cells in culture. Examples of such assays include luciferase reporter gene assays and calcium flux assays. Biochemical assays are usually easier to perform and are generally less prone to artifacts than conventional cell-based assays.
  • Biochemical assays typically function according to a desired mechanism, decreasing the amount of follow-up experimentation required to confirm a compound's status as a "hit.”
  • a major disadvantage of biochemical assays is the lack of biological context. Compound “hits” from biochemical screens do not have to traverse a plasma membrane or other structures to reach and affect the target protein. Consequently, biochemical assays tend to be far less predictive of a compound's activity in an animal than cell-based assays.
  • Cell-based assays preserve much ofthe biological context of a molecular target. Compounds that cannot pass through the plasma membrane or that are toxic to the cell are not pursued. This context, however, adds complexity to the assay. Therefore conventional cell-based assays are much more prone to artifact or false positive results than are biochemical assays. Compounds that trigger complex toxic reactions or trigger apoptosis are particularly troublesome. Much ofthe labor devoted to conventional cell-based high throughput screening is directed to follow-up assays that detect false hits or hits that work by undesirable mechanisms.
  • screening assays are conducted using cells comprising at least one targeted DNA-binding domain (e.g., an engineered zinc finger protein) and at least one CME.
  • the screening assays described herein allow for high throughput screening of candidate compounds and can be accomplished while reducing false positives. As a result, discovery will be more efficient and compounds identified using the screening methods disclosed herein will have greater specificity and, consequently, will be prone to fewer potential side effects than those identified by prior methods.
  • cell-based assays can utilize a cell line which expresses (either constitutively or inducibly) a chimeric protein comprising a fusion ofthe catalytic domain of CME with a targeted DNA binding domain (e.g., an engineered ZFP) that binds one or more reporter genes.
  • the fusion protein(s) are able to up- or down-regulate transcription of reporter gene(s), which can be either endogenous or exogenous.
  • the cells can be transiently or stably transfected with a polynucleotide encoding the fusion molecule.
  • a ZFP-CME fusion represses transcription of a reporter gene to which the ZFP is targeted, in cells in which the fusion protein is expressed.
  • Compounds which, when contacted with such cells, result in increased reporter gene expression are modulators ofthe CME.
  • Some cells are designed to express a sequence encoding a ZFP-CME fusion protein in operable linkage to an inducible promoter.
  • inducible promoters are available that can be regulated by small molecules or other stimuli such as heat. Operable linkage to an inducible promoter allows activation of a ZFP-CME fusion, and thereby modulation of a molecular target by the expressed fusion protein, to be controlled by supplying the cell with the appropriate small molecule or other stimulus.
  • Use of inducible promoters is useful for achieving transient modulation of cellular proteins whose permanent over- or under-expression would result in lethality to the cell. Inducible expression is also advantageous in reducing secondary effects due to modulation of an intended cellular protein.
  • regulation of one cellular protein can directly or indirectly results in changes in the relative abundance of many others proteins within the cell.
  • a ZFP-CME fusion protein By inducing a ZFP-CME fusion protein shortly before an assay is performed, such secondary changes are minimized. Accordingly, differences in response between a test cell and a control cell having a molecular target or other protein subject to regulation are entirely or substantially entirely due to interaction between the compound and the molecular target rather than secondary effects caused by regulation ofthe target.
  • the present methods and compositions allow for a direct transcriptional readout of the activities of chromatin modifying catalytic activities in vivo. This in turn, allows high-throughput screening for potential inhibitors and activators of these activities.
  • the direct targeting of chromatin (e.g., histone) modifications to a particular reporter gene, via fusions ofthe catalytic domain of a CME to a targeted DNA-binding domain (e.g., an engineered ZFP) results, in certain embodiments, in transcriptional regulation an endogenous gene. Changes in such targeted gene regulation can be utilized as an activity assay at the RNA or protein level, providing a direct, rapid assay system for use in screening.
  • expression of a ZFP-CME fusion is regulatable, for example by an inducible system such as TREx (Invitrogen, Carlsbad, CA; see also US Patent No. 4,833,080), allowing for specific control ofthe expression of ZFP-CME fusion proteins.
  • TREx Invitrogen, Carlsbad, CA; see also US Patent No. 4,833,080
  • transcription of a polynucleotide sequence encoding a ZFP-CME fusion is positively regulated by provision of tetracycline or a tetracycline analogue, such as, for example, doxycycline.
  • modulators of individual enzymes such as, for example, modulators of enzymes in the histone H3 lysine 9 methyltransferases G9A, SETDB1, Suv39Hl and H2, histone H3 Lysine 4, 27 or 36 methyltransferases, and H4 lysine 20 methyltransferases can be identified. Fusion proteins can also be designed and synthesized to assay for inhibitors of histone acetyltransferases, histone deacetylases, arginine methyltransferases such as CARMl or PRMTl, or any transcriptional modulator which functions via modification of chromatm structure.
  • the methods and compositions disclosed herein allow for screening of in vivo modulators (e.g., activators, inhibitors) of specific DNA methyltransferases such as DNMTl, DNMT3A and DMNT3b.
  • the cells and cell lines described herein are suitable for the subsequent confirmation of the action of modulators identified via conventional methodologies (e.g., immunoprecipitation), further enhancmg the functionality of these cell lines and streamlining the validation of potential modulators, and strengthening the utility ofthe disclosed methods and compositions.
  • Cells comprising fusions of ZFPs and CMEs may be generated by any method known in the art.
  • a cell or cell line can be stably transfected or transiently transfected with nucleic acids encoding the fusions.
  • test and control cells are used in which the test cell is substantially identical (e.g., isogenic) to the control cell except for the presence ofthe fusion molecule (and, possibly, a low incidence of random mutations resulting from environmental factors).
  • An "isogenic cell” is a cell that, with the possible exception of a few random mutations due to environmental factors, contains identical genetic material to that of another cell.
  • test and control cells typically >99% or 99.9 or 99.99% ofthe genetic material of one cell is identical to that of another.
  • the phenotype of the test and controls cell populations will differ only i regard to the levels ofthe fusion protein(s).
  • test and control cells will differ only with respect to the compounds to which they are exposed during testing, hi additional embodiments, test and control cells will both comprise similar or identical levels of a ZFP-CME fusion protein, but will differ in that the test cells are exposed to a compound and the control cells are not.
  • the cells can be individual cells or a population, the latter being more usual.
  • the cell types can be cell lines or natural (e.g., isolated) cells. Cell lines are available, for example from the Anerican Type Culture Collection (ATCC), or can be generated by methods l ⁇ iown in the art, as described for example in Sambrook et al., supra. Similarly cells can be isolated by methods l ⁇ iown in the art.
  • Other non-limiting examples of cell types include cells that have or are subject to pathologies, such as cancerous cells, or pathogenically infected cells; stem cells; fully differentiated cells; partially differentiated cells; immortalized cells and the like.
  • prokaryotic e.g., bacteria
  • eukaryotic e.g., yeast, plant, insect, fungal, piscine and mammalian cells
  • Mammalian (human and non-human) cell types are particularly preferred. The choice of cell type depends in part on the intended recipient of a drug. For example, human cell types are advantageous for screening drugs intended for use in human, and feline cell types are advantageous for screening drugs intended for use in cats.
  • Suitable mammalian cells include CHO (Chinese hamster ovary) cells, HEP-G2 cells, BaF-
  • eukaryotic cells include, for example, insect (e.g., sp.frugiperda), yeast (e.g., S. cerevisiae, S.
  • Bacterial cell types include E. coli, B. subtilis and S. typhimurium.
  • Cells can be transiently or stably transfected or transformed with a ZFP-CME fusion molecule (or polynucleotide encoding the fusion molecule). Methods of transfecting cells are l ⁇ iown in the art and described, for example, in Ausubel et al, supra.
  • two different cell populations can, for example, be put in two different vessels.
  • a solution of a candidate compound can be added sequentially to both vessels and transcription of a reporter gene detennined. Transcription can be detennined in any number of ways, for example, by measuring mRNA levels and/or expression ofthe protein using techniques l ⁇ iown in the art and described herein. When there is a significant difference (z ' .e., outside the scope of experimental error) between values of reporter gene expression for the respective cell populations, one determines the candidate compound to be a "hit" in the assay.
  • a control cell population expresses a ZFP-CME fusion but is not contacted with a compound.
  • a first test cell population also expresses the ZFP-CME fusion and is contacted with the compound.
  • a second test cell population, not expressing the ZFP-CME fusiori, is also contacted with the compound.
  • the first test cell population is compared with the control population to determine whether the compound modulates the regulation ofthe reporter gene by the ZFP-CME fusion. If an effect ofthe compound on the reporter gene regulation is observed, expression ofthe reporter gene in the second test cell population is examined.
  • an effect ofthe compound on reporter gene expression in the second test cell population is an indication of non-specificity ofthe compound and would therefore disqualify the compound as a hit.
  • control cells and the first test cell population is exposed to inducer and the second test cell population is not exposed to inducer.
  • analysis of cellular response in test and control cells is performed in parallel. In other methods, analysis of cellular response in test cells is compared with historical controls. In some methods, cellular response in presence of a compound in either test or control cells is compared with the response of like cells in absence ofthe compound.
  • compounds are screened individually. In other methods, many compounds are screened in parallel. Microtiter plates and robotics are particularly useful for parallel screening of many compounds. Optical detection can be employed for rapidity and automation. Hundreds, thousands or even millions of compounds can be screened per week.
  • derivatives ofthe compound can be made to maximize its ability to interact with its molecular target.
  • Derivatives can be produced using conventional techniques such as self-consistent field (SCF) analysis, configuration interaction (Cl) analysis, and normal mode dynamics analysis. Computer programs for implementing these techniques are readily available. See Rein et al., Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss, New York, 1989). Compound derivatives are subjected to rescreening in the cell-based assay to select the one(s) that demonstrate the best interaction profile with the molecular target.
  • SCF self-consistent field
  • Cl configuration interaction
  • Normal mode dynamics analysis Computer programs for implementing these techniques are readily available. See Rein et al., Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss, New York, 1989). Compound derivatives are subjected to rescreening in the cell-based assay to select the one(s) that demonstrate the best interaction profile with the mole
  • compounds to be screened in the present methods can be obtained from combinatorial libraries of peptides or small molecules, can be hormones, growth factors, and cytokines, can be naturally occurring molecules or can be from existing repertoires of chemical compounds synthesized by the pharmaceutical industry.
  • Combinatorial libraries can be produced for many types of compound that can be synthesized in a step-by-step fashion.
  • Such compounds include polypeptides, beta-turn mimetics, polysaccharides, nucleic acids, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines and oligocarbamates.
  • a variety of different cellular and/or biochemical responses can also be measured and compared in the methods described herein.
  • the cellular response to administration of a compound can be quantified as a value or level of a cellular property, such as cell growth, neovascularization, hormone release, pH changes, changes in intracellular second messengers such as GMP, receptor binding and the like.
  • the units ofthe value depend on the property.
  • the units can be units of absorbance, photon count, radioactive particle count or optical density.
  • a compound that interacts with it When the molecular target is intracellular, a compound that interacts with it must traverse the cell membrane.
  • a compound contacted with a cell can cross the cell membrane in a number of ways. If the compound has suitable size and charge properties, it can be passively transported across the membrane. Other processes of membrane passage include active transport (e.g., receptor mediated transport), endocytosis and pinocytosis. Where a compound cannot be effectively transported by any ofthe preceding methods, microinjection, biolistics or other methods can be used to deliver it to the internal portion ofthe cell.
  • the compound to be screened is a protein
  • a nucleic acid encoding the protein can be introduced into the cell and expressed within the cell.
  • the zinc finger protein-CME fusion to be tested must be introduced into the cell. Typically such is achieved by introducing either the ZFP-CME molecule or a nucleic acid encoding the ZFP-CME into the cell resulting in expression ofthe fusion protein within the cell.
  • Nucleic acids can be introduced by ' conventional means including viral based methods, chemical methods, lipofection and microinjection. The introduced nucleic acid can integrate into the host cliromosome, persist in episomal form or can have a transient existence in the cytoplasm.
  • an exogenous protein can be introduced into a cell in protein form.
  • the zinc finger protein can be introduced by lipofection, biolistics, or microinjection or through fusion to membrane translocating domains.
  • the compositions described herein can be provided to the target cell in vitro or in vivo.
  • the compositions can be provided as polypeptides, polynucleotides or combination thereof.
  • compositions are provided as one or more polynucleotides.
  • a zinc finger protein-containing composition can be designed as a fusion between a polypeptide zinc finger and one or more functional domains (e.g., CMEs, activation domains and/or repression domains), that is encoded by a fusion nucleic acid.
  • the nucleic acid can be cloned into intennediate vectors for transforaiation into prokaryotic or eukaryotic cells for replication and/or expression.
  • Intermediate vectors for storage or manipulation ofthe nucleic acid or production of protein can be prokaryotic vectors, (e.g., plasmids), shuttle vectors, insect vectors, or viral vectors for example.
  • a nucleic acid encoding a zinc finger protein can also cloned into an expression vector, for administration to a bacterial cell, fungal cell, protozoal cell, piscine cell, plant cell, piscine cell, or animal cell, preferably a mammalian cell, more preferably a human cell.
  • a cloned nucleic acid is typically subcloned into an expression vector that contains a promoter to direct transcription.
  • Suitable bacterial and eukaryotic promoters are well l ⁇ iown in the art and described, e.g., in Sambrook et al, supra; Ausubel et al, supra; and Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990).
  • Bacterial expression systems are available in, e.g., E. coli, Bacillus sp., and Salmonella. Palva et al. (1983) Gene 22:229-235. Kits for such expression systems are commercially available.
  • Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available, for example, from Invitrogen, Carlsbad, CA and Clontech, Palo Alto, CA.
  • the promoter used to direct expression ofthe nucleic acid of choice depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification. In contrast, when a protein is to be used in vivo, either a constitutive or an inducible promoter is used, depending on the particular use ofthe protein. In addition, a weak promoter can be used, such as HSV TK or a promoter having similar activity.
  • the promoter typically can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tet-regulated systems and the RU-486 system.
  • elements that are responsive to transactivation e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tet-regulated systems and the RU-486 system.
  • elements that are responsive to transactivation e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tet-regulated systems and the RU-486 system.
  • an expression vector typically contains a transcription unit or expression cassette that contains additional elements required for the expression ofthe nucleic acid in host cells, either prokaryotic or eukaryotic.
  • a typical expression cassette thus contains a promoter operably linked, e.g., to the nucleic acid sequence, and signals required, e.g., for efficient polyadenylation ofthe transcript, transcriptional termination, ribosome binding, and/or translation termination. Additional elements ofthe cassette may include, e.g., enhancers, and heterologous spliced intronic signals.
  • inducible promoters e.g., operably linked to control expression ofthe ZFP- CME
  • inducible promoters can be used, for example using the tet-repressor system described in Gossen et al. Science (1995) 268:1766-1769, describe fusion of a tetracycline resistance gene repressor to a viral transcription activation domain in order to induce rapid, greatly amplified gene expression in the presence of tetracycline. It is a modification of a preexisting system in which low levels of tetracycline prevented gene expression.
  • the gene that codes for the tetracycline resistance gene repressor was mutagenized and a mutant fusion protein was created that depended on tetracycline for activation was identified.
  • the construct can provide an on/off switch for high expression of a gene.
  • activator/promoter sequences l ⁇ iown in the art may also be used in construction of transactivator plasmids and plasmids in accordance with the present invention. These include, but are not limited to: (1) the T7 lac promoter construct activated by T7 RNA polymerase as the transactivator (Dubendorfs & Studier, J. Mol. Biol., 219: 45-49, 1991); (2) the Lex A (binding domain)/Gal4 transcriptional activator-for the Lex A promoter (Brent & Ptashne, Cell 43: 729-736, 1985); (3) Gal4/VP16 (Carey et al, J- Mol. Biol.
  • T3 lac constructs activated by T3 RNA polymerase as the transactivator (Deuschle et al., Proc. Natl. Acad. Sci. USA 86: 5400-5404, 1989); and (7) glucocorticoid inducible mouse mammary tumor virus promoter system, (Lee et al., Nature 294: 228-232, 1981; Huang et al., Cell 27: 245-256, 1981; Ostrowski et al., Mol Cell. Biol. 3: 2045-2057, 1983).
  • the tet operator/eCMV promoter exemplified herein also may be modified to comprise the vaccinia virus promoter (Fuerst et al., 1987, supra) instead ofthe eCMN promoter.
  • the particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use ofthe resulting ZFP polypeptide, e.g., expression in plants, animals, bacteria, fungi, protozoa etc.
  • Standard bacterial expression vectors include plasmids such as pBR322, pBR322-based plasmids, pSKF, pET23D, and commercially available fusion expression systems such as GST and LacZ.
  • Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, for monitoring expression, and for monitoring cellular and subcellular localization, e.g., c-myc or FLAG.
  • Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SN40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus.
  • Other exemplary eukaryotic vectors include pMSG, pAN009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSNE, and any other vector allowing expression of proteins under the direction ofthe SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or otlier promoters shown effective for expression in eukaryotic cells.
  • Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase.
  • High-yield expression systems are also suitable, such as baculovirus vectors in insect cells, with a nucleic acid sequence coding for a ZFP as described herein under the transcriptional control ofthe polyhedrin promoter or any other strong baculovirus promoter.
  • Elements that are typically included in expression vectors also include a replicon that functions in E. coli (or in the prokaryotic host, if other than E.
  • coli a selective marker, e.g., a gene encoding antibiotic resistance, to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the vector to allow insertion of recombinant sequences.
  • Standard transfection methods can be used to produce bacterial, mammalian, yeast, insect, or other cell lines that express large quantities of zinc finger proteins, which can be purified, if desired, using standard techniques. See, e.g., Colley et ⁇ /. (1989) /. Biol. Chem. 264:1 619-1 622; and Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed.) 1990.
  • Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques. See, e.g., Morrison (1977) /. Bacterial 132:349-351; Clark-Curtiss et al. (1983) in Methods in Enzymology 101:347-362 (Wu et al, eds).
  • Aiy procedure for introducing foreign nucleotide sequences into host cells can be used. These include, but are not limited to, the use of calcium phosphate transfection, DEAE-dextran- mediated transfection, polybrene, protoplast fusion, electroporation, lipid-mediated delivery (e.g., liposomes), microinjection, parti ⁇ le bombardment, introduction of naked D ⁇ A, plasmid vectors, viral vectors (both episomal and integrative) and any ofthe other well known methods for introducing cloned genomic D ⁇ A, cD ⁇ A, synthetic D ⁇ A or other foreign genetic material into a host cell (see, e.g., Sambrook et al, supra).
  • Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • nucleic acids include lipofection, microinjection, ballistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked D ⁇ A, artificial virions, and agent-enhanced uptake of D ⁇ A. Lipofection is described in, e.g., U.S. Patent ⁇ os.
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424 and WO 91/16024. Nucleic acid can be delivered to cells (ex vivo administration) or to target tissues (in vivo administration).
  • lipid:nucleic acid complexes including targeted liposomes such as immunolipid complexes
  • RNA or DNA virus-based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
  • Viral vectors can be administered directly to subjects (in vivo) or they can be used to treat cells in vitro, wherein the modified cells are administered to subjects (ex vivo).
  • Conventional viral based systems for the delivery of ZFPs include retroviral, lentiviral, poxviral, adenoviral, adeno-associated viral, vesicular stomatitis viral and herpes viral vectors.
  • Lentiviral vectors are retroviral vector that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral nucleic acid delivery system would therefore depend on the target cell and/or tissue. Retroviral vectors have a packaging capacity of up to 6-10 kb of foreign sequence and are comprised of cw-acting long terminal repeats (LTRs). The lTunimum cz ' s-acting LTRs are sufficient for replication and packaging ofthe vectors, which are then used to integrate the exogenous gene into the target cell to provide permanent transgene expression.
  • LTRs long terminal repeats
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLN), simian immunodeficiency virus (SIN), human immunodeficiency virus (HIN), and combinations thereof.
  • MiLV murine leukemia virus
  • GaLN gibbon ape leukemia virus
  • SIN simian immunodeficiency virus
  • HIN human immunodeficiency virus
  • Adeno-associated virus (AAV) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo applications. See, e.g., West et al. (1987) Virology 160:38-47; U.S. Patent No. 4,797,368; WO 93/24641; Kotin (1994) Hwm. Gene Ther. 5:793-801; and Muzyczka (1994) /. Clin. Invest. 94:1351. Construction of recombinant AAV vectors are described in a number of publications, including U.S. Patent No.
  • Recombinant adeno-associated virus vectors based on the defective and nonpathogenic parvovirus adeno-associated virus type 2 are a promising nucleic acid delivery system.
  • Exemplary AAV vectors are derived from a plasmid containing the AAV 145 bp inverted terminal repeats flanking a transgene expression cassette. Efficient transfer of nucleic acids and stable transgene delivery due to integration into the genomes ofthe transduced cell are key features for this vector system.
  • Wagner et al. (1998) Lancet 351 (9117): 1702-3; andKearns et al. (1996) Gene Ther. 9:748-55.
  • pLASN and MFG-S are examples are retroviral vectors that have been used in clinical trials.
  • PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al. (1995) Science 270:475- 480. Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. Ellem et ⁇ /. (1997) Immunol Immunother.
  • Adenoviral-based systems are useful.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and are capable of infecting, and hence delivering nucleic acid to, both dividing and non- dividing cells. With such vectors, high titers and levels of expression have been obtained.
  • Adenovirus vectors can be produced in large quantities in a relatively simple system.
  • Ad vectors can be produced at high titer and they readily infect a number of different cell types. Most adenovirus vectors are engineered such that a transgene replaces the Ad Ela, Elb, and/or E3 genes; the replication defector vector is propagated in human 293 cells that supply the required El functions in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing, differentiated cells such as those found in the liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity for inserted DNA. Al example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection.
  • Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ⁇ 2 cells or PA317 cells, which package retroviruses.
  • Viral vectors used in nucleic acid delivery are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the protein to be expressed. Missing viral functions are supplied in trans, if necessary, by the packaging cell line. For example, AAV vectors used in nucleic acid delivery typically only possess ITR sequences from the AAV genome, which are required for packaging and integration into the host genome.
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line is also infected with adenovirus as a helper.
  • the helper virus promotes replication ofthe AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g, heat treatment, which preferentially inactivates adenoviruses.
  • a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface ofthe virus.
  • the ligand is chosen to have affinity for a receptor known to be present on the cell type of interest.
  • Han et al. (1995) Proc. Natl. Acad. Sci. USA 92:9747-9751 reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor.
  • filamentous phage can be engineered to display antibody fragments (e.g., F a or F v ) having specific binding affinity for virtually any chosen cellular receptor.
  • F a or F v antibody fragments
  • Such vectors can be engineered to contain specific uptake sequences thought to favor uptake by specific target cells.
  • Vectors can be delivered in vivo by administration to a subject, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described infra.
  • vectors can be delivered to cells ' ex vivo, such as cells explanted from a subject (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation ofthe cells into a subject, usually after selection for cells which have incorporated the vector.
  • a subject e.g., lymphocytes, bone marrow aspirates, tissue biopsy
  • hematopoietic stem cells e.g., hematopoietic stem cells
  • Ex vivo cell transfection (e.g., for diagnostics, research, or for gene therapy such as via reinfusion ofthe transfected cells into the host organism) is well l ⁇ iown to those of skill in the art.
  • cells are isolated from the subject organism, transfected with a nucleic acid (gene or cDNA), and re-infused back into the subject organism (e.g., patient).
  • a nucleic acid gene or cDNA
  • Various cell types suitable for ex vivo transfection are well l ⁇ iown to those of skill in the art. See, e.g., Freshney et al, Culture of Animal Cells, A Manual of Basic Technique, 3rd ed., 1994, and references cited therein, for a discussion of isolation and culture of cells from patients.
  • hematopoietic stem cells are used in ex vivo procedures for cell transfection and nucleic acid delivery.
  • the advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow.
  • Methods for differentiating CD34+ stem cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN- ⁇ and TNF- ⁇ are l ⁇ iown. Inaba et al. (1992) /. Exp. Med. 176:1693-1702.
  • Stem cells are isolated for transduction and differentiation using l ⁇ iown methods.
  • stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), and lad (differentiated antigen presenting cells). See Inaba et al, supra.
  • Vectors e.g., retroviruses, adenoviruses, liposomes, etc.
  • nucleic acids can be also administered directly to the organism for transduction of cells in vivo.
  • naked DNA can be administered.
  • Administration is by any ofthe routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. Suitable methods of administering such nucleic acids are available and well l ⁇ iown to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions described herein. See, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989.
  • fusion proteins are administered directly to target cells.
  • the target cells are cultured in a medium containing one or more CMEs (or functional fragments thereof) fused to one or more ofthe ZFPs described herein.
  • fusion proteins can be administered to cells or tissues in vivo or ex vivo.
  • polypeptide compounds An important factor in the administration of polypeptide compounds is ensuring that the polypeptide has the ability to traverse the plasma membrane of a cell, or the membrane of an intracellular compartment such as the nucleus.
  • Cellular membranes are composed of lipid-protein bilayers that are freely permeable to small, nonionic lipophilic compounds and are inherently impemieable to polar compounds, macromolecules, and therapeutic or diagnostic agents.
  • proteins, lipids and otlier compounds which have the ability to translocate polypeptides across a cell membrane, have been described.
  • membrane translocation polypeptides have amphiphilic or hydrophobic amino acid subsequences that have the ability to act as membrane-translocating carriers.
  • homeodomain proteins have the ability to translocate across cell membranes.
  • the shortest internalizable peptide of a homeodomain protein, Antennapedia was found to be the third helix ofthe protein, from amino acid position 43 to 58. Prochiantz (1996) Curr. Opin. Neurobiol. 6:629-634.
  • Another subsequence, the h (hydrophobic) domain of signal peptides was found to have similar cell membrane translocation characteristics. Lin et al. (1995) /. Biol. Chem. 270:14255-14258.
  • Examples of peptide sequences which can be linked to a zinc finger polypeptide (or fusion containing the same) for facilitating its uptake into cells include, but are not limited to: an 11 amino acid peptide ofthe tat protein of HTV; a 20 residue peptide sequence which corresponds to amino acids 84-103 ofthe pl6 protein (see Fahraeus et al. (1996) Curr. Biol 6:84); the third helix ofthe 60-amino acid long homeodomain of Aitennapedia (Derossi et al. (1994) /. Biol. Chem.
  • a signal peptide such as the Kaposi fibroblast growth factor (K-FGF) h region (Lin et al, supra); and the VP22 translocation domain from HSV (Elliot et al. (1997) Cell 88:223-233).
  • K-FGF Kaposi fibroblast growth factor
  • VP22 translocation domain from HSV
  • Toxin molecules also have the ability to transport polypeptides across cell membranes. Often, such molecules (called “binary toxins”) are composed of at least two parts: a translocation or binding domain and a separate toxin domain. Typically, the translocation domain, which can optionally be a polypeptide, binds to a cellular receptor, facilitating transport ofthe toxin into the cell.
  • Clostridium perfringens iota toxin diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus anthracis toxin, and pertussis adenylate cyclase (CYA)
  • DT diphtheria toxin
  • PE Pseudomonas exotoxin A
  • PT pertussis toxin
  • Bacillus anthracis toxin Bacillus anthracis toxin
  • pertussis adenylate cyclase CYA
  • Such subsequences can be used to translocate polypeptides, including the polypeptides as disclosed herein, across a cell membrane. This is accomplished, for example, by derivatizing the fusion polypeptide with one of these translocation sequences, or by fonning an additional fusion of the translocation sequence with the fusion polypeptide.
  • a linker can be used to link the fusion polypeptide and the translocation sequence.
  • Aiy suitable linker can be used, e.g., a peptide linker.
  • a suitable polypeptide can also be introduced into an animal cell, preferably a mammalian cell, via liposomes and liposome derivatives such as immunoliposomes.
  • liposome refers to vesicles comprised of one or more concentrically ordered lipid bilayers, which encapsulate an aqueous phase.
  • the aqueous phase typically contains the compound to be delivered to the cell.
  • the liposome fuses with the plasma membrane, thereby releasing the compound into the cytosol.
  • the liposome is phagocytosed or taken up by the cell in a transport vesicle.
  • the liposome is either degraded or it fuses with the membrane ofthe transport vesicle and releases its contents.
  • the liposome ultimately becomes permeable and releases the encapsulated compound at the target tissue or cell.
  • active drug release involves using an agent to induce a permeability change in the liposome vesicle.
  • Liposome membranes can be constructed so that they become destabilized when the environment becomes acidic near the liposome membrane. See, e.g., Proc.
  • DOPE Dioleoylphosphatidylethanolamine
  • liposomes typically comprise a fusion polypeptide as disclosed herein, a lipid component, e.g., a neutral and/or cationic lipid, and optionally include a receptor-recognition molecule such as an antibody that binds to a predetermined cell surface receptor or ligand (e.g., an antigen).
  • a lipid component e.g., a neutral and/or cationic lipid
  • a receptor-recognition molecule such as an antibody that binds to a predetermined cell surface receptor or ligand (e.g., an antigen).
  • Suitable methods include, for example, sonication, extrusion, high pressure/homogenization, microfluidization, detergent dialysis, calcium- induced fusion of small liposome vesicles and ether-fusion methods, all of which are well known in the art.
  • targeting moieties that are specific to a particular cell type, tissue, and the like.
  • targeting moieties e.g., ligands, receptors, and monoclonal antibodies
  • Examples of targeting moieties include monoclonal antibodies specific to antigens associated with neoplasms, such as prostate cancer specific antigen and MAGE. Tumors can also be diagnosed by detecting gene products resulting from the activation or over-expression of oncogenes, such as ras or c-erbB2.
  • fetal tissue such as the alphafetoprotein (AFP) and carcinoembryonic antigen (CEA).
  • Sites of viral infection can be diagnosed using various viral antigens such as hepatitis B core and surface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virus antigens, human immunodeficiency type-1 virus (HIV-1) and papilloma virus antigens.
  • Inflammation can be detected using molecules specifically recognized by surface molecules which are expressed at sites of inflammation such as integrins (e.g., VCAM-1), selectin receptors (e.g., ELAM-1) and the like.
  • Standard methods for coupling targeting agents to liposomes are used. These methods generally involve the incorporation into liposomes of lipid components, e.g., phosphatidylethanolamine, which can be activated for attachment of targeting agents, or incorporation of derivatized lipophilic compounds, such as lipid derivatized bleomycin.
  • Antibody targeted liposomes can be constructed using, for instance, liposomes which incorporate protein A. See Renneisen et al. (1990) /. Biol. Chem. 265: 16337-16342 and Leonetti et al. (1990) Proc. Natl. Acad. Sci. USA 87:2448-2451.
  • kits for performing any ofthe above methods typically contains cells comprising a ZFP-CME fusion polypeptide and/or a nucleic acid encoding a ZFP- CME fusion polypeptide for use in the above methods, or components for making such cells.
  • kits contain pairs of test and control cells differing in that one cell population is transformed with an exogenous nucleic acid encoding a ZFP-CME fusion protein designed to regulate expression of a molecular target or other protein within the test cells.
  • Some kits contain a single cell type and other components that allow one to produce control cells from that cell type. Such components can include a vector encoding a zinc finger protein or the zinc finger protein itself.
  • Additional kits contain nucleic acids which encode one or more ZFP-CME fusion proteins.
  • the kits can also contain buffers for transformation of cells, culture media for cells, and/or buffers for performing assays.
  • the kits also contain a label indicating that the cells are to be used for screening compounds.
  • a label includes any material such as instructions, packaging or advertising leaflet that is attached to or otherwise accompanies the other components ofthe kit.
  • compositions and methods described herein allow for the identification of whether specific modifications act as dominant signals to regulate gene expression z>z vivo, which, in turn, functionally validates the importance of specific enzymes in gene regulation.
  • the identification of compounds that modulate chromatin modifying enzymatic activities can be therapeutically applied, for example using CME activators to stimulate the differentiation of a specific cell lineage, as an anti-proliferation/cancer therapy or in the treatment of specific diseases.
  • CME activators to stimulate the differentiation of a specific cell lineage, as an anti-proliferation/cancer therapy or in the treatment of specific diseases.
  • Acetylation ofthe tat protein of HIN via p/CAF, GC ⁇ 5
  • the identification of compounds that inhibit tat acetylation allows for the development of drugs that prevent and/or treat HIV infection.
  • the disclosed fusion molecules can be used in vitro to test for the effect of a compound on the catalytic activity ofthe CME portion ofthe fusion. See Example 2.
  • Histone H3 and H4 are methylated at lysine and arginine residues (7). These modifications have been associated with both transcriptionally active and repressed genes z z vivo. Methylation of specific arginine residues by the coactivator-associated proteins CARMl and PRMTl has been associated with transcriptional activation (8-10). Methylation of lysine 4 of histone H3, however, has been implicated in both transcriptional activation (11, 12) and in telomeric silencing (13, 14), but primarily the methylation of residues within histone H3 has been linked to transcriptional repression.
  • H3K9 histone 3
  • SUN39H1 and SUV39H2 15, 16
  • SUN39H1 and SUV39H2 are close homologues ofthe Drosophila heterochromatin associated protein Su(var)3-9, a modifier of position effect variegation (17, 18), and the S. pombe silencing factor Clr4 (19) and thus formally connect HMTs with the regulation of chromatin structure (20).
  • PPAR ⁇ (29).
  • ZFPs wliich regulate the expression of the endogenous NEGF-A gene (28) we show here that direct targeting ofthe minimal catalytic HMT domain of either SUN39H1 or G9A alone is sufficient to effect the local methylation of H3K9 at the promoter of this locus and the consequent transcriptional repression ofthe NEGF-A gene.
  • ZFP-HMT mediated repression is enhanced when coupled with histone deacetylase recruitment, supporting genetic studies which connect H3K9 HMT activity with histone deacetylation.
  • HMT Histone Methyltransferase
  • G9A is a significantly more potent HMT than SUV39H1 in this assay.
  • a western blot ofthe immunoprecipitates employed in the in vitro HMTase assay shown hi Fig.2A (Panel II) confirms that this difference in activity between the G9A and Suv Del 76 chimeras is not a consequence of differential expression levels.
  • ZFP- A which has been used tliroughout this study thus far, binds between a pair of binding sites for a second engineered ZFP (VZ +42/ +530 referred to as ZFP-B).
  • these two proteins have binding sites which are separated by less than a nucleosome length, thus ensuring that the catalytic activities recruited to either location should act upon the same local region of chromatin (Fig. 3A).
  • Fig. 3A To recruit HDAC activity to the promoter we employed the v-ErbA repression domain.
  • v-ErbA is a viral relative of avian thyroid honnone receptor (TR) that constitutively recruits the ⁇ CoR/SMRT corepressor complex, wliich utilizes associated HDAC activities to repress transcription ( see [33]) and references therein).
  • Repression of NEGF-A by v-ErbA (Fig. 1) is associated with consequent deacetylation ofthe promoter proximal histone tails.
  • Fig. 3B shows that G9A, Suv Del 76 and vErbA function to repress the endogenous NEGF-A locus when fused to either ofthe two ZFPs.
  • GFP fusions of all three domains or ZFP alone constructs fail to affect the transcription of this locus.
  • recruitment of both G9A and v-ErbA simultaneously to the VEGF-A promoter resulted in a significant increase in the level of repression, beyond that of either functional domain alone (Fig.3C and Experimental Procedures).
  • no increase in repression was observed by the use of combinations of ZFPs fused to the same domain (Fig.3D).
  • HMT domains is enhanced by the simultaneous delivery of histone deacetylase function, confirming the functional link between these chromatin modifications in gene repression in vivo.
  • Fig. 4A The results of this analysis are shown in Fig. 4A.
  • the level of H3K9 methylation signal is enriched -2-3 fold by ZFP-Suv Del 76 or ZFP-G9A compared to either the mock transfected or the ZFPA no functional domain control samples. No signal was obtained from control samples prepared in the absence ofthe antibody.
  • transfection with the ZFP linked to the catalytically null HMT mutant abolished the enrichment of dimethly-H3K9 leaving only a residual signal when compared to the control samples (Fig. 4A).
  • Control primers for exon 1 of the pi 6 locus show no enrichment upon introduction ofthe ZFP-HMT fusions again demonstrating that the enrichment is specific to the VEGF-A promoter (Fig. 4A).
  • direct targeting of HMT activity to the VEGF-A promoter via a designed zinc-finger transcription factor results in the specific methylation of H3K9 within nucleosomes proximal to the ZFP binding site in vivo.
  • Methylation of H3K9 is postulated to act as a signal tag for the recruitment of HP1, which through self-association and interaction with SUV39H1 is thought to result in the spread ofthe methylation signal away from the original methylated H3K9 residue/HP- 1 binding site [21].
  • ZFP-HMT chimeras we re-analyzed the above samples with primer sets specific for regions increasingly distant from the ZFP binding site, i.e. +1, and -500, relative to the transcription start site. The results, shown in Figs.
  • novel ZFP transcription factors can be engineered to direct functional domains and/or catalytic activities to precise locations within the human genome to regulate endogenous gene expression.
  • this unique capability allows the study ofthe molecular processes involved in both transcriptional regulation and in the modulation of chromatin structure directly on endogenous genes i.e. within their native chromatin architecture.
  • the direct targeting of enzymatic activities such as the H3K9 HMTs described here, able to epigenetically mark a region of chromatin for transcriptional silencing provides both a powerful research tool and potential therapeutic avenue in the study and treatment of human disease.
  • HEK293 cells were grown in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum in a 5% C02 incubator at 37 C.
  • HEK293 cells were plated in 12-well plates at a density of 250,000 cells/well and transfected 1 day later using Lipofectamine 2000 reagent (Gibco-BRL, MD) according to manufacturers recommendations, using 9 ⁇ l of Lipofectamine 2000 reagent and 1.5 ⁇ g of ZFP plasmid DNA per well. The medium was removed and replaced with fresh medium 6-12 h after transfection.
  • Fig. 3 to compensate for the difference in apparent Kd of each ZFP for its binding site [27], 250ng ofthe ZFP-A constructs were combined with 1.25 ⁇ g ofthe ZFP-B constructs tliroughout.
  • Whole cell lysates were pre-cleared using 40ul bed volume of Protein G Agarose beads for 30 minutes at 4°C with agitation.
  • the lysates were spun at lOOOrpm for 1 minute and the clarified lysate removed and used in an immunoprecipitation with 5ul/ of anti-HA epitope tag antibody (sc- 7392 Santa Cruz) or an IgG control antibody and incubated at 4°C for 2hrs with agitation. 20ul bed volume of Protein G Agarose beads was then added and the samples incubated for an additional hour at 4 C with agitation.
  • Samples were spun down (lOOOrpm for 1 minute) and washed x3 in wash buffer (20mM HEPES pH7.9, 75mM KCl, 2.5mM MgCl 2j ImM DTT, 0.5mM PMSF). Samples were then re-suspended in HMT assay buffer (50mM TRIS pH8.0, 20mM KCl, 250mM Sucrose, lOmM MgCl 2 , ImM DTT) containing lO ⁇ g bulk histones (Sigma) and lul S-adenosyl - (methyl- 3 H)-L-methionine (80 Ci/mM; PerkinElmer life sciences) as methyl donor.
  • HMT assay buffer 50mM TRIS pH8.0, 20mM KCl, 250mM Sucrose, lOmM MgCl 2 , ImM DTT
  • ZFP DNA-binding domains were designed and synthesized according to co-owned WO 00/41566, WO 00/42219 and WO 02/46412. See also Zhang et al. (2000)/. Biol. Chem 275: 33,850-33,860 and Liu et al. (2001) / Biol. Chem 276:11323-11334.
  • rtPCR cloning of SUV39H1 and G9A cDNA was prepared and pooled from RNA derived from HEK293, MCF-7 and U20S cell lines using the thennoscript RT-PCR system (GibcoBRL) according to manufacturers recommendations. The DNA encoding human G9A catalytic domain and full length SUN39H1 were generated via PCR from the cD ⁇ A pool using Platinum Taq High Fidelity D ⁇ A polymerase (Invitrogen) using the indicated primers. Constructs generated via PCR were sequence confirmed. SUN39H1 deletions 76 and 149 were generated via PCR using Full length SUV39H1 as template.
  • VEGF-A in the tissue culture media by transfected HEK293 cells was assayed after 48 hrs using a human VEGF-A ELISA kit (R&D systems) in duplicate according to manufacturers recommendations.
  • HEK293 cells were lysed and total RNA prepared using the high pure RNA isolation kit
  • RNA 25 ng was used in real-time quantitative RT-PCR analysis using TaqMan chemistry in a 96-well format on an ABI 7700 SDS machine (PerkiiiElmer Life Sciences) as described previously [27]. Briefly, reverse transcription was performed at 48 °C for 30 min using MultiScribe reverse transcriptase (Perl ⁇ nElmer Life Sciences). Following a 10-min denaturation at 95 °C, PCR amplification using AnpliGold DNA polymerase was conducted for 40 cycles at 95 °C for 15 s and at 60 °C for 1 min. Primer/probes used were as described previously [28]. The results were analyzed using SDS Version 1.6.3 software.
  • Chromatin immunoprecipitation ChlP
  • Cliromatin immunoprecipitation was performed using the ChlP assay kit according to the manufacturer's instructions (Upstate Biotechnology, NY). Briefly, approximately 10 million cells were transfected with expression plasmid and utilized 72 hrs post transfection. Samples were cross-linked with 1% formaldehyde for 10 min at 37°C, washed with PBS, and re-suspended in lysis buffer. The cell lysate was sonicated on ice for a total of 2 minutes (in 5 second pulses), resulting in an average DNA fragment length of approximately 500 bp.
  • immunoprecipitation was performed in ChlP dilution buffer overnight with anti-dimethyl-Histone H3K9 (Upstate Biotechnology) with agitation. Salmon spenn DNA/Protein A agarose slurry was added and incubated for lhr at 4°C with agitation. The antibody-agarose complex was centrifuged and washed x5, and the immunoprecipitated fraction eluted. The cross-linking was reversed by incubation at 65°C for 4 hours in the presence of 200 mM NaCl.
  • the DNA was recovered by phenol/chloroform extraction, precipitated and the abundance of specific sequences quantitated using real time PCR (TaqMan) as described above, omitting the reverse transcription reaction step. Relative abundances ofthe various NEGF-A genomic primers were calculated relative to an internal GAPDH genomic probe set.
  • GAPDH genomic forward primer ACATCAAGAAGGTGGTGAAG (SEQ ID ⁇ :27), reverse primer AGCTTGACAAAGTGGTCGTTG (SEQ ID NO:28), VEGF +400 region forward primer CAGCGAAAGCGACAGGGG (SEQ ID NO:29), reverse primer GTCAGCTGCGGGATCCC (SEQ ID NO:30), VEGF -500 region forward primer GGCCACCACAGGGAAGCT (SEQ ID NO:3l), reverse primer ACACAGACACACACGTCCTCACT (SEQ ID NO:32), VEGF start site (+1 region) forward primer
  • TREx U20S cell populations were stably transfected with a modified, TET regulated, CMV based expression vector (pCDNA4/TO, Invitrogen) encoding the ZFP fusion proteins; ZFP-A G9A cat domain or ZFP-A Suv39Hl Del76, respectively.
  • Cells stably transfected with a ZFP-CME are plated at a specified cell density in a 96 well format.
  • Example 8 Assay for modulators of oncogenic fusion proteins cDNA is prepared from RNA extracted from leukemic cell lines which contain and express the chimeric translocation of interest (e.g. PML/RAR ⁇ ), using the thermoscript RT-PCR system (GibcoBRL) according to manufacturers recommendations.
  • RNA extracted from leukemic cell lines which contain and express the chimeric translocation of interest (e.g. PML/RAR ⁇ ), using the thermoscript RT-PCR system (GibcoBRL) according to manufacturers recommendations.
  • the DNA encoding the PML/RAR ⁇ fusion protein is generated via PCR from the cDNA pool using Platinum Taq High Fidelity DNA polymerase (Invitrogen) and sequence confirmed. Using PCR and standard molecular biology techniques, the DNA binding domain of RAR ⁇ portion of this chimera is replaced with a ZFP DNA binding domain, which is itself targeted to one or more endogenous reporters. Using transient transfections of expression plasmids of this construct into human cell lines, the repressive activity of this construct upon the transcriptional activity ofthe endogenous reporter gene(s)can be confirmed via RNA or protein based assays. These ZFP chimeras can then also be used to create stable, inducible cell lines for screening purposes.
  • Example 9 Targeted antagonism of nuclear hormone receptor-activated gene expression by a ZFP-PAD V fusion protein
  • Peptidylarginine deiminase V deiminates arginine residues, thereby converting them to citrulline, in histones H3 and H4.
  • the particular arginine residues acted upon by PAD V are normally methylated (in nucleosomes located in or near the gene) by CARMl and/or PRMTl during the process of transcriptional activation of gene expression mediated by nuclear hormone receptors (NHRs) such as, for example, ER alpha.
  • NHRs nuclear hormone receptors
  • the zinc finger binding domains were targeted to the human vascular endothelial growth factor (NEGF) gene and are denoted NOP32E and NOP 30A. See co-owned US Patent application US2003/0021776 for their amino acid sequences and D ⁇ A target sites. Methods for the design and synthesis of zinc finger binding domains and fusion proteins are disclosed, for example, in co- owned US Patent No. 6,453,242.
  • NEGF vascular endothelial growth factor
  • a fusion between the ER alpha ligand binding domain (amino acids 308-595) and a VEGF- targeted zinc finger binding domain (VOP 32B) was constructed and shown to activate VEGF transcription in a beta-estradiol-dependent fashion. See Figure 11, lane 4 compared to lanes 7 (empty vector control) and 9 (mock-transfected cells).
  • a PAD V fusion to a different VEGF- targeted ZFP NOP 30
  • estradiol- stimulated activation of NEGF transcription was reduced. See Figure 11, lane 5.
  • NEGF-targeted ZFP-PAD N fusions in the absence of ER alpha, had no effect on NEGF transcription (compare lanes 1 and 2 with lanes 7-9).
  • a ZFP-PAD N fusion targeted to a sequence other than the NEGF gene had no effect on either basal NEGF transcription (lane 3) or ER alpha-stimulated activation of VEGF transcription (lane 6). Accordingly, ⁇ HR-stimulated activation of gene expression can be reduced or blocked by targeting the activity of a histone modifying enzyme such as, for example, PAD V to the gene.
  • a histone modifying enzyme such as, for example, PAD V
  • Nishioka K Chuikov S, Sarma K, Erdjument-Bromage H, Allis CD, Tempst P, Reinberg D: Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation. Genes Dev 2002; 16:479-489. 12. Strahl BD, Ohba R, Cook RG, Allis CD: Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena. Proc Natl Acad Sci USA 1999; 96:14967-14972.
  • Histone H3 lysine 4 methylation is mediated by Setl and required for cell growth and rDNA silencing in Saccharomyces cerevisiae. Genes Dev 2001; 15:3286-3295.
  • Tachibana M Sugimoto K, Fukus ima T, Shinkai Y: Set domain-containing protein, G9a
  • G9a Set domain-containing protein

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Abstract

L'invention concerne des compositions et un procédé utilisés pour identifier un composé capable de moduler l'activité d'une enzyme de modification de la chromatine. Ce procédé implique des fusions entre un domaine de liaison à l'ADN ciblé et une enzyme de modification de la chromatine (ou un fragment fonctionnel de celle-ci), des polynucléotides codant de telles fusions, ainsi que des procédés d'utilisation.
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WO2006132666A1 (fr) 2005-06-06 2006-12-14 Decision Biomarkers, Inc. Epreuves fondees sur des agencements d'ecoulement liquide
EP3789492A1 (fr) * 2019-09-05 2021-03-10 Universität Stuttgart Analyse cellulaire basée sur la fluorescence pour la détection de l'activité régulatrice de gènes de protéines associées à la chromatine et complexes de protéines
US20230159927A1 (en) * 2020-05-08 2023-05-25 Duke University Chromatin remodelers to enhance targeted gene activation

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CA2308377A1 (fr) * 1997-11-10 1999-05-20 The Salk Institute For Biological Studies Methodes d'utilisation d'inhibiteurs de co-represseurs dans le cadre du traitement de maladies neoplasiques
WO1999045132A1 (fr) * 1998-03-02 1999-09-10 Massachusetts Institute Of Technology Proteines a poly-doigts de zinc a sequences de liaison ameliorees
US6599692B1 (en) * 1999-09-14 2003-07-29 Sangamo Bioscience, Inc. Functional genomics using zinc finger proteins
CA2316036A1 (fr) * 1999-08-27 2001-02-27 Keqiang Wu Regulation de l'expression genetique chez des vegetaux
WO2002092002A2 (fr) * 2001-05-11 2002-11-21 The Burnham Institute Methodes de criblage, methodes diagnostiques et therapeutiques relatives a riz

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Publication number Priority date Publication date Assignee Title
WO2006132666A1 (fr) 2005-06-06 2006-12-14 Decision Biomarkers, Inc. Epreuves fondees sur des agencements d'ecoulement liquide
US8986983B2 (en) 2005-06-06 2015-03-24 Courtagen Life Sciences, Inc. Assays based on liquid flow over arrays
EP3789492A1 (fr) * 2019-09-05 2021-03-10 Universität Stuttgart Analyse cellulaire basée sur la fluorescence pour la détection de l'activité régulatrice de gènes de protéines associées à la chromatine et complexes de protéines
WO2021043871A1 (fr) * 2019-09-05 2021-03-11 Universität Stuttgart Dosage cellulaire à base de fluorescence pour la détection de l'activité régulatrice de gène de protéines et de complexes protéiques associés à la chromatine
US20230159927A1 (en) * 2020-05-08 2023-05-25 Duke University Chromatin remodelers to enhance targeted gene activation

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