CA2529677A1 - Method of modulating gene transcription - Google Patents

Abstract

It is an aspect of the present invention to provide a method of modulating nucleosome remodelling or gene transcription in a cell, said method comprising modulating in the cell an interaction of a methyl-CpG-binding protein with one or more subunits of a nucleosome remodelling complex.

Description

METHOD OF MODULATING GENE TRANSCRIPTION
TECHNICAL FIELD

The present invention relates to the field of gene transcription, more specifically to methylation-mediated transcriptional repression.

BACKGROUND
Appropriate differentiation and development of higher organisms requires precisely regulated expression of multiple genes. The primary control for most genes is exerted at the level of transcription. This involves the combinatorial action of tissue-specific and ubiquitous transcription factors acting on regulatory sequences that are proximal (promoters) or distal (enhancers, insulators, silencers, and locus control regions [LCRs]) to a gene. The existence of functionally distinct cis-acting elements indicates that the high degree of regulation involved in coordinated gene expression within a complex organism requires more intricate pathways than a simple promoter can provide to turn genes on and off. A critical aspect of such pathways and coordination is the regulation imposed upon genes within a complex nuclear environment.

The human genome is composed of about 3.3x109 base pairs (bp) and each human cell contains within its nucleus two copies of this DNA. To reach this high level of compaction, eukaryotic DNA is organized into chromatin, a highly organised package of DNA within the eukaryotic nucleus which plays a critical role in regulating gene expression and other nuclear processes. The basic structural unit of chromatin is the nucleosome, which consists of approximately 146 bp of DNA
wrapped in 1.75 superhelical turns around a histone octamer containing two molecules each of histone subunits H2A, H2B, H3 and H4. This unit is repeated once every 200 bp or so as a nucleosomal array in chromosomal DNA. The array is further compacted into a higher-order structure by the association of histone with nucleosomes within the array.

The functional consequence of chromatin packaging, in general, is to restrict access of the DNA to a variety of DNA-binding proteins that regulate gene activity.
Biochemical and genetic evidence demonstrate that nucleosomes are normally repressive for transcription. Several elegant mechanisms have evolved, however, that modulate chromatin structure to increase the accessibility of DNA for protein interaction. These pathways involve distinct protein complexes that function either as motors to disrupt nucleosomes (ATP-dependent nucleosome remodelling complexes) or as enzymatic machinery to chemically modify histones (histone acetyltransferases and deacetylases). These mechanisms appear to be critical in activating or repressing gene transcription in a particular cell type or to be poised for expression at a specific stage of development or in response to environmental signals.

The proteins responsible for nucleosome remodelling work together in large multi-protein complexes. However, little is currently known about the way in which these nucleosome remodelling complexes carry out their role in increasing access to the genome.

What is clear is that multiple levels of control are involved in regulated gene transcription, from the activation of the chromosomal domain, in which a nucleic acid resides, to the formation of a basal initiation complex on a given promoter within the domain. Nevertheless, questions still remain as to how tissue- or developmental-state-specific transcription is established and how coordinate transcription of multiple genes is achieved. In addition, the mechanism by which critical DNA control elements, often acting at long-range, such as enhancers, insulators, silencers, and LCRs, regulate transcription is still poorly understood.
Thus, there remains a need to elucidate the mechanisms implicated in the regulation of gene transcription, more particularly gene repression, so that improved strategies can be developed for modulating the activation or repression of selective genes and thereby provide effective treatment regimes for debilitating diseases or conditions such as cancer, Fragile X Syndrome, Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, alpha-thalassemia/mental retardation syndrome, X-linked (ATRX), Beckwith-Wiedemann syndrome (BWS), Prader-Willi syndrome (PWS) and Angelman syndrome (AS).

It is an aspect of the present invention to provide improved methods for the modulation of gene transcription, more particularly modulation if methylation-mediated transcriptional repression.

SUMMARY OF THE INVENTION

It is therefore an aspect of the present invention to provide a method of modulating gene transcription in a cell in vitro, the method comprising exposing the cell to a compound capable of modulating an interaction of a methyl-CpG-binding protein with one or more subunits of a nucleosome remodelling complex.

In a preferred embodiment of the present invention, the methyl-CpG-binding protein is MeCP2.

In another preferred embodiment of the present invention, the nucleosome remodelling complex is an ATP-dependent SWI/SNF nucleosome remodelling complex. More preferably, the one or more subunits of the nucleosome remodelling complex comprises Brm, PRMT, Rb protein, BAF57 or INI1. Even more preferably, the compound to which the cell is exposed is capable of modulating an interaction of the methyl-CpG-binding protein with Brm and BAF
of the nucleosome remodelling complex.
In a preferred embodiment of the present invention, the compound is capable of inhibiting the interaction of the methyl-CpG-binding protein with one or more subunits of the nucleosome remodelling complex. Alternatively, the compound is capable of enhancing the interaction of the methyl-CpG-binding protein with one or more subunits of the nucleosome remodelling complex.

In a preferred embodiment of the present invention, the methyl-CpG-binding protein and the one or more subunits of a nucleosome remodelling complex are associated with a region of a nucleic acid molecule. The region may be a regulatory region of a DNA molecule, such as those selected from the group consisting of a promoter, enhancer, insulator, silencer, or a locus control region. In a more preferred embodiment of the present invention, the promoter is selected from the group consisting of the ABCB1 promoter, the THBS1 promoter and the FMRI promoter.

It is also an aspect of the present invention to provide a method of treating or preventing a disease or disorder associated with aberrant gene transcription, the method comprising administering to a patient in need thereof a pharmaceutically effective amount of a compound capable of modulating the interaction of a methyl-CpG-binding protein with one or more subunits of a nucleosome remodelling complex, as herein described.

In a preferred embodiment of the present invention, the disease or disorder is selected from the group consisting of colon cancer, liver cancer, lung cancer, ovarian cancer, Wilms' tumour, Beckwith-Wiedemann syndrome, Prader-Willi syndrome, Angelman syndrome, Albright hereditary osteodystrophy, pseudohypoparathyroidism type Ia, pseudohypoparathyroidism type Ib, transient neonatal diabetes mellitus, Fragile X Syndrome mental retardation, systemic lupus erythematosus, Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome and Alpha-thalassemia/mental retardation syndrome, X-linked (ATRX).

It is yet another aspect of the present invention to provide a method of screening for a compound capable of modulating nucleosome remodelling or gene transcription in a cell, the method comprising exposing the ce!l to a test compound, identifying an interaction of a methyl-CpG-binding protein with one or more subunits of a nucleosome remodelling complex in the presence of the test compound and comparing the interaction to an interaction of the methyl-CpG-binding protein with the one or more subunits of the nucleosome remodelling complex in the absence of the test compound.
Also provided is a compound identified by the screening method of the present invention as herein described.

Throughout the description and the claims of this specification the word "comprise"
and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

5 The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention before the priority date of each claim of this application.

IN THE FIGURES:

Figure 1: MeCP2 associates with Brm in vivo. (a) MeCP2 cofractionates with Brm-based SWI/SNF complex. Western blot analysis using the indicated antibodies.
The input lane represents 20 mg of NIH3T3 nuclear extract. (b) MeCP2, Brm and BAF57 co-immunoprecipitate. Glycerol-gradient fractions 17-20 were pooled and immunoprecipitated using preimmune serum or antibodies to Brm or MeCP2.
Bound proteins were analyzed by western blotting using antibodies to MeCP2, BAF57 or Brm. (c) MeCP2 directly interacts with Brm and INil. Approximately 1-mg of GST, GST-Sin3A/PAHII or GST-MeCP2 were immobilized on GST beads, and their ability to interact with 35S-labeled SWI/SNF subunits was tested.
GST and GST fusion proteins were visualized by western blotting using antibody to GST.
Input represents 10% of the total amount of labelled protein used in each reaction.
(d) MeCP2, Brm and HDAC2 colocalize in mouse interphase nuclei. NIH3T3 cells were fixed, immunostained with antibodies to HDAC2 (top panel) or MeCP2 (bottom panel) and detected with Alexa 488-labeled secondary antibodies (green).
Antibodies to Brm were detected with Alexa 594-labelled secondary antibodies (red). Nuclei were counterstained with DAPI (blue or black). Panels show triple and split images and arrows indicate regions of nuclear co-localization. (e) Western-blot analyses show that expression of Brm and MeCP2 does not change in cells treated with the epigenetic modifiers 5-azacytidine (5aC) and TSA. (f) RT-PcR analysis (top panel) of ABCBI expression in CEM-CORP and CEM-A7R cells. Western-blot analyses (bottom panel) show comparable levels of MeCP2 and Brm in CEM cells.

(g) Endogenous MeCP2 immunoprecipitates with Brm. Antibodies against MeCP2 and Brm immunoprecipitate soluble Brm and MeCP2, respectively, consistent with the existence of a complex that contains MeCP2 and Brm. Preimmune sera and no-antibody controls did not immunoprecipitate any detectable protein.
Immunoprecipitations (IF) and unbound flow-through (U) were analyzed as indicated.

Figure 2: High-resolution ChIP map of the ABCBI promoter shows a similar distribution pattern for MeCP2 and Brm binding and movement in response to treatment with the epigenetic modifiers 5-azacytidine (5aC) and TSA. (a) Different regions of the ABCBI promoter in the CpG island were analyzed by PCR using primer pairs A-I. (b) Assessment of MeCP2 binding on the ABCBI promoter by ChIP analyses. Controls include the input chromatin fraction, no antibody (-Ab) control and unbound fraction after immunoprecipitation with antibody to MeCP2.
The bound MeCP2 fraction shows binding to the ABCBI promoter. Soluble chromatin was carefully prepared and sonicated to shear DNA fragments less than or equal to 0.3 kb in length. Sheared chromatin fractions above this upper limit were not processed for ChIP mapping of the ABCBI promoter. (c) Quantification of the MeCP2 and Brm ChIP fractions on the ABCBI promoter by PCR analysis for the CpG island. ChIP quantification was plotted as a ratio of bound Brm ChIP
(no antibody control - bound) to input signal and expressed as bound Brm on they axis. Error bars show the standard deviations of three independent assays. (d) Bisulfite sequencing of the ABCS1 CpG island was done as previously described and confirms that the epigenetic modifier 5-azacytidine, but not TSA, demethylates the ABCBI CpG sequence (see El-Osta et al. Mol. Cell. Biol. 22:1844-57 [2002], the contents of which are incorporated herein by reference).. Each lane represents an individual sequenced clone. Combined treatment with 5-azacytidine and TSA
does not further potentiate demethylation of methylated CpG sites compared with treatment with 5-azacytidine alone. Methylated CpG sites are shown in black;
unmethylated CpG sites, in white.

Figure 3: Assembly of the MeCP2 and Brm corepressor complex on soluble chromatin, as shown by reciprocal ChIP-ReChIP analysis of the ABCb1 promoter.
(a) The ABC81 promoter was analyzed for Brm and MeCP2 recruitment by ChIP.
(b) The methylation status of ABCBI specifies transcriptional activity. ABCB9 is hypermethylated and repressed in CEM-CCRF cells but is hypomethylated and active in CEM-A7R cells. (c) ChIP-ReChIP analysis of CEM-CCRF cells. Soluble chromatin was prepared from CEM-CCRF cells and divided into two chromatin aliquots (CAl and CA2), which were immunoprecipitated (1 ChIP) with antibodies to MeCP2 and Brm, respectively. Immunocomplexes were collected and eluted, and soluble chromatin fractions CAl and CA2 were reimmunoprecipitated (2 ChIP) with reciprocal antibodies against Brm and MeCP2, respectively. (d) Real-time amplification kinetics illustrating the enrichment of Brm and MeCP2 by ChIP
and ChIP-ReChIP. Kinetic curves for primaly (1) and secondary (2) ChIP are shown.
(e) Brm is part of the HDACI corepressor complex enriched on the silent ABCBI
allele. ChIP-ReChIP analysis was done as described in (c) on soluble chromatin prepared from CEM-CCRF and CEM-A7R cells and immunoprecipitated with antibodies to either HDAC1 or acetylated histone H3, respectively. (f) Comparison of Brm (left panel) and BAF57 (right panel) recruitment on ABCB1 chromatin in CEM-CCRF (silent ABCBI) and CEM-A7R (active ABCBI) cells. (g) The Brm-containing MeCP2 corepressor complex includes HDACI and Sin3a on soluble chromatin, as shown by reciprocal ChIP-ReChIP analysis of inactive ABCBI. G6PD
shows reduction in MeCP2, Sin3a, Brm and HDAC1 complexes by ChIP and ReChIP. Primary ChIP was done using antibody to acetylated H4 as an internal control for antibody specificity and as a positive control on active G6PD. (h) Real-time amplification kinetics show the enrichment of corepressor determinants on silent and active ABCBI and G6PD, respectively. Kinetic curves for primary (1) and secondary (2) ChIP are shown.

Figure 4: Recruitment of the MeCP2 and Brm corepressor complex is specified by promoter methylation. Soluble chromatin was prepared from CEM-CCRF cells and divided into two chromatin aliquots, which were immunoprecipitated with antibodies to MeCP2 and Brm, respectively. Immunocomplexes were collected and eluted, and the soluble chromatin fractions were reimmunoprecipitated (ReChIP) with reciprocal antibodies against Brm and MeCP2, respectively. Controls for the ChIP
and ReChIP fractions include the input chromatin, no antibody (-Ab) control and unbound fraction after immunoprecipitation with antibodies to MeCP2 or Brm.
(a) The DCK CpG island was analyzed for Brm and MeCP2 recruitment by ChIP.
Primers amplify a region from +101 to +270 relative to the transcription initiation start site (+1). (b) The MeCP2 and Brm corepressor complex is directiy recruited to the THBSI promoter, as shown by reciprocal ChIP-ReChIP analysis. Primers amplify a region from -218 to -137 relative to the transcription initiation start site (+1).

Figure 5: Somatic knockdown of Brm by siRNA reactivates silent ABCBI. The hallmark of ABCBI silencing is hypermethylation of the CpG island that is enriched with the MeCP2-HDAC corepressor complex, which is reversible by the combined inhibition of genomic methylation and histone deacetylase activity. (a) Knock-down of DNMTI by siRNA reactivates ABCBI, confirming that repression of ABCB1 transcription is mediated by methylation. (b,c) MeCP2-mediated silencing of the ABCBI promoter can be alleviated by depleting MeCP2 and Brm by RNA
interference. PCR analysis and quantification used cDNA primer pairs specific for ABCB1. The relative level of ABCB1 mRNA was normalized against HPRT. Error bars show the standard deviations of three independent assays. Immunoblot analysis shows protein products for MeCP2 and Brm in control (-) and siRNA-treated (+) cells. (d) Brm is required for stable association of MeCP2 on ABCBI.
RNA interference was done in CEM-CCRF cells to knock-down MeCP2 or Brm, and ChIP experiments were done to assay relevant genomic binding. Soluble chromatin was prepared and immunopurified using antibodies to Brm, BAF57 or MeCP2. Association of binding was quantified using real-time PCR analysis.
Reciprocal experiments were done examining (e) MeCP2 and (f) BAF57 association on ABCBI. Error bars show the standard errors of three independent experiments.

Figure 6: The applicability of the MeCP2-containing Brm corepressor complex on methylated gene silencing can be extended to FMR1. Two cell lines were used to test the hypothesis that Brm and MeCP2 are on FMR1 chromatin when the gene is silent. (a) Diagram of FMRI and G6PD positioned on the X chromosome and representation of the gene regions used in the ChIP analyses. (b) Real-time PCR
analysis of poly(A)+ mRNA for FMRI expression in normal and FRAXA cells. (c) Example of real-time amplification kinetics of Brm and MeCP2 ChIP binding on FMRI in normal and FRAXA cells. (d) Quantification of Brm and MeCP2 chromatin assembly on the FMRI promoter in normal and FRAXA cells. Schematic representation of the FMRI gene region analyzed by real-time PCR is shown as a thick black line, TM3, relative to the transcription initiation start site (+1). (e) Demethylation by 5-azacytidine (5ac) reactivates silent FMRI in FRAXA cells but does not change gene expression in normal cells. (f) Corepressors Brm and MeCP2 are released from the FMRI promoter after 5-azacytidine (5ac)-induced demethylation. (g) FMRI silencing in FRAXA cells can be partly alleviated by MeCP2 and Brm siRNA knock-down. Real-time quantification was. done using primer pairs specific for FMRI. The relative level of FMRI mRNA was normalized against HPRT. Similar results were obtained in independent experiments. (h) BAF57 is enriched on repressed FMRI in FRAXA cells. Representative gel images show input controls for the ChIP assay, including the bound and unbound fractions after immunoprecipitation with antibody to BAF57.

Figure 7: Silent and active FMRI assemble distinct complexes in normal and FRAXA cells. Soluble FMR1 chromatin was prepared from normal (a) and FRAXA
(b) cells and immunoprecipitated with antibodies against histone H3 methylated at Lys4 (H3K4), Set7, acetylated histones H3 and H4 and HDAC1. Real-time PCR
was used to quantify the level of bound chromatin. Input amplification is shown in black; antibody-bound, in red. G6PD is transcriptionally active and was used as an internal control for immunopurification of chromatin. The corepressor complex (MeCP2, Brm and HDAC1) was not associated with G6PD chromatin in normal (c) and FRAXA (d) cells.

DETAILED DESCRIPTION OF THE INVENTION

It is an aspect of the present invention to provide a method of modulating gene transcription in a cell, said method comprising exposing the cell to an exogenous compound that modulates an interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex.

The present invention is based on the present inventors' findings that during methylation-mediated gene silencing, methyl-CpG-binding domain (MBD) proteins are recruited on repressed promoters and associate with nucleosome remodelling complexes.

Methyl CpG-Binding Proteins 5 Of the methyl CpG-binding domain (MBD) family (e.g. MBD1, MBD2, MBD3, MBD4, Kaiso, MeCP2), MeCP2 was the first member to be described at the molecular level. MeCP2 is a multidomain protein that has been shown to be associated with the methylation of histone H3 at Lys9. It has been found to localize to densely methylated regions (major satellite DNA) of the mouse genome, 10 although only a small portion of the MeCP protein is devoted to selective recognition of methyl CpG. MeCP2 also contains a transcriptional repression domain (TRD) that overlaps a nuclear localization signal.

The methyl-CpG-binding protein that may be targeted in accordance with the method of the present invention may be a methyl-CpG-binding protein of any species, including, but not limited to, human (methyl CpG binding protein 2 [MeCP2]; also known as HGNC:6990, DKFZp686A24160, MRX16, MRX79, PPMX, RTS and RTT), mouse (methyl CpG binding protein 2 [Mus musculus], also known as MGI:99918, 1500041 B07Rik, Mbd5 and WBPIO), rat (methyl CpG binding protein 2 [Rattus norvegicus], also known as RGD:3075), zebrafish (methyl CpG
binding protein 2 [Danio rerio], also known as ZDB-GENE-030131-7190, MGC111857, fk96a04, wu:fk96a04 and zgc:111857), ovine, bovine, equine and bovine. In a preferred embodiment of the present invention, the methyl-CpG-binding protein is human MeCP2.
Nucleosome Remodelling Complexes and Subunits Thereof The discovery of nucleosome remodelling activities has been key in deciphering the principles that enable cells to both organize their genomes into compact chromatin and ensure that the genetic information remains accessibie to regulatory factors and enzymes within the confines of the nucleus. These nucleosome remodelling complexes weaken the tight wrapping of DNA around the histone octamers, thereby facilitating the sliding of histone octamers to neighbouring DNA
segments, their displacement to unlinked DNA, and the accumulation of patches of accessible DNA on the surface of nucleosomes, thus allowing the recruitment of activator determinants to gene sequences. It is presumed that the collective action of these enzymes endows chromatin with dynamic properties that govern all nuclear functions dealing with chromatin as a substrate.

The nucleosome remodelling complex that may be targeted in accordance with the method of the present invention includes the ATP-dependent nucleosome remodelling complexes, or subunits thereof. ATP-dependent nucleosome remodelling activity is believed to play a key role in the regulation of transcription by RNA polymerase and it has been proposed to be a prerequisite for a variety of other cellular processes that require access to the chromatin template. In addition to ATP-dependent nucleosome remodeling, multisubunit complexes that can acetylate or methylate histone and nonhistone proteins also have the potential to directly modify chromatin structure and function.

A host of ATP-dependent chromatin remodeling complexes has been identified via biochemical fractionation of cell extracts, yeast genetics, or genome database mining. A hallmark of these multisubunit complexes is that they contain a member of the SWI2/SNF2 subfamily of DNA-stimulated ATPases. At least seventeen members of the SWI2/SNF2 family have been identified in the yeast genome, four of which have been purified as subunits of distinct chromatin remodeling complexes (e.g. SWI-SNF, RSC, ISW1 and ISW2). Additional ATP-dependent remodeling complexes have been identified in Drosophila (ACF, CHRAC, NURF and Brm), humans (hSWI-SNF, NURD and RSF) and frogs (Mi-2). Each of these complexes appears to catalyze a reaction in which the energy of ATP hydrolysis is used to weaken histone-DNA interactions which leads to an increase in nucleosomal DNA
accessibility. In the case of the yeast SWI-SNF, Drosophila Brm, and human SWI-SNF complexes, it has been suggested that this reaction is required for transcriptional regulation of target genes in vivo.

Examples of ATP-dependent nucleosome remodelling complexes, and subunits thereof, which may be targeted in accordance with the methods of the present invention, are illustrated in the figure below:

S%4'I2JSNF2 fmnulv ISWI fanity MI-2 family ATPase 8ramo AiPase PHO ATPasp SAW .~...-,=.....-...,........,.ARW i domain ~...-=- ~ r ~-m~~~~~1 , k. . _ ...
~", az~ ,~.,.....'~,,,.? = .~..............R.,.......... :.
domatn SWUSNIF RSC
ISWI TSM
'4N11= ..ar Y " ~~ /
Pi.W
~ ~.
i 1et~~Jl Yeast 1rp7 (ystt, y,i,.;
""" ~rEr .w~ ., . P !- -.."~SP:);cc,~?'1 ~_../ ~/!
NuRll hS1~'USNF RSF hr1CF/ hCHRAC
= ~~. =~- %ktn~i~ ':
w ~ r[p_nrt~ i~i~n~ 1VC.RF
N~} ~ t ltb1~ ,lli-? ~Tl~ls Human ~~3~ rixP}~-~ -S'~To j Yi tt~l l r~~ ~ ~ /( Jti tt~P9Ti iUU' inil dS11'I)rNF
_.. ~ NURF CHRAC t1CF
~iiti=, -_ Drosophiln ,n~~tiA~~liftj In a preferred embodiment of the present invention, the nucleosome remodelling complex is an ATP-dependent SWI/SNF nucleosome remodelling complex.
The subunit of the nucleosome remodelling complex which may be targeted in accordance with the method of the present invention may be determined by any means known to a person skilled in the art, and may include any single subunit or any combination of subunits, as depicted in the figure above. They include subunits of the yeast SWI/SNF remodelling complex (including SWI1, SWI2/SNF2, SWI3, SNF11, Swp29, Swp73, Swp82, SNF5, SNF6, Arp7 and Arp9), subunits of the yeast RSC remodelling complex (including RSC1-10, RSC14, RSC15, STH1 and SFH1), subunits of the yeast ISW1 and ISW2 remodelling complexes (including ISW1, ISW2, p74, p110, p105 and p140), subunits of the human SWI/SNF remodelling complex (including Brgl, Brm, Ini1, BAF47, BAF50, BAF60a-c, BAF155, BAF170 and BAF250), subunits of the human RSF remodelling complex (including p325 and SNF2h), subunits of the human ACF/WCRF
remodelling complex (including ACF/WCRF180 and SNF2h), subunits of the human CHRAC remodelling complex (including ACF/WCRF180, p15, p17 and SNF2h), subunits of the human NuRD remodelling complex (including Mi-2-CHD3/4, HDAC1, HDAC2, MTA1, MTA2, RbAp48, RbAp46 and MBD3), subunits of the Drosophila remodelling complex SWI/SNF (including Brm, BAP155, BAP60, BAP55, BAP74, BAP47, BAP111 and Snr), subunits of the Drosophila remodelling complex NURF (including ISWI, NURF301, p55 and p38), subunits of the Drosophila remodelling complex CHRAC (including ISWI, ACF1, p14 and p16) and subunits of the Drosophila remodelling complex ACF (including ISWI, ACF1 and ACF1 [170]).

In a preferred embodiment, the subunit of the nucleosome remodelling complex includes Brm, BAF57, protein arginine methyltransferase (PRMT), retinoblastoma (Rb) protein, or a combination thereof. In a more preferred embodiment of the present invention, the subunit of the nucleosome remodelling complex is Brm or BAF57, or a combination thereof.

As used herein, the term "gene" encompasses all regulatory and coding sequence contiguously associated with a single hereditary unit with a genetic function.
Genes can include non-coding sequences that modulate the genetic function that include, but are not limited to, those that specify polyadenylation, transcriptional regulation, DNA conformation, chromatin conformation, extent and position of base methylation and binding sites of proteins that control all of these. Genes encoding proteins are comprised of "exons" (coding sequences), which may be interrupted by "introns" (non-coding sequences). In some instances complexes of a plurality of protein or nucleic acids or other molecules, or of any two of the above, may be required for a gene's function. On the other hand a gene's genetic function may require only RNA expression or protein production, or may only require binding of proteins and/or nucleic acids without associated expression. In certain cases, genes adjacent to one another may share sequence in such a way that one gene will overlap the other. A gene can be found within the genome of an organism, in an artificial chromosome, in a plasmid, in any other sort of vector, or as a separate isolated entity.
As used herein, the phrase "modulate gene transcription" includes, without limitation, up- and down-regulation of initiation of gene transcription, rate of gene transcription, and/or gene transcription levels.

The exogenous compound to which the cell is exposed in accordance with the methods of the present invention will modulate the interaction of the methyl-CpG-binding protein to the subunit of a nucleosome remodelling complex by either inhibiting said interaction or enhancing said interaction. Where the interaction of the methyl-CpG-binding protein to the subunit of a nucleosome remodelling complex is enhanced, the desired effect of said modulation is the down-regulation of initiation of gene transcription, rate of gene transcription, and/or gene transcription levels (e.g., gene silencing).

By contrast, where the interaction of the methyl-CpG-binding protein to the subunit of a nucleosome remodelling complex is inhibited, the desired effect of said modulation is the up-regulation of initiation of gene transcription, rate of gene transcription, and/or gene transcription levels. In a preferred embodiment, the effect of said inhibition is a reversal of transcriptional repression (e.g., initiation of transcription of a silenced gene).

Modulating an interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodeliing complex in accordance with the present invention may occur before said methyl-CpG-binding protein and nucleosome remodelling complex are bound to a nucleic acid molecule. Alternatively, modulation may occur after either or both of said methyl-CpG-binding protein and subunit of a nucleosome remodelling complex is bound to a nucleic acid. The nucleic acid molecule which may be the target of the methyl-CpG-binding protein/nucleosome remodelling complex interaction includes a regulatory region of a DNA molecule, such as a promoter, enhancer, insulator, silencer, or a locus control region.
Preferably, the regulatory region is selected from the group consisting of the ABCBI promoter, the THBSI promoter and the FMRI promoter.

If it is desirable to achieve an up-regulation of initiation of gene transcription, rate of gene transcription, and/or gene transcription levels in a cell, several approaches are available. In one preferred approach, the compound to which the cell is exposed in accordance with the methods of the present invention (hereinafter described as the "compound') will inhibit the interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex. For example, the compound may bind to the methyl-CpG binding protein and inhibit its association (e.g., binding) with a subunit of the nucleosome remodelling complex or, alternatively, the compound may bind to a subunit of the nucleosome remodelling complex and inhibit its association (e.g. binding) with a methyl-CpG binding protein.

5 If it is desirable to achieve down-regulation of gene transcription, rate of gene transcription and/or gene transcription levels, several approaches are also available. For example, the compound may bind to a methyl-CpG binding protein and promote or enhance its association (e.g., binding) with a subunit of the nucleosome remodelling complex or, alternatively, the compound may bind to a 10 subunit of the nucleosome remodelling complex and promote or enhance its association (e.g., binding) with a methyl-CpG binding protein.

It would be understood by the skilled addressee that the degree of modulation of an interaction of a methyl-CpG-binding protein with a subunit of a nucleosome 15 remodelling complex may be dependent, in part, upon the amount of compound (i.e., concentration) to which the cell is exposed.

The term "exogenous compound" as used herein includes compounds that are not normally found in nature and which may be partly or wholly synthetically produced (e.g., using recombinant DNA techniques, chemical synthesis, etc.). The term also includes naturally occurring compounds that have been substantially isolated and/or purified from their natural environment. For example, the compound may be isolated and/or purified away from some or all of the proteins and compounds with which it is normally associated in nature, and thus may be substantially pure.
For example, an isolated compound is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total sample.
A sample of substantially pure compound comprises at least about 75% by weight of the total sample, with at least about 80% being preferred, and at least about 90%
being particularly preferred. The definition also includes the production of a compound from one organism in a different organism or host cell.
Alternatively, the compound may be made at a significantly higher concentration than is normally seen, through the use of an inducible promoter or high expression promoter, such that the compound is made at increased concentration levels.

Exemplary exogenous compounds include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries.
An isolated and/or purified compound refers to a compound that (1) has been separated from at least about 50% of polynucleotide, protein, lipid, carbohydrate, or other material with which it is naturally found when isolated from a natural source, (2) is not linked (by covalent or noncovalent interaction) to all or a portion of a component to which the isolated and/or purified compound is linked in nature, (3) is operably linked (by covalent or noncovalent interaction) to a component with which it is not linked in nature, or (4) does not occur in nature. Preferably, the isolated polypeptide is substantially free from any other contaminating polypeptides or other contaminants that are found in its natural environment.

The term "exogenous compound" also includes peptides endogenously expressed within the cell following the introduction therein of an exogenous nucleic acid molecule. Thus, in certain embodiments, the cell endogenously produces a recombinant test compound which includes, but is not limited to, a test polypeptide, a test nucleic acid and/or a test carbohydrate which is capable of modulating an interaction of a methyl-CpG-binding proteiri with a subunit of a nucleosome remodelling complex in accordance with the present invention. Thus, in another approach, the compound for use in accordance with the methods of the present invention may be expressed endogenously in a cell by means of the insertion, into the cell's genetic material, of a nucleic acid molecule (e.g, DNA and/or RNA) which is capable of driving the expression of said compound, as hereinbefore described.
Thus, in a preferred embodiment of the present invention, there is provided a method of modulating the interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex in a cell, the method comprising exposing the cell to a nucleic acid molecule which is capable of driving the expression of a compound in the cell and wherein said compound is capable of modulating the interaction of the methyl-CpG-binding protein with the subunit of the nucleosome remodelling complex in a cell.

The compound used in the methods according to the present invention may include antibodies or antigen-binding fragments thereof (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab')2 and FAb expression library fragments, scFV molecules, and epitope-binding fragments thereof) or other small molecules that are capable of binding to a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex, thereby modulating the interaction between said methyl-CpG-binding protein and subunit of a nucleosome remodelling complex, as herein described.

The modulation of an interaction of a methyl-CpG-binding protein with a subunit of the nucleosome remodelling complex in a cell may be determined by the skilled addressee by any or a combination of diagnostic or prognostic assays known in the art. For example, a cell which has been exposed to the compound in accordance with the method of the present invention may be analysed directly for a change in gene expression by measuring the level of RNA in the cell and comparing that level of RNA with the level of RNA in a cell which has not been exposed to said compound. The level of gene expression (i.e., RNA levels) may be analysed by using any or a combination of means known in the art, including, but not limited to Northern blot analysis and reverse transcription-polymerase chain reaction.
The cell which has been exposed to the compound in accordance with the method of the present invention may also be analysed indirectly for a change in gene expression by measuring a surrogate marker of gene transcription, such as the concentration of a protein that is derived from the corresponding gene (e.g., via Western blotting, enzyme-linked immunosorbent assays, HPLC, etc).

The compound used in accordance with the method of the present invention may be a recombinant methyl-CpG-binding protein or recombinant nucleosome remodelling complex (or a subunit thereof), or a combination thereof, capable of promoting or enhancing the interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex. The compounds used in accordance with the method of the present invention include fragments and variants of methyl-CpG-binding proteins and/or subunits of a nucleosome remodelling complex which are capable of promoting or enhancing the interaction of the methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex.

The compound used in accordance with the method of the present invention may also comprise antisense and/or ribozyme molecules that are capable of inhibiting the expression of a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex in a cell, thereby inhibiting their effect on gene silencing within the cell. Still further, triple helix molecules can be utilized in reducing the level of said methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex in a cell.

As used herein, the term "antisense" refers to a nucleotide sequence that is complementary to a nucleic acid encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex, as herein described. For example, the antisense molecule may be complementary to the coding strand of the double-stranded cDNA molecule or complementary to the mRNA sequence encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex. The antisense nucleic acid is preferably complementary to an entire methyl-CpG-binding protein coding strand and/or the entire coding strand of a subunit of a nucleosome remodelling complex, or to only a portion thereof. In a further embodiment, the antisense nucleic acid molecule is antisense to a "non-coding region" of the coding strand of a nucleotide sequence encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex (e.g., the 5' and 3' untransiated regions).
An antisense nucleic acid can be designed such that it is complementary to the entire coding region of a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or non-coding region of an RNA
molecule encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex. . For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the RNA
encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the moiecule or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

In a further embodiment of the present invention, short interfering nucleic acid molecules (siRNA) that inhibit gene expression of a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex can also be used in accordance with the method of the present invention to reduce the level of gene expression of a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex.

The terms " short interfering nucleic acid", "siNA", " short interfering RNA", "siRNA", " short interfering nucleic acid molecule", "short interfering oligonucleotide molecule", or "chemically-modified short interfering nucleic acid molecule", as used herein, preferably refer to any nucleic acid molecule capable of inhibiting or down-regulating the expression of a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex, for example, by mediating RNA interference ("RNAi") or gene silencing in a sequence-specific manner. Chemical modifications can also be applied to any siNA sequence of the present invention. For example, the siNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to a nucleotide sequence encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex and the sense region having a nucleotide sequence corresponding to a nucleotide sequence encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex. The siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example, wherein the double stranded region is about 19 base pairs); the antisense strand comprises nucleotide sequence that is complementary to a nucleotide sequence encoding a methyl-CpG-binding protein and/or a subunit of a 5 nucleosome remodelling complex and the sense strand comprises nucleotide sequence corresponding a nucleotide sequence encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex. Alternatively, the siNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid 10 based or non-nucleic acid-based linker(s).

The siNA can be a polynucleotide with a hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to a nucleotide sequence in 15 a separate target nucleic acid molecule or a portion thereof and the sense region having a nucleotide sequence corresponding to a nucleotide sequence encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex.

20 The siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to a nucleotide sequence encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex and the sense region having a nucleotide sequence corresponding to a nucleotide sequence encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi.

The siNA can also comprise a single stranded polynucleotide having a nucleotide sequence complementary to a nucleotide sequence encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex (for example, where such siNA molecule does not require the presence within the siNA
molecule of a nucleotide sequence corresponding to a nucleotide sequence encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5'-phosphate or a 5',3'-diphosphate.

In a preferred embodiment, the siNA molecule of the present invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der Waals interactions, hydrophobic interactions, and/or stacking interactions. In a further embodiment, the siNA
molecule of the present invention comprises a nucleotide sequence that is complementary to a nucleotide sequence encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex.

In another embodiment, the siNA molecule of the present invention interacts with a nucleotide sequence encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex in a manner that causes inhibition of expression of the methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex. As used herein, siNA molecules need not be limited to those molecules containing only RNA, but further encompasses molecules comprising chemically-modified nucleotides or those in combination with non-nucleotides.

In certain preferred embodiments, the siNA molecule of the present invention lacks 2'-hydroxy (2'-OH) containing nucleotides. Such siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can, however, have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups.
Optionally, siNA molecules of the present invention can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions.
The modified siNA molecules of the invention can also be referred to as short interfering modified oligonucleotides "siMON." As used herein, the term siNA
is preferably meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), translational silencing, and others. In addition, as used herein, the term RNAi is preferably meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, or epigenetics. For example, siNA
molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic regulation of the gene expression of a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex by siNA molecules of the present invention can result from siNA-mediated modification of the chromatin structure to alter the gene expression of a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex.

The antisense and short interfering RNA molecules of the present invention are typically exposed directly to a cell, or generated in situ such that they hybridise with or bind to cellular mRNA and/or genomic DNA encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex to thereby inhibit expression of said methyl-CpG-binding protein and/or subunit of a nucleosome remodelling complex (e.g., by inhibiting transcription and/or translation).
The antisense and short interfering RNA molecules can also be delivered to a cell using vectors, or by viral mechanisms (such as retroviral or adenoviral infection delivery).
To achieve sufficient intracellular concentrations of the molecules, vector constructs in which the molecule is placed are under the control of an appropriate promoter.
In yet another embodiment, the antisense nucleic acid molecule of the present invention is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual a-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et a/. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. A ribozyme having specificity for a methyl-CpG-binding protein-encoding nucleic acid molecule and/or a subunit of a nucleosome remodelling complex-encoding nucleic acid molecule can include one or more sequences complementary to the nucleotide sequence of the corresponding cDNA sequence, and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). For example, an RNA molecule encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA
molecules (see, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418).

In a further embodiment, the expression of a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the corresponding gene in the cell (see generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y.
Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15). The potential sequences that can be targeted for triple helix formation can be increased by creating a so-called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

The antisense molecules may also be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecule can be modified to generate peptide nucleic acids (see Hyrup B. et al.
(1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms "peptide nucleic acid" or "PNA" refers to a nucleic acid mimic, e.g., a DNA
mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et a/. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93:14670-675.
In other embodiments, the antisense molecules may comprise other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc.
Natl. Acad.
Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA
84:648-652;
PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT
Publication No. W089/10134). In addition, antisense molecules can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

Another method by which nucleic acid molecules may be utilized to modulate an interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex in a cell is through the use of aptamer molecules specific for said methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to protein ligands (see, e.g., Osborne, et al. (1997) Curr. Opin. Chem. Biol. 1(1):5-9; and Patel, D. J. (June 1997) Curr. Opin.
Chem. Biol.
1(1):32-46). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic protein molecules may be, aptamers offer a method by which the interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex may be specifically modulated without the introduction of drugs or other molecules which may have pluripotent effects.
The designing of mimetics to a known pharmaceutically compound is also a known approach to the development of an compound for use in accordance with the method of the present invention. This might be desirable where the compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of use (e.g., peptides are generally unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal). Mimetic design, synthesis, and testing are generally used to avoid large-scale screening of molecules for a target property.

When designing a mimetic, it is desirable to firstly determine the particular regions of 5 the compound that are critical and/or important in determining the target property. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide (e.g., by substituting each residue in turn). These parts or residues constituting the active region of the compound are known as its "pharmacophore". Once the pharmacophore has been found, its structure is modelled 10 according to its physical properties (e.g., stereochemistry, bonding, size, and/or charge), using data from a range of sources (e.g., spectroscopic techniques, X-ray diffraction data, and NMR). Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms), and other techniques can be used in this modelling process.
In a variant of this approach, the three dimensional structure of the compound and its binding partner are modelled. This can be especially useful where the compound and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic. A template molecule is then selected, and chemical groups that mimic the pharmacophore can be grafted onto the template.
The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, does not degrade in vivo, and retains the biological activity of the lead compound. The mimetics found are then screened to ascertain the extent they exhibit the target property, or to what extent they inhibit it. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

Screening Assays It is yet another aspect of the present invention to provide a method of screening for a compound which modulates nucleosome remodelling or gene transcription in a cell, said method comprising exposing the cell to a test compound, identifying an interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex in the presence of the test compound and comparing said interaction to an interaction of the methyl-CpG-binding protein with the subunit of a nucleosome remodelling complex in the absence of the test compound. Any or a combination of methods of identifying an interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex may be used as known in the art, such as those hereinbefore described.

The compounds used in accordance with the methods of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including, but not limited to, biological libraries, peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; e.g., Zuckermann et al., 1994, J. Med.
Chem.
37:2678-2685), spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the 'one-bead one-compound' library method and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are generally limited to peptide libraries, while the other four approaches are generally applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (see Lam, 1997, Anticancer Drug Des. 12:145).
Examples of methods for the synthesis of molecular libraries have been described (e.g., DeWitt et a/., 1993, Proc. Natl. Acad. Sci. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al., 1994, J. Med. Chem.
37:2678; Cho et al., 1993, Science 261:1303; Carrell et al., 1994, Angew.
Chem.
Int. Ed. Engl. 33:2059; Carell et a/., 1994, Angew. Chem. Int. Ed. Engl.
33:2061;
and Gallop et al., 1994, J. Med. Chem. 37:1233).

Libraries of compounds can be presented in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. No. 5,223,409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci.
USA
89:1865-1869) or on phage (Scott et al., 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; U.S. Pat. No. 5,223,409).

In such embodiments, a culture of such cells will collectively provide a library of potential effector molecules and those members of the library which either enhance or inhibit an interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex can be selected and identified.
In one embodiment, an assay is a cell-based assay in which a cell is exposed to a test compound, and the ability of the test compound to modulate an interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex is determined, as hereinbefore described. The cell may be derived from any species, though preferably of mammalian origin.

In yet another embodiment of the present invention, a cell-free assay is provided in which a test compound is evaluated for its ability to bind to a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex. Without being limited by theory, the ability of a compound to bind to a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex is likely to suggest that the compound is thereby capable of modulating the interaction of the methyl-CpG-binding protein with the subunit of the nucleosome remodelling complex in a cell to which the compound is exposed. For example, soluble forms of a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex (or fragments thereof) can be used in a cell-free assay of the present invention.
Cell-free assays may involve preparing a reaction mixture of a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex and the test compound under conditions and for a time sufficient to allow the methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex to interact and bind to the test compound, thus forming a complex that can be removed and/or detected, as hereinbefore described.

Determining the ability of a test compound to bind to a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex can be determined by any or a combination of methods known in the art. For example, the ability of a test compound to bind to a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex can be determined using real-time biomolecular interaction analysis (BIA; e.g., Sjolander et al., 1991, Anal. Chem. 63:2338-2345;
Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705). "Surface plasmon resonance" (SPR) or "BIA" detects biospecific interactions in real time, without labelling any of the interactants (e.g., BlAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of SPR), resulting in a detectable signal that can be used as an indication of real-time reactions between biological molecules.

The test compound may be anchored onto a solid phase and a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex applied thereto.
The binding of the anchored test compound to a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex can then be detected at the end of the reaction, as herein described. Alternatively, the methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labelled, either directly or indirectly, with detectable labels discussed herein.

Binding of the test compound to a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. A fusion protein can also be provided which adds a domain that allows one or both of the components (i.e. the methyl-CpG-binding protein and a subunit of a nucleosome remodelling complex) to be bound to a matrix. For example, glutathione-S-transferase/3700 fusion proteins or glutathione-S-transfera- se/target fusion proteins can be adsorbed onto glutathione Sepharose.TM. beads (Sigma Chemical, St. Louis, Mo.) or glutathione-derivatized microtiter plates, which are then combined with the test compound or the methyl-CpG-binding protein and/or subunit of a nucleosome remodelling complex, and the mixture incubated under conditions conducive for complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, and the complex determined directly or indirectly, for example, as herein described. Alternatively, the complexes can be dissociated from the matrix, and the level of binding of the methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex with the test compound determined using standard techniques known in the art.

The present invention also provides a compound identified by the screening method of the present invention as being capable of modulating the interaction of the methyl-CpG-binding protein with the subunit of a nucleosome remodelling complex and the use of such compounds for therapeutic and/or prophylactic purposes, as herein described.

Pharmaceutical Compositions In yet another aspect of the present invention, there is provided a pharmaceutical composition comprising a compound which is capable of modulating the interaction of the methyl-CpG-binding protein to a subunit of a nucleosome remodelling complex, as herein described (also referred to herein as the "compound"), together with a pharmaceutically acceptable carrier, excipient, diluent and/or adjuvant.
Pharmaceutical compositions of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

As used herein, the term "pharmaceutically acceptable carrier' includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
Supplementary compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components:
a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid;
buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic 5 water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for 10 example, water, ethanol, polyol (for example, glycerol, propylene glycol, or liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion or by the use of surfactants. Prevention of the action of microorganisms can be achieved by 15 incorporation of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, or sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent 20 which delays absorption, for example, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions 25 are prepared by incorporating the compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously 30 sterile-filtered solution thereof.

Oral compositions generally comprise an inert diluent or an edible carrier.
For the purpose of oral therapeutic administration, the compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as aart of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurised container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished with nasal sprays or suppositories. The compounds can be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
For transdermal administration, the compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

In one embodiment, the compound is prepared with a carrier that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, coliagen, polyorthoesters, and polylactic acid.
Methods for preparation of such formulations will be apparent to those skilled in the art.
The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.
4,522,811.
It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein preferably refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred.
While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the present invention, the therapeutically effective dose can be estimated initially from a cell culture assay. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful doses in humans.
Levels in plasma may be measured, for example, by high performance liquid chromatography.

The skilled artisan will appreciate that a therapeutically effective amount of an compound according to the present invention (i.e., an effective dosage) may be influenced by a number of factors, such as the dosage and timing required to effectively treat a subject, the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, the degree of modulation required, the severity of the disease or disorder, previous treatments and other diseases present. The frequency of delivery of the compound can also be determined by the skilled addressee.

Where the compound is an antibody or a biologically active fragment thereof, the dosage may be 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg).
However, if the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg may be appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Methods of Therapeutic and/or Prophylactic Treatment It is another aspect of the present invention to provide a method of treating or preventing a disease or disorder associated with aberrant gene transcription, said method comprising administering to a patient in need thereof a pharmaceutically acceptable amount of a compound capable of modulating the interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex as hereinbefore described.

For example, the compound may inhibit the interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex or alternatively, the compound may enhance the interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex.

It will be understood by the skilled addressee that the methods of the present invention may be used to treat or prevent any disease or disorder which is associated with aberrant gene transcription, such as cancer, Fragile X
Syndrome, Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, Alpha-thalassemia/mental retardation syndrome, X-linked (ATRX), Beckwith-Wiedemann syndrome (BWS), Prader-Willi syndrome (PWS) and Angelman syndrome (AS).

Where the disease or disorder to be treated or prevented in accordance with the methods of the present invention is a cancer, it may include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head and neck cancer, leukaemia, lung cancer, lymphoma, melanoma, non-small-cell lung cancer, ovarian cancer, prostate cancer, testicular cancer, uterine cancer, cervical cancer, thyroid cancer, gastric cancer, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma, iastoma~-ependyr~ , ' ~e~ily ~m . , rr , gerria~eff fu~o~~, extracrania( cancer, Hodgkin's disease, leukaemia, acute lymphoblastic leukaemia, acute myeloid leukaemia, liver cancer, medulloblastoma, neuroblastoma, brain tumours generally, non-Hodgkin's lymphoma, osteosarcoma, malignant fibrous histiocytoma of bone, retinoblastoma, rhabdomyosarcoma, soft tissue sarcomas generally, supratentorial primitive neuroectodermal and pineal tumours, visual pathway and hypothalamic glioma, Wilms' tumour, acute lymphocytic leukaemia, adult acute myeloid leukaemia, adult non-Hodgkin's lymphoma, chronic lymphocytic leukaemia, chronic myeloid leukaemia, oesophageal cancer, hairy cell leukaemia, kidney cancer, multiple myeloma, oral cancer, pancreatic cancer, primary central nervous system lymphoma, skin cancer, small-cell lung cancer, among others. The cancer may be malignant or benign in nature.
In a preferred embodiment, the prophylactic or therapeutic method according to the present invention comprises the steps of administering an compound, as herein described, to a subject who has a disease, a symptom of disease or predisposition toward a disease associated with aberrant gene transcription, for the purpose to cure, heal alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of the disease, or the predisposition towards the disease.

In another preferred embodiment of the present invention, the prophylactic or therapeutic method comprises the steps of administering an compound, as herein described, to an isolated tissue or cell obtained from a subject who has a disease, a symptom of disease or predisposition toward a disease associated with aberrant gene transcription, as hereinbefore described, and reintroducing said tissue or cell into the subject for the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of the disease, or the predisposition towards the disease.

The "compound" according to the present invention includes, but is not limited to, small molecules, peptides, antibodies, ribozymes, and antisense oligonucleotides, as herein described. With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained 5 from the field of pharmacogenomics. "Pharmacogenomics", as used herein, preferably refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More preferably, the term refers to the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or 10 "drug response genotype"). Thus, another aspect of the present invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with an compound in accordance with the methods of the present invention, as herein described (for the purpose of modulating gene transcription in that individual), according to that individual's drug response genotype. Pharmacogenomics allows a 15 clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

If it is desirable to achieve an up-regulation of initiation of gene transcription, rate of 20 gene transcription, and/or gene transcription levels in a cell, several therapeutic and/or prophylactic approaches are available. In one preferred approach, the compound, as hereinbefore described, is administered to the subject and inhibits the interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex. For example, the compound may bind to the methyl-CpG binding protein and 25 inhibit its association (e.g., binding) with a subunit of the nucleosome remodelling complex or, alternatively, the compound may bind to a subunit of the nucleosome remodelling complex and inhibit its association (e.g. binding) with a methyl-CpG
binding protein. The compound is typically administered to the subject in need thereof, together with a pharmaceutically acceptable carrier, in an amount effective to up-30 regulate the initiation of gene transcription, rate of gene transcription, and/or gene transcription levels and thereby cure, heal alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of the disease, or the predisposition towards the disease.

35 In another approach, the compound for use in accordance with the methods of the present invention may be introduced into the subject by way of gene therapy;
that is, by expressing the compound endogenously in a cell of the subject by means of administering to the subject a nucleic acid molecule (e.g, DNA and/or RNA) which is capable of driving the expression of said compound. Thus, in a preferred embodiment of the present invention, there is provide a method of treating or preventing a disease or disorder associated with aberrant gene transcription, said method comprising administering to a subject in need thereof a nucleic acid molecule which is capable of expressing in a cell of the patient an compound that is capable of modulating the interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex as hereinbefore described.

Conditions in which gene transcription is repressed, and where it is therefore desirable to inhibit the interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex, may be identified by a skilled addressee by any or a combination of diagnostic or prognostic assays known in the art. For example, a biological sample obtained from a subject (e.g. blood, serum, plasma, urine, saliva, and/or cells derived therefrom) may be analysed for a surrogate marker of gene transcription, such as the concentration of a particular protein which is usually derived from the corresponding gene (e.g., via Western blotting, enzyme-linked immunosorbent assays, HPLC, etc). Alternatively, a skilled addressee may analyse gene expression directly by measuring the level of RNA in a biological tissue or cell derived from a subject using any or a combination of means known in the art (e.g. Northern blot analysis, reverse transcription-polymerase chain reaction, etc).

In a preferred embodiment of the present invention, the disease or disorder to be treated or prevented in accordance with the methods of the present invention includes Fragile X Syndrome, Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, Alpha-thalassemia/mental retardation syndrome, X-linked (ATRX), Beckwith-Wiedemann syndrome (BWS), Prader-Willi syndrome (PWS) and Angelman syndrome (AS) and various cancers, including, but not limited to bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head and neck cancer, leukaemia, lung cancer, lymphoma, melanoma, non-small-cell lung cancer, ovarian cancer, prostate cancer, testicular cancer, uterine cancer, cervical cancer, thyroid cancer, gastric cancer, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma, glioblastoma, ependymoma, Ewing's sarcoma family of tumours, germ cell tumour, extracranial cancer, Hodgkin's disease, .

leukaemia, acute lymphoblastic leukaemia, acute myeloid Ieukaemia, liver cancer, medulloblastoma, neuroblastoma, brain tumours generally, non-Hodgkin's lymphoma, osteosarcoma, malignant fibrous histiocytoma of bone, retinoblastoma, rhabdomyosarcoma, soft tissue sarcomas generally, supratentorial primitive neuroectodermal and pineal tumours, visual pathway and hypothalamic glioma, Wilms' tumour, acute lymphocytic leukaemia, adult acute myeloid leukaemia, adult non-Hodgkin's lymphoma, chronic lymphocytic leukaemia, chronic myeloid leukaemia, oesophageal cancer, hairy cell Ieukaemia, kidney cancer, multiple myeloma, oral cancer, pancreatic cancer, primary central nervous system lymphoma, skin cancer, small-cell lung cancer, among others. The cancer may be malignant or benign in nature.

If it is desirable to achieve down-regulation of gene transcription, rate of gene transcription and/or gene transcription levels, several therapeutic and/or prophylactic approaches are also available. For example, the compound may bind the methyl-CpG binding protein and promote or enhance its interaction (e.g., binding) with a subunit of the nucleosome remodelling complex or, alternatively, the compound may bind to a subunit of the nucleosome remodelling complex and promote or enhance its interaction with a methyl-CpG binding protein. The compound is typically administered to the subject in need thereof, together with a pharmaceutically acceptable carrier, in an amount effective to down-regulate the initiation of gene transcription, rate of gene transcription, and/or gene transcription levels and thereby cure, heal alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of the disease, or the predisposition towards the disease.

In another approach, the compound for use in accordance with the methods of the present invention may be expressed endogenously in a cell by means of the insertion, into the cell's genome, of a nucleic acid molecule (e.g, DNA and/or RNA) which is capable of driving the expression of said compound, as hereinbefore described.

Conditions in which gene transcription is active, and where it is desirable to promote or enhance the interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex so as to silence gene transcription, may be o w -identified by those skilled in the art by any or a combination of diagnostic or prognostic assays known in the art, as hereinbefore described.

In a preferred approach, the compound administered to a subject is a recombinant methyl-CpG-binding protein and/or subunit of a nuc(eosome remodelling complex identified by the aforementioned screening assays, which is/are capable of promoting or enhancing the interaction of an endogenous methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex, thus promoting gene silencing. Preferred embodiments of such compounds include methyl-CpG-binding proteins, and fragments and variants thereof, which are capable of binding native subunits of a nucleosome remodelling complex and thereby resulting in gene silencing. Alternatively, such compounds include one or more subunits of a nucleosome remodelling complex, and fragments and variants thereof, which are capable of binding native methyl-CpG-binding proteins and thereby resulting in gene silencing.

Alternatively, gene therapy may be employed to modulate the endogenous expression of a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex in a cell of a subject in need of such therapy, including, but not limited to, rats, mice, dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. For example, a retroviral vector that is capable of driving the expression of a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex, or a biologically active fragment thereof, in a cell may be administered to a subject for engineering cells in vivo to express the recombinant methyl-CpG-binding protein and/or subunit of the nucleosome remodelling complex in vivo. For overview of gene therapy, see, for example, Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, Strachan T. and Read A. P., BIOS
Scientific Publishers Ltd (1996).
Further, antisense, siRNA, ribozyme, and/or triple helix molecules that are capable of inhibiting the expression of and reducing the level of gene expression of a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex, as hereinbefore described, may also be used in accordance with the therapeutic and/or prophylactic methods of the present invention.

Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by aberrant gene expression is through the use of aptamer molecules specific for a methyl-CpG-binding protein and/or a subunit of a nucleosome remodelling complex, as hereinbefore described.

In conjunction with the treatment and/or prevention of diseases or conditions associated with aberrant gene expression in accordance with the methods of the present invention, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may also be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a therapeutic agent to modulate the interaction of a methyl-CpG-binding protein with a subunit of a nucleosome remodelling complex, as well as tailoring the dosage and/or therapeutic regimen of such treatment.

It would also be well appreciated by one skilled in the art that the methods of treatment hereinbefore described could be used in any number of combinations with each other, or with other treatment regimes currently employed in the art.

Examples of the procedures used in the present invention will now be more fully described. It should be understood, however, that the following description is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

EXAMPLES
1. Methods 1.1 Cell culture drup treatments.
Cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum and gentamicin solution at 37 C in a 5% CO2 atmosphere. Cells with TSA
or 5-azacytidine as previously described (EI-Osta et al. Mol. Cell. Biol. 22:1844-[2002], the contents of which are incorporated herein by reference).
1.2 Antibodies, immunoprecipitation and immunoblot analysis.
Antibodies against Brm, 0-tubulin (Santa Crux), acetylated histones H3 and H4, 5 HDAC1, Sin3a and histone H3 methylated at Lys4 (Upstate Biotechnology) were used for immunoprecipitation, ChIP and immunoblot analyses. Cells were lysed in 1% SDS, 10 mM EDTA and 50mM Tris-HCI (pH 8.1) with a cocktail of protease inhibitors at 4 C for 10 min. The lysate was precleared with protein A/G
agarose (Santa Cruz) for 30 min and subjected to immunoprecipitation overnight at 4 C.
10 Immunoprecipitations were carried out in dilution buffer containing 167 mM
NaCI
and subsequently washed in low-salt (150 mM NaCi) and high-salt (500 mM NaCI) solutions before elution. Immunocomplexes were collected with protein A/G
agarose beads, washed, size-fractionated by SDS-PAGE and transferred to nylon membrane using standard techniques. Approximately 1 mg of NIH3T3 nuclear 15 extract was fractionated on a 22-50% linear glycerol (5 ml) gradient.
Twenty-five 200 ml fractions were collected and 10% of each fraction was analysed by western blotting. Immunoprecipitated complexes were washed with buffer containing 40 mM
Tris (pH 8.0), 100 mM NaC1 and 0.5% Nonidet P-40.

20 1.3 Coinmunoprecipitation and GST pull-down assays.
To immobilize GST fusion proteins, approximately 200-500 g of induced bacterial extracts were incubated with 30 l of GST beads on ice for 30 min. Bound proteins were washed with buffer STE-100 (20 mM Tris-HCI (pH 7.6), 5 mM MgCI2i 100 mM
NaCi and 1 mM EDTA) supplemented with 0.5% Nonidet P-40 and 1% bovine 25 serum albumin. The washed beads were then blocked in 250 I of buffer STE-containing 1 mg/mI of uninduced bacterial extract, 1% bovine serum albumin, 0.5%
Carnation milk and 100 mg/mI ethidium bromide. Approximately 8 x 104 c.p.m. of in vitro-translated and 35S-labeled SWI/SNF subunits were added to immobilized GST
fusion proteins and incubated at 4 C for 12-46 hours. The beads were washed 30 three times with 300 l of STE-100 containing 1% bovine serum albumin and 0.5%
Carnation milk and then twice with STE-100 containing 150 mM NaCl. Bound proteins were then separated by SDS-PAGE and detected by autoradiography.

1.4 Bisulfite DNA seguencin_g.
35 Genomic DNA was prepared and subjected to bisulfite conversion in the presence of 0.3 M NaOH as previously described (EI-Osta et a/. MoL Cell. Biol. 22:1844-[2002], the contents of which are incorporated herein by reference). Bisulfite-treated DNA was desulfonated in 0.3 M NaOH at 37 C. After neutralizing it with ammonium acetate, DNA was prepared for PCR amplification and cloning with TOPO TA vector in accordance with the manufacturer's protocol (Invitrogen).

1.5 Immunofluorescence microscopy.
Immunofluorescence analysis was carried out on mouse NIH3T3 cells grown on slides and fixed with 4% formaldehyde as previously described (Wheatley et a!.
.10 Curr. Biol. 11:886-90 [2001], the contents of which are incorporated herein by reference). The following primary and secondary antibodies were used in these experiments: rabbit antibody to Brm (Abram) followed by chicken antibody to rabbit conjugated to Alexa 594 (Invitrogen); rabbit antibody to HDAC2 (Merck) followed by donkey antibody to goat conjugated to Alexa 488 (Invitrogen); goat antibody to Brm (Santa Crux) followed by donkey antibody to goat conjugated to Alexa 594 (Invitrogen); and rabbit antibody to MeCP2 (Upstate) followed by donkey antibody to goat conjugated to Alexa 488 (Invitrogen). Images were captured using a confocal microscope (Leica) and analyzed for colocalization over single optical sections.
1.6 siRNA knock-down and transfections.
siRNAs were synthesised and purified for Brm, MeCP2 and DNMTI using the Silencer siRNA Construction Kit (Ambion) in accordance with the manufacturer's instructions. Sense and antisense oligonucleotides (sequences available on request) were designed using the online Whitehead Institute siRNA selection program against RNA target sequences: Brm, nucleotide positions 1,770-1,792;
MeCP2, nucleotide positions 1,019-1,041; and DNMT1, nucleotide positions 4,036-4,058. Cells were transfected with siRNA using either oligofectamine or lipofectamine 2000 (Invitrogen) for as long as 3 days in Opti-MEMI
(invitrogen).
Control cells were transfected with siRNAs alone (minus oligofectamine vehicle) and without siRNAs (vehicle alone). The online Whitehead Institute siRNA
sdection program is available at http://jura.wi.mit.edu/biocfsiRNA/home.php.

1.7 RNA isolation and first-strand cDNA synthesis.
Poly(A)+ mRNA was prepared following the protocol recommended by the manufacturer (RNAture) and treated it with DNase (Ambion). Purified mRNA was reverse transcribed in TE buffer (10 mM Tris-HCI (pH 8.0) and 1 mM EDTA) using first-strand cDNA synthesis in accordance with the manufacturer's instructions (Life Technologies).
The primers used for cDNA amplification of the MDR1 amplicon had the nucleotide sequences: 5' AAGCCACGTCAGCTCTGGATA and 3' CGGCCTTCTCTGGCTTT
GT.

The primers used for cDNA amplification of the HPRT amplicon had the nucleotide sequences: 5' TGACACTGGCAAAACAATGCA and 3' GGTCCTTTTCACCAGCA
AGCT.

The primers used for cDNA amplification of the FMR1 amplicon had the nucleotide sequences: 5' GGAACAAAGGACAGCATCGC and 3' CTCTCCAAACGCAACTG
GTCT.

Resuspended DNA was amplified by PCR using the following MDRI primer sequences; ChIP region A (5' GTCCTGTAGTTATATGGATA and 3' CGGATTGACTGAATGCTGAT); ChIP region B(5' ACTTGCCCTTTCTAGAGAGG
and 3' CGGATTGACTGAATGCTGAT); ChIP region C (5' GGCCGGGAGCA
GTCATCTGT and 3' CTTCCTGTGGCAAAGAGAGC); ChIP region D (5' CCTGAGCTCATTCGAGTAGC and 3' GGAAGAAGATACTCCGACTT); ChIP
region E(5' AAAATTTCACGTCTTGGTGG and 3' ATCTGAAAGCCTGACACTTG);
ChIP region F (5' TTCCTGAACTTGGTCTTCAC and 3' GTCTAGATCTAACCCC
ACTT); ChIP region G (5' TCAGGAGCTCCTGGAGCAGC and 3' GGGCTC
AGAGAGCAGGTCCC); ChIP region H (5' GGGACCTGCTCTCTGAGCCC and 3' GTCTCCAGCATCTCCACGAA) and ChIP region 1(5' CAACTCTGCCTTCG
TGGAGA and 3' GAGCGCCCGCCGTTGATGCC).
The primer sequences for the PCR amplification of the TM3 region of the fragile X
mental retardation gene 1(FMR1) by ChIP were 5' CGCCCAAAATCTGGTGAGAG
and 3' AGTGGCAACCAGGGTGACC.

The primer sequences for the PCR amplification of the dCK gene by ChIP were 5' CTCCCAGCCCTCTTTGCC and 3' GCCTTGCGTCCCACATTT.

The primer sequences for the PCR amplification of the THBS1 by ChIP were 5' GGGCACCGACTTCTCTGAGA and 3' CGCGCAACTTTCCAGCTAG.
The primer sequences for the PCR amplification of the G6PDH gene by ChIP were 5' ACACGCTGTTTGTTGTGCTTG and 3' CACAATGACCTGGAGCATGG.

Gene expression was quantified using the ABI Prism 7700 Sequence Detection System. PCR amplification was carried out in 96-well optical plates with a 20 l reaction volume consisting of 5 pmol of forward and reverse primers, lx Platinum SYBR Green qPCR SuperMix-UDG and Rox Reference Dye (Invitrogen).
Reactions were first incubated for 2 min at 50 C and then for 10 min at 95 C
and then carried over 40-50 cycles at 95 C for 15 seconds and 60 C for 60 seconds.
1.8 ChIP and ChIP-ReChIP.
Cell treatment with 5-azacytidine and TSA, DNA sonication and ChIP were carried out as previously described (EI-Osta et al. Mol. Cell. Biol. 22:1844-57 [2002], the contents of which are incorporated herein by reference) for enrichment of MeCP2-HDAC corepressor complex on the ABCBI locus. Soluble chromatin fractions were resuspended in IP dilution buffer (0.01% SDS, 1.1% Triton X 100, 1.2 mM EDTA, 16,7 mM Tris-HCI (pH 8.1) and 167 mM NaCI) for immunoprecipitation as previously described for the methyl-CpG binding domain protein family (EI-Osta et al. Mol. Cell. Biol. 22:1844-57 [2002], the contents of which are incorporated herein by reference). ChIP analysis was carried out for Brm with protein A/G agarose beads, washed and the immunocomplexes separated from DNA by elution with 1%
SDS and 0.1 M NaHCO3. Resuspended DNA was amplified by PCR with the primers hereinbefore desribed and PCR amplifications were quantified as previously described for the ABCBI promoter (EI-Osta et 61. Mol. Cell. Biol.
22:1844-57 [2002]; El-Osta et al. Mol. Biol. Rep. 28:209-15 [2001] and El-Osta and Wolffe Biochem.Biophys. Res. Commun. 289:733-37 [2001], the contents of which are each incorporated herein by reference). The cycling parameters on a GeneAmp PCR System (PE Applied Biosystems) were as follows: 95 C for 60 seconds, followed by 32 cycles at 95 C for 60 seconds, 58 C for 60 seconds and 72 C for seconds, and an extension at 72 C for 10 min. Samples were size-fractionated by PAGE and stained with ethidium bromide. Quantitative analyses were carried out using Quantity One imaging and analysis software (Bio-Rad). Primer sequences for PCR amplification of FMRI, DCK, THBSI and G6PD are hereinbefore described.
PCR amplification was carried out in 96-well plates using 10-20 pmol of forward and reverse primers, lx Platinum SYBR Green qPCR SuperMix-UDC and Rox Reference Dye (Invitrogen). ABCBI and FMRI amplification reactions were incubated for 2 min at 50 C and then for 10 min at 98 C and carried over 40-50 cycles at 98 C for 20 seconds and 64 C for 40 seconds. To analyze the reciprocal association between Brm and MeCP2 immunoprecipitates, soluble chromatin fractions derived from cross-linked cells were divided into two aliquots. The first chromatin aliquot was immunoprecipitated with antibody to MeCP2, washed, the bound immune-DNA complexes released in 20 mM dithiothreitol solution for 30 min at 37 C and resuspended in one volume of IP dilution buffer for ReChIP with antibody to Brm. The second chromatin aliquot was treated identically, except that it was immunoprecipitated with antibody to Brm before ReChIP with antibody to MeCP2. Control ChiPs were also carried out to test antibody specificity with either a no antibody control or an irrelevant antibody control.

1.9 siRNA knock-down and ChIP assay.
CEM cells were seeded 16 hours before transfection at 40% confluence in Opti-MEMI media. Cells were transfected two to three times every 24 hours with 5 M
each of the MeCP2 or Brm siRNA constructs using 0.08 mg lipofectamine 2000 (Invitrogen) diluted in OptiMEMI media. Control cells were treated with 0.08 mg of lipofectamine 2000. Cells were collected 24 hours after the final transfection. ChIP
assays were carried out using 4-8 x 106 cells with either 6 pg of Brm (sc-6450) or l of MeCP2 and BAF57 antibodies. Soluble chromatin was immunopurified and real-time PCR analyses carried out essentially as described for the ChIP
platform described above. ABCBI ChIP primers amplified a region from +181 to +247 from the transcription start site.

2: Results 2.1 MeCP2 associates with the Brm SWI/SNF complex We investigated whether components of the SWI/SNF family associate with endogenous MeCP2. To define the components of the corepressor complex, we carried out glycerol-gradient sedimentation experiments using nuclear extract from NH3T3 cells. MeCP2 cofractionated with Brm, which is presumed to be part of the 5 larger SWI/SNF complex (Figure 1a). Western-blot analysis of nuclear extracts also showed that BAP57 and IN11 cofractionated with MeCP2, suggesting that MeCP2 associates in vivo with the Brm-containing SWI/SNF complex. The SWI/SNF
complex contains components of the Sin3a-HDAC corepressor complex. Therefore, we tested the MeCP2-Brm glycerol-gradient fractions for the presence of Sin3a and 10 HDAC2. Consistent with previous findings, both Sin3a and HDAC2 coeluted with MeCP2 and components of the Brm complex. To confirm this association, we carried out reciprocal immunopurifications using glycerol-gradient fractions followed by western-blot analysis using antibodies to MeCP2 and Brm (Figure 1 b).
Results of these analyses are consistent with the existence of a complex containing 15 MeCP2, Bun and BAF57.

To examine further whether MeCP2 directly interacts with Brm, we carried out glutathione S-transferase (GST) pull-down experiments using bacterially expressed MeCP2 and in vitro-translated recombinant SWI/SNF subunits. MeCP2 specifically 20 interacted with Brm and INI1 (Figure 1 c), consistent with the proposed existence of a soluble corepressor containing MeCP2 and the Brm-containing SWI/SNF
complex. To validate these protein interaction results, we used immunofluorescence to investigate the localization of Brm and MeCP2 in NIH3T3 cells. Staining for the protein determinants showed considerable, but not precise, 25 overlap for Brm and HDAC2 and colocalization of Brm and MeCP2 (Figure 1d).
We counterstained chromatin with DAPI to confirm that the colocalized signals were present in the nucleus. These results, together with the coelution data, suggest that a substantial proportion of MeCP2 stably associates with Brm in solution and that the overlap between the relevant species is substantial, though incomplete.
The methylated gene ABCBI (also called MDR1; OMIM 171050) is enriched for the MeCP2-HDAC repressor complex, and transcriptional activation correlates with a change in MeCP2 association. We reasoned that 5-azacytidine-induced demethylation should result in the release of MeCP2 and Brm complex from the ABCBI promoter. We used inhibitors of DNA methyltransferase (5-azacytidine) and HDAC (trichostatin A, TSA) to bring about changes in chromatin remodeling and examined resulting protein expression. Treatment with 5-azacytidine or TSA
did not alter protein expression levels (Figure le). Expression of ABCBI is inversely correlated with the methylation status of the promoter. CEM-CCRF
cells do not express ABCBI, and its silencing is directly linked with promoter methylation. In contrast, the active gene is hypomethylated in CEM-A7R cells, as determined by RT-PCR (Figure 1 f). Changes in gene expression are specified by CpG methylation of the ABCB1 promoter rather than global changes in 2 protein expression. MeCP2 and Brm are comparably expressed, as measured by western blotting in both CEM cell lines (Figure If). Using a modified version of the co-immunoprecipitation technique, we crosslinked cells with formaldehyde, lysed them and carried out immunoprecipitations for MeCP2 and Brm followed by western-blot analysis. Antibody to Brm immunoprecipitated MeCP2 from soluble cross-linked protein extracts (Figure Ig); conversely, antibodies to MeCP2 immunoprecipitated Brm. We then attempted to determine whether an interaction between Brm and MeCP2 could be observed using a conventional co-immunoprecipitation approach.
MeCP2 was present in immunoprecipitates of antibody to Brm, as shown by immunoblot assay using an MeCP2-specific antibody (Figure 1g). In a reciprocal assay, MeCP2 was co-purified from Brm isolates, suggesting that the two corepressors interact specifically, consistent with the existence of a soluble complex containing MeCP2 and Brm (Figures la-d).

2.2 SWI/SNE subunits are presenf on MeCP2-repressed genes Having shown that Brm and MeCP2 could form a complex, we next tested whether Brm is associated with hypermethylated ABCB1. In all experiments, we used input and unbound chromatin controls, including specificity or no-antibody controls, and compared these with bound Brm chromatin fractions. We quantified ChIP binding by two-fold titration of immunoprecipitated DNA and analyzed it by PCR. ChIP
shows enrichment of Brm on hypermethylated ABCBI, and 5-azacytidine-induced demethylation remodelled recruitment (Fig. 8). We previously showed in the same cell line that MeCP2 was not localized to the unmethylated gene deoxycytidine kinase (DCK), suggesting that binding is dependent on CpG methylation. These experimental findings and other results from our laboratory support a model in which the Brm-containing MeCP2 repressor complex is associated with the methy-lated ABCB1 promoter.

The capacity of Brm and MeCP2 to bind to the methylated ABCBI promoter, coupled with their release mediated by DNA methylation inhibitors led us to examine whether the binding pattern of MeCP2 specified on the ABCB9 CpG island closely resembled that of Brm. We modified our ChIP platform to map the binding patterns of Brm and MeCP2 with high resolution (Fig. 2a) and analyzed the change in CpG methylation of the ABCBI promoter from the same cells. MeCP2 was associated with the hypermethylated promoter, and its pattern of association was similar to that of Brm (Fig. 2b). Treatment with TSA did not substantially alter MeCP2 or Brm association, but 5-azacytidine-induced demethylation consistently released Brm and MeCP2 from ABCB1 chromatin (Fig. 2c). The combination of 5-azacytidine and TSA did not potentiate the release of MeCP2 and Brm more than treatment with 5-azacytidine alone.

We carried out bisulfite sequencing in the absence and the presence of TSA and 5-azacytidine to verify that hypomethylation of the ABCBI promoter correlates with the release of Brm and MeCP2.

The inhibition of DNA methylation by 5-azacytidine significantly reduced CpG
methylation (P < 0.001; Fig. 2d). Consistent with previous observations, TSA
did not markedly alter the methyl-CpG pattern, and the extent of hypomethylation was not potentiated by combined treatment with 5-azacytidine and TSA (EI-Osta et a/.
Mol. Cell. Biol. 22:1844-57 [2002], the contents of which are incorporated herein by reference). Notably, the Brm and MeCP2 complex seems to be released from the target promoter after treatment with 5-azacytidine, suggesting that induced demethylation interferes with the recruitment of Brm and MeCP2.

2.3: Defining the Brm-containing MeCP2 complex by ChIP-ReChIP
Our results support a model in which Brm and MeCP2 complexes jointly operate to silence ABCB1 expression. To verify binding to the promoter, we carried out primary ChIP followed by a secondary immunoprecipitation (ReChIP) to resolve whether the determinants are enriched on similar regions of the ABCB1 promoter or whether the two complexes are recruited independently of each other. In the ChIP-ReChIP technique, we divided soluble chromatin fractions derived from cross-linked cells into two aliquots. We immunoprecipitated the first chromatin aliquot with antibody to MeCP2, washed it, released the bound immune-DNA complexes by elution and prepared them for immunopurification (or ReChIP) with antibody to Brm. We treated the second chromatin aliquot identically, except that we immunoprecipitated it with antibody to Brm before carrying out ReChIP with antibody to MeCP2 (Fig. 3a). We amplified precipitated DNA by PCR using amplimers that recognize the region from +296 to +595 of the ABCBI promoter relative to the transcription initiation (+1) start site. The ABCBI promoter has a weak association with MeCP2 in drug-resistant CEM-A7R cells, which correlates with the hypomethylated CpG status, hyperacetylation of histones and transcriptional activity of the gene in these cells (EI-Osta et al. Mol. Cell.
Biol.
22:1844-57 [2002], the contents of which are incorporated herein by reference) (Fig. 3b). The ReChIP experiments using reciprocal antibodies showed binding of endogenous Brm on MeCP2 immunoprecipitates and MeCP2 association on Brm immunoprecipitates (Fig. 3c,d). This binding was specific because ReChIP
experiments using an irrelevant antibody control specific for [3-tubulin did not immunoprecipitate Brm or MeCP2 fractions. To exclude the possibility that Brm recruitment to the ABCBI promoter was independent of MeCP2 recruitment, we carried out ChIP assays on CEM A7R cells. Primary ChIP experiments showed that HDAC1 was not associated with hypomethylated ABCBI chromatin in CEM-A7R
cells but was associated with the methylated gene in CEM-CCRF cells.
Furthermore, ReChIP assays (Fig. 3e) showed that Brm was not associated with hypomethylated ABCB1 in CEM-A7R cells.

To validate the finding that Brm and HDAC1 binding was specific to the methylated ABCBI promoter, we used antibodies to acetylated histone H3 in the primary ChIP
to assess directly the transcriptional activity of ABCBI and serve as a control for specificity of protein binding to active chromatin. ReChIP experiments with Brm and HDAC1 did not reimmunoprecipitate hyperacetylated histone H3, suggesting that the binding of Brm and HDAC1 on the methylated ABCBI promoter in CEM-CCRF
cells was specific (Fig. 3e). ChIP showed a differential association for Brm and BAF57 in both cell lines, as quantified by real-time PCR, and illustrated the strong correlation for Brm and BAF57 binding on the methylated ABCB1 promoter in CEM-CCRF cells (Fig. 3f). To show that the Brm-containing MeCP2 complex is part of a larger corepressor network, we repeated the ChIP-ReChIP experiments using antibodies specific for Sin3a and HDAC1 in addition to MeCP2 and Brm (Fig.
3g,h).

These experiments confirmed that the MeCP2 corepressor complex was enriched on the hypermethylated promoter with Brm, Sin3a and HDAC1. These results demonstrate a multifaceted association between MeCP2 and Brm-containing SWI/SNP on the hypermethylated ABCBI locus (Fig. 9).
MeCP2 is not associated with the unmethylated DCK promoter sequence in CEM-CCRF cells (see El-Osta et al. Mol. Biol. Rep. 28:209-15 [2001]). ChIP-ReChIP
analysis of unmethylated DCK did not immunoprecipitate Brm or MeCP2, consistent with the hypomethylation status of the promoter (Fig. 4a). To determine whether the MeCP2-Brm silencing complex could target other methylated sequences, we tested thrombospondin-I (THBSI), a matrix glycoprotein that is transcriptionally repressed by hypermethylation in CEM-CCRF cells. Antibodies to Brm and MeCP2 immunopurified THBS1 chromatin, suggesting that both complexes were recruited (Fig. 4b). Similarly, ReChIP analysis of primary eluted MeCP2 and Brm fractions confirmed that Brm and MeCP2 were localized on the THBSI promoter. These results show that assembly of MeCP2 and Brm does not target methylated ABCBI in CEM-CCRF cells and suggests that the repression complex may bind more broadly to methylated sequences.

2.4: SW!/SNF function is required for MeCP2 association To examine the functional importance of SWUSNF factors on ABCB1 silencing, we constructed small molecule inhibitors to downregulate expression of specific genes by introducing homologous double-stranded RNA (siRNA). Knock-down of DNMTI by RNA interference reactivated ABCBI expression (Fig. 5a), indicating that our transcriptional model of ABCBI repression is dependent on DNA
methylation. To test the hypothesis that Brm is a component of the MeCP2 repression complex, we transfected CEM-CCRF cells with siRNA targeting either Brm or MeCP2. Transfection of CEM-CCRF cells with no siRNA (vehicle only) did not cause any transcription, whereas knock-down of MeCP2 reactivated expression of methylation silenced ABCBI (Fig. 5b). We obtained similar results when Brm expression was targeted by siRNA (Fig. 5c). These results suggest that MeCP2 is part of a corepressor complex involving Brm. To assess whether MeCP2 is functionally required for stable Brm association, we transfected cells with specific siRNA molecules and examined binding to relevant genomic regions of the ABCB1 promoter. We also carried out the reciprocal experiment for Brm to examine the association of MeCP2 by ChIP. Knock-down of Brm by siRNA substantially reduced enrichment of both determinants on methylated ABCBI (Fig. 5d). Converse experiments using MeCP2 siRNA showed a similar association between MeCP2 and Brm (Fig. 5e). We also observed considerable changes in BAF57 binding on 5 the ABCBI promoter using our siRNA strategy (Fig. 51). These data suggest that the chromatin determinants are part of a corepressor complex involved in gene silencing.

2.5: MeCP2-containing SWI/SNF subunits determine FMRI silencinq 10 To confirm that Brm assembly with the MeCP2 complex was not unique to ABCB1 or THBSI in cancer cells, we examined the association of these chromatin determinants at methylated FMRI in a model of fragile X mental retardation (OMIM
3009550). Fragile X syndrome is the most commonly inherited mental retardation disorder and is caused by the expansion and methylation of a polymorphic CCC
15 triplet repeat at the 5' untranslated end of FMRI. The molecular mechanisms underlying the specificity of FMRI silencing are unknown.

We carried out ChIPs to determine whether MeCP2 assembles on repressed FMRI
chromatin. We used the X-linked gene glucose 6-phosphate dehydrogenase 20 (G6PD) as an internal control (Fig. 6a). We used cell line models of fragile X
syndrome in which FMRI is transcriptionally active in normal (C49) cells and silent in fragile X RJKI412 (FRAXA) cells (Fig. 6b). ChIP analysis indicated a marked difference in Brm and MeCP2 association with FMR1 chromatin in the different cell lines (Fig. 6c). FMR1 is transcriptionally active in normal cells, correlating with the 25 reduction in association of the corepressor complex. Notably, MeCP2 and Brm are assembled on the FMRI promoter in FRAXA cells, suggesting that these components are involved in FMRI silencing (Fig. 6d). To delineate the mechanism of methylation-mediated repression, we treated normal and FRAXA cells with 5-azacytidine and found that the demethylating agent could derepress silent FMR1 in 30 FRAXA cells (Fig. 6e). Because the mechanism underlying 5-azacytidine reactivation of FMR1 is unknown, we assayed for the specificity of corepressor binding on FMRI and found, by ChIP that Brm and MeCP2 were released from the FMRI promoter after treatment of FRAXA cells with 5-azacytidine (Fig. 6f).

35 To assess the functional relevance of MeCP2 and Brm on FMR1, we examined whether RNA interference could alleviate silencing. Knockdown of Brm and MeCP2 by siRNA reactivated FMRI transcription in FRAXA cells, suggesting that the determinants are part of the repressive silencing complex (Fig. 6g). Double knock-down experiments with MeCP2 and Brm resulted in a further increase in mRNA
abundance, suggestive of a tight cooperation between MeCP2 and Brm corepressor complexes. ChIP analysis showed that BAF57 was also associated with the FMRI promoter on the repressed gene in FRAXA cells (Fig. 6h).

These results indicate, for the first time to our knowledge, that the MeCP2-containing Brm-SWI/SNF complex silences transcription on methylated-repressed genes in distinct disease models.

2.6: Distinct histone modifications associated with FMRI chromatin Having shown that MeCP2 and Brm cooperate to silence FMRI, we sought to understand the chromatin landscape of the FMR1 promoter. We compared the binding of the histone methyltransferase Set7 and methylation of histone H3 at Lys4, together with acetylation of histones H3 and H4 and HDAC1, in normal (Fig.
7a,c) and FRAXA (Fig. 7b,d) cells. Chromatin localization was most notable for Set7, methylation of histone H3 at Lys4 and enrichment of acetylated histones on the active FMRI promoter in normal cells. In contrast, these chromatin determinants and modifications were under-represented in FRAXA cells.
Furthermore, the FMR1 promoter in FRAXA cells was enriched with HDAC1, further supporting our model of silencing mediated by the MeCP2 remodelling complex. To confirm the specificity of binding, we also examined chromatin binding on the active G6PD allele in normal and FRAXA cells. G6PD was enriched with methylation of histone H3 at Lys4, Set7 and acetylated histones H3 and H4 in both normal (Fig. 7a,c) and FRAXA (Fig. 7b,d) cells. In contrast, association of HDAC1, MeCP2 and Brm was substantially weaker. These results indicate that the MeCP2-containing remodelling complex is enriched on deacetylated chromatin, a hallmark of silent genes.

Claims (27)

1. A method of modulating gene transcription in a cell, the method comprising exposing the cell to an exogenous compound capable of modulating an interaction of a methyl-CpG-binding protein with one or more subunits of a nucleosome remodelling complex.

2. The method according to claim 1, wherein the methyl-CpG-binding protein is MeCP2.

3. The method according to claim 1 or claim 2, wherein the nucleosome remodelling complex is an ATP-dependent SWI/SNF nucleosome remodelling complex.

4. The method according to claim 1 or claim 2, wherein the one or more subunits of the nucleosome remodelling complex comprises Brm, PRMT, Rb protein, BAF57 or INI1.

5. The method according to claim 1 or claim 2, wherein the compound is capable of modulating an interaction of the methyl-CpG-binding protein with Brm and BAF57.

6. The method according to any one of claims 1 to 5, wherein the compound inhibits the interaction of the methyl-CpG-binding protein with one or more subunits of the nucleosome remodelling complex.

7. The method according to any one of claims 1 to 5, wherein the compound enhances the interaction of the methyl-CpG-binding protein with one or more subunits of the nucleosome remodelling complex.

8. The method according to any one of claims 1 to 7, wherein the methyl-CpG-binding protein and the one or more subunits of a nucleosome remodelling complex are associated with a region of a nucleic acid molecule.

9. The method according to claim 8, wherein the region is a regulatory region of a DNA molecule.

10. The method according to claim 9, wherein the regulatory region is selected from the group consisting of a promoter, enhancer, insulator, silencer, or a locus control region.

11. The method according to claim 10, wherein the promoter is selected from the group consisting of the ABCB1 promoter, the THBS1 promoter and the FMR1 promoter.

12. A method of treating or preventing a disease or disorder associated with aberrant gene transcription, the method comprising administering to a patient in need thereof a pharmaceutically effective amount of a compound capable of modulating the interaction of a methyl-CpG-binding protein with one or more subunits of a nucleosome remodelling complex.

13. The method according to claim 12, wherein the methyl-CpG-binding protein is MeCP2.

14. The method according to claim 12 or claim 13, wherein the nucleosome remodelling complex is an ATP-dependent SWI/SNF nucleosome remodelling complex.

15. The method according to claim 12 or claim 13, wherein the one or more subunits of a nucleosome remodelling complex comprises Brm, PRMT, Rb protein, BAF57 or INI1.

16. The method according to claim 12 or claim 13, wherein the compound is capable of modulating an interaction of the methyl-CpG-binding protein with Brm and BAF57.

17. The method according to any one of claims 12 to 16, wherein the compound inhibits the interaction of the methyl-CpG-binding protein with the one or more subunits of the nucleosome remodelling complex.

18. The method according to any one of claims 12 to 16, wherein the compound enhances the interaction of the methyl-CpG-binding protein with the one or more subunits of the nucleosome remodelling complex.

19. The method according to any one of claims 12 to 18, wherein the disease or disorder is selected from the group consisting of colon cancer, liver cancer, lung cancer, ovarian cancer, Wilms' tumour, Beckwith-Wiedemann syndrome, Prader-Willi syndrome, Angelman syndrome, Albright hereditary osteodystrophy, pseudohypoparathyroidism type Ia, pseudohypoparathyroidism type Ib, transient neonatal diabetes mellitus, Fragile X Syndrome mental retardation, systemic lupus erythematosus, Immunodeficiency, centromeric instability and facial anomalies (CIF) syndrome and Alpha-thalassemia/mental retardation syndrome, X-linked (ATRX).

20. A method of screening for a compound capable of modulating nucleosome remodelling or gene transcription in a cell, the method comprising exposing the cell to a test compound, identifying an interaction of a methyl-CpG-binding protein with one or more subunits of a nucleosome remodelling complex in the presence of the test compound and comparing the interaction to an interaction of the methyl-CpG-binding protein with the one or more subunits of the nucleosome remodelling complex in the absence of the test compound.

21. The method according to claim 20, wherein the methyl-CpG-binding protein is MeCP2.

22. The method according to claim 20 or claim 21, wherein the nucleosome remodelling complex is an ATP-dependent SWI/SNF nucleosome remodelling complex.

23. The method according to claim 20 or claim 21, wherein the one or more subunits of the nucleosome remodelling complex comprises Brm, PRMT, Rb protein, BAF57 or INI1.

24. The method according to claim 20 or claim 21, wherein the compound is capable of modulating an interaction of the methyl-CpG-binding protein with Brm and BAF57

25. The method according to any one of claims 20 to 24, wherein the compound inhibits the interaction of the methyl-CpG-binding protein with one or more subunits of the nucleosome remodelling complex.

26. The method according to any one of claims 20 to 24, wherein the compound enhances the interaction of the methyl-CpG-binding protein with one or more subunits of the nucleosome remodelling complex.

27. A compound identified by the screening method according to any one of claims 20 to 26 as being capable of modulating the interaction of the methyl-CpG-binding protein with the subunit of a nucleosome remodelling complex.