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J. Biol. Chem., Vol. 275, Issue 45, 34963-34967, November 10, 2000

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The Minimal Repression Domain of MBD2b Overlaps with the Methyl-CpG-binding Domain and Binds Directly to Sin3A^*

Joern Boeke, Ole Ammerpohl, Simone Kegel, Udo Moehren, and Rainer Renkawitz

From the Genetisches Institut der Justus-Liebig-Universität Gießen, Heinrich-Buff-Ring 58, D-35392 Gießen, Germany

Received for publication, July 6, 2000, and in revised form, August 18, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Different mechanisms mediating methylation-dependent repression have been demonstrated. Two of these mechanisms play a role in the context of the granulocyte/macrophage-specific lysozyme gene: direct interference with DNA binding of the transcription factor GA-binding protein and deacetylation of histones. Besides enhancement in the unmethylated state, and transcriptional repression upon DNA methylation, the lysozyme downstream enhancer confers tissue-specific demethylation. Because both demethylation activity and repression ability have been attributed to the methyl-CpG-binding domain-containing protein MBD2, we analyzed this protein. The short form MBD2b binds to the methylated lysozyme enhancer and mediates transcriptional repression. MBD2b is capable of binding to the transcriptional repressor Sin3A. The interaction domain of Sin3A required for binding to MBD2b contains the paired amphipathic helix 3. We identified a minimal functional domain that confers both transcriptional repression as well as the interaction to Sin3A. In contrast to the functionally related proteins MeCP2 and MBD1, the repression domain of MBD2b overlaps with the methyl-CpG-binding domain.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cytosine methylation of CpG dinucleotides is found at most CpGs in the genomes of vertebrates. In the case of the human genome, about 85% are methylated, whereas the majority of the nonmethylated CpG elements are found in so called CpG islands (for review see Refs. 1 and 2). Although higher eukaryotic genomes without CpG methylation are known (3, 4), methylation of CpGs is essential for mouse development. Mice lacking the functional DNA methyltransferase required for maintenance methylation die early in embryonic development (5-7). An important connection between methylation of tumor suppressor genes and cancer progression has also been shown (8, 9). In many of the cases analyzed, transcriptional repression was functionally connected with the state of methylation of the gene or of the promoter (for review see Ref. 1). Gene repression and imprinting is another example of the impact of methylation and its functional connection to repression (for review see Ref. 10). Furthermore, differential methylation and demethylation has been shown for many tissue-specific genes. During embryogenesis, housekeeping genes remain unmethylated, whereas tissue-specific genes become methylated. Transcriptional induction of tissue-specific genes is correlated with demethylation (for review see Ref. 11). Different mechanisms mediating methylation-dependent repression have been demonstrated. One mechanism is by directly interfering with the DNA binding of transcription factors (for review see Ref. 12). Another mechanism involves deacetylation of histones, which leads to transcriptional repression (12). That both of these mechanisms play a role in the context of a single gene has been shown for the granulocyte macrophage-specific lysozyme gene (13). Furthermore, mechanisms of methylation-dependent repression that do not involve histone deacetylation have also been demonstrated (14, 15).

A connection between proteins with methyl-CpG-binding domains and gene repression has been documented for the methyl-CpG-binding domain (MBD)¹-containing protein MBD2. This factor can interact with the Mi-2/NuRD histone deacetylase complex without being an integral component of this complex (16). Furthermore, MBD2 together with histone deacetylases, HDAC1, HDAC2, and RbAp46/48 are components of the MeCP1 complex (17). These connections with histone deacetylases are functionally supported because MBD2-mediated repression is sensitive to trichostatin A, an inhibitor of histone deacetylases (17). Repression and direct binding to the Sin3A histone deacetylase complex has been demonstrated for MeCP2, another methyl-CpG-binding protein (reviewed in Ref. 12). Here we wanted to identify a transcriptional repression domain (TRD) within MBD2b, an N-terminal truncation of MBD2. In addition we asked whether binding of Sin3A plays a role in the transcriptional repression mediated by MBD2. We determined that MBD2b binds to the methylated lysozyme enhancer sequence. Furthermore, we identified a minimal functional domain of MBD2, which confers transcriptional repression, binds to Sin3A, and overlaps with the methyl-CpG-binding domain.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmids-- For expression of GST-MBD2b fusions in bacteria, pGEX-2T MBD2b was generated by in-frame insertion of the Cfr9I/EcoRI fragment from pcDNA3.1 MBD2b (generously provided by Dr. M. Szyf) into the BamHI site of pGEX-2T (Amersham Pharmacia Biotech). The C-terminal deletions of GST-MBD2b were created by digestion of pGEX-2T MBD2b with EcoRI and the internal sites with HindIII (aa 1-211), NcoI (aa 1-163), Acc65I (aa 1-45), and AccI (aa 1-27), followed by religation. The N-terminal deletions of GST-MBD2b were cloned by cutting pGEX-2T MBD2b with BamHI and the internal sites with Acc65I (aa 45-262), NcoI (aa 163-262), and HindIII (aa 211-262), and religation. The GST fusion of the internal fragment of MBD2b (aa 163-211) was created by excision of the NcoI/HindIII fragment of pGEX-2T MBD2b and in-frame insertion into pGEX-2T digested with BamHI/EcoRI. The GST fusion of the internal fragment of MBD2b (aa 27-82) was generated by polymerase chain reaction using Pfu polymerase (Stratagene) and specific primers using pGEX-2T MBD2b as a template. GST fusions of mSin3A were described previously (18).

For expression of lexA-MBD2b fusions in yeast, pEG202 MBD2b was constructed by excision of the Cfr9I/XhoI fragment of pcDNA3.1 MBD2b and in-frame insertion into pEG202 digested with EcoRI and XhoI. The VP16 fusion of mSin3A (aa 54-724) in pVP2 was kindly provided by A. Baniahmad.

Mammalian expression vectors for Gal-MBD2b, Gal-MBD2b (aa 145-262), Gal-MBD2b (aa 163-262), and Gal-MBD2b (aa 27-82) were created by excision of the corresponding MBD2b sequences out of pGEX-2T MBD2b or pGEX-2T MBD2b (aa 27-82) and the in-frame insertion into pABGal94 linker (19).

Cell Culture and Transfection-- HeLa and CV1 cells were grown at 37 °C under 5% CO₂ in Dulbecco`s modified Eagle's medium plus10% fetal calf serum and transfected using the calcium phosphate method (20). Transient transfection of HeLa cells was carried out using 3 pmol of reporter plasmid, ptkCATH/N, or pHS3.2-tkCATH/N (21) (either mock methylated or methylated with M.HpaII methylase) and 1.5 pmol of pcDNA3.1 MBD2b expression vector or the empty vector. For delineation of the repression domain, CV1 cells were transfected using 1.5 pmol of 17-mer 6xs upstream activating sequence tkCATH/N reporter (22) and 0.1-3.0 pmol of Gal-MBD2b fusion expression plasmids or the empty vector. Cells were harvested 36-48 h after transfection and CAT assays were carried out as described before (23).

GST Pull-down of in Vitro Translated Proteins-- GST and GST fusion proteins were expressed in Escherichia coli BL21. GST pull-downs were carried out essentially as described earlier (24). Bacteria were induced with 0.2 mM isopropyl-D-thiogalactopyranoside for 3 h at 20 °C. Recombinant proteins were purified with glutathione-Sepharose beads (Amersham Pharmacia Biotech) and analyzed on SDS-polyacrylamide gel electrophoresis to normalize protein amounts. Equivalent amounts of GST fusion proteins were incubated with [³⁵S]methionine-labeled mSin3A or hMBD2b proteins, produced by the T7/T3 TNT-coupled transcription/translation system (Promega) in 200 µl of binding buffer (100 M NaCl, 20 mM Tris-HCl, (pH 8.0), 1 mM EDTA, 0.5% Nonidet P-40, 5 µg of ethidium bromide, 100 µg of bovine serum albumin). After 0.5 h of incubation at room temperature, the beads were washed eight times with 1 ml of binding buffer without ethidium bromide and bovine serum albumin. The bound proteins were eluted with SDS sample buffer, fractionated on SDS-polyacrylamide gel electrophoresis, and, after treatment with sodium salicylate, visualized by fluorography. Binding efficiency was quantified by densitometry and analysis with the TINA raytest program. The percentage of binding was calculated relative to the input after subtraction of the GST signal. Yeast two-hybrid experiments were performed as described previously (25, 26) using MBD2b f.l as bait (fused to the DNA-binding domain of lexA) and mSin3A as prey (fusion to the VP-16 activation domain).

Gel Mobility Shift Assays-- GST fusion proteins expressed in bacteria were purified with glutathione-Sepharose beads and eluted using 10 mM reduced glutathione in gel shift binding buffer (10 mM Tris-HCl (pH 8.0), 3 mM MgCl₂, 50 mM NaCl, 0.1 mM EDTA, 0.1% Nonidet P-40, 2 mM dithiothreitol, 5% glycerol, and 0.4 mg/ml bovine serum albumin) (27). Mobility shift assays were performed using equivalent amounts of recombinant protein and ³²P radiolabeled HS3.2 fragment (21).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

MBD2b Binds to the Methylated Mouse M-lysozyme Downstream Enhancer-- The M-lysozyme gene is inactive in nonmyeloid cells as well as in myeloid precursor cells but is activated during granulocyte/macrophage differentiation. The downstream enhancer (HS3.2) is methylated in inactive cell types and is demethylated during differentiation (28-30). Recently we have shown that repression of the mouse M-lysozyme gene involves both hindrance of enhancer factor binding to the methylated enhancer and histone deacetylation (13). For the HS3.2 enhancer several functions have been determined and at least partially characterized. Besides enhancement in the unmethylated state, repression and induction of demethylation have also been demonstrated (21). Both demethylation activity and repression ability have been attributed to one of the MBD containing proteins, MBD2. MBD2 mediates transcriptional repression when tested with a reporter gene containing a methylated artificial CpG cluster proximal to the promoter (17). For MBD2b, which shows a 152-amino acid N-terminal truncation in comparison with MBD2, a demethylation activity has been shown (31). Therefore, we analyzed whether MBD2b is able to bind to the methylated HS3.2 enhancer and whether binding mediates repression. We expressed MBD2b and truncated mutants as GST fusion proteins and performed bandshift experiments with methylated as well as unmethylated HS3.2 DNA (Fig. 1). Clearly there is a methylation-dependent binding of MBD2b and two of the truncated MBD2b derivatives. This specificity of binding for methylated DNA was seen with a wide range of protein concentrations. Deletion of a C-terminal part of the methyl-CpG-binding domain (MBD2b 1-45) leads to loss of DNA binding.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   MBD2b binds to the methylated HS 3.2 fragment. GST fusion proteins of MBD2b containing the intact MBD but not GST alone form a complex with the 32P-labeled HS 3.2 oligonucleotide when methylated (+CH3) but not with the unmethylated fragment (CH3). Neither GST alone nor the GST-MBD2b fusion (aa 1-45) with a deletion within the MBD was able to bind to this fragment.

If this in vitro DNA binding of MBD2b is of functional relevance, one would predict that expression of MBD2b would increase repression of an HS3.2-containing reporter. Therefore, we placed the HS3.2 fragment upstream of a tkCAT reporter and transfected this construct after methylation with the M.HpaII methylase into HeLa cells.

Negative control transfections show a minimal repressive effect by MBD2b on the unmethylated tkCAT reporter (Fig. 2, lanes 1 and 2). Methylation of the reporter shows a significant effect on repression because of many methylated CpG/HpaII sites throughout the reporter plasmid. Interestingly the repressed activity is not changed by MBD2b (lanes 3 and 4). The presence of the HS3.2 enhancer leads to strong enhancement (not shown), which is set to 1 and not changed by MBD2b (lanes 5 and 6). Methylation reduces the enhancement (about 8-10-fold repression; lanes 5 and 7), whereas MBD2b significantly increases the repression of the enhancer more than 60-fold (Fig. 2, lane 8).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   MBD2b increases repression on the methylated HS 3.2-tkCAT reporter. HeLa cells were transfected with the tkCAT or HS 3.2tkCAT reporter either after methylation with M.HpaII methylase (CH3) or after mock methylation (). Cotransfections were carried out using equal amounts of pcDNA3.1 MBD2b or the empty vector. Fold repression on both reporter constructs was calculated relative to the CAT activity after transfection of the empty vector and unmethylated reporter.

MBD2b Binds Directly to the Paired Amphipathic Helix 3 Domain of Sin3A-- MBD2 has been shown to be associated with histone deacetylases in the MeCP1 repressor complex (17, 32). Histone deacetylases have been found to be components of two major complexes: the Sin3A/HDAC complex and the Mi-2/NuRD complex (27, 33). Although it is not an integral part of the Mi-2/NuRD complex, MBD2 has been shown to interact in vitro with this complex. We wanted to know whether this is the only histone deacetylase complex MBD2 that can bind to or whether binding to Sin3A may also be possible. Therefore, we carried out GST pull-down assays to test for an interaction between MBD2b and Sin3A. The GST-MBD2b fusion protein binds in vitro translated Sin3A (Fig. 3B). Furthermore, a GST-Sin3A fusion also binds in vitro translated MBD2b (Fig. 3B). Then we asked whether a particular region of Sin3A mediates the interaction with MBD2b. Several truncations of the GST-Sin3A fusion were tested for interaction with in vitro translated MBD2b (Fig. 3A). Specific binding could be seen for the Sin3A truncation containing amino acids 404-545. This region contains the paired amphipathic helix 3 domain, which has been shown to mediate different protein-protein interactions (18). Because these pull-down assays only demonstrate a potential interaction tested in vitro, we wanted to examine a MBD2b/Sin3A interaction within a living cell. Therefore, we utilized the yeast two-hybrid assay designed such that interaction of Sin3A with the DNA bound lexA-MBD2b fusion would result in gene activation mediated by the VP16-Sin3A fusion. Negative controls (lexA or VP16) showed no gene activation, as did the lexA-MBD2b fusion. In contrast, the cotransfection of lex MBD2b with a VP16-Sin3A fusion results in a strong activation of the reporter indicating a specific interaction of MBD2b and Sin3A (Fig. 4).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   MBD2b binds to the paired amphipathic helix 3 of Sin3A. A, schematic overview on the Sin3A protein fusions to GST indicating its characteristic paired amphipathic helix domains (18). B, left panel, binding of GST-MBD2b to Sin3A in vitro. Right panel, binding of GST-Sin3A deletions to MBD2b in vitro. The indicated GST fusions were expressed in E. coli, purified, and incubated with in vitro translated, 35S-labeled Sin3A or MBD2b. Lane 1 contains 10% of the input used for precipitation. The percentage of binding was determined by densitometry and is indicated below the lanes.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   MBD2b interacts with Sin3A in vivo. Yeast two-hybrid experiments were performed with MBD2b f.l. as bait (lexA fusion) and Sin3A as prey (fused to the VP16 activation domain).

MBD2b-mediated Repression Colocalizes with the Sin3A Interaction Domain-- If the direct interaction between MBD2b and Sin3A demonstrated above plays a role in vivo, one would predict that a region that interacts with Sin3A should also be capable of transcriptional repression. To determine which region of MBD2b binds to Sin3A, we generated a detailed set of truncations of the GST-MBD2b fusion (Fig. 5A) that were used for GST pull-down assays with in vitro translated Sin3A. These experiments identified a region within MBD2b that mediates interaction with Sin3A (Fig. 5). This region (amino acids 27-82) can apparently be subdivided into two parts because MBD2b aa 1-45, as well as MBD2b aa 45-262, are able to bind Sin3A. Interestingly, the Sin3A interaction region overlaps with the methyl-CpG-binding domain but is not able to bind to DNA (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 5.   A small region of MBD2b (aa 27-82) is sufficient for binding to Sin3A. A, schematic representation of GST-MBD2b constructs, indicating the MBD and the minimal interaction region. Interaction of these constructs with Sin3A in GST pull-down assays is shown on the right. , no interaction; +, interaction; ++, strong interaction. For exact binding efficiencies see B. B, pull-down experiments with GST-MBD2b fusions and in vitro translated Sin3A. 35S-Labeled full-length Sin3A was incubated with GST alone or with the indicated truncations fused to GST. The input shown is 10% of the in vitro translated product used in the assay; the percentage of binding indicated below the lanes was determined by densitometry.

To avoid interference of compromised DNA binding of MBD2b truncations versus functional repression, we generated fusions of full-length MBD2b and of truncations with the GAL4 DNA-binding domain. These fusions were tested in transient transfections for the activity to repress transcription of a tkCAT reporter containing GAL DNA-binding elements. GAL-MBD2 strongly represses the activity of the tkCAT reporter. Furthermore, regions of MBD2b (aa 27-82 or 45-262) that bind to Sin3A also efficiently repress transcription, whereas a region (aa 163-262) that does not bind Sin3A showed no ability to repress transcription (Fig. 6).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Repression function of MBD2b is contained within the Sin3A interaction region. CV1 cells were transiently cotransfected using the reporter construct 17-mer 6xs tkCAT and expression plasmids for Gal-MBD2b full-length or the indicated truncations. Fold repression was calculated relative to the CAT activity obtained after expression of the Gal DNA-binding domain.

Gene repression by co-expressed effectors can be either due to DNA binding of the effector and thereby modulating the nearby transcriptional initiation or due to squelching co-factors that are required for gene activation. To distinguish between these two mechanisms we tested whether DNA binding of the MBD2b repression domain is required to confer functional repression. We used the GAL MBD2b fusion and two types of reporter genes, one harboring GAL-binding sites and the basal reporter, which has no GAL-binding elements (Fig. 7). The results clearly show that DNA binding is required for repression mediated by GAL MBD2b, because the basal tkCAT reporter was not affected by any concentration of GAL-MBD2b, whereas repression of 6xs upstream activating sequence tkCAT by GAL MBD2b was dose-dependent. Thus, DNA binding via the GAL4 DNA-binding domain is required for repression of GAL MBD2b.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Repression mediated by the minimal repression domain of MBD2b is dose- and DNA binding-dependent. CV1 cells were transfected using ptkCAT or 17-mer 6xs tkCAT as reporter. Cotransfection of varying amounts (0.1, 0.25, and 0.5 pmol) of Gal-MBD2b (aa 27-82) represses transcription only in the context of the upstream activating sequence-containing reporter. Fold repression was normalized to the CAT activity after transfection of the Gal DNA-binding domain.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A family of nuclear proteins has been identified that has a MBD in common (34). For some of these proteins (MeCP2, MBD1, and MBD2) transcriptional repression has been shown (17, 35, 36), thereby offering a functional connection for the observed correlation that CpG methylation is often found in inactive genes or inactive promoter regions (12). For MBD2 two in-frame potential start codons are found, the upstream AUG allowing translation of MBD2 and an AUG 152 codons downstream, giving rise to MBD2b, which is fully contained within MBD2 (34). On the protein level, the in vivo presence has been shown for MBD2 in contrast to MBD2b (17), both of which confer transcriptional repression (Ref. 17 and this paper). In addition to repression, a DNA demethylase activity of MBD2b has been reported (31). We became interested in this protein because the lysozyme downstream HS3.2 enhancer also shows such a dual activity. In the methylated state, this enhancer is repressed (13, 28) and provides the cis-acting sequences leading to tissue-specific demethylation (21). Nevertheless, extensive analysis of the demethylase activity of MBD2b failed to show any demethylation (data not shown). Overexpressed or in vitro translated MBD2b did not demethylate a 1.2-kilobase enhancer fragment containing the HS3.2 sequence nor an artificial CpG-island in an in vitro demethylation assay performed accordingly to Bhattacharya et al. (31). Additionally cotransfected MBD2b failed to activate the CAT activity of a methylated HS3.2 reporter gene construct in transient transfections.

The repression activity of MBD2 was shown to involve HDACs, because repression was sensitive to trichostatin A treatment (17). Consequently, MBD2 has been found to be associated with HDACs or to interact with HDAC containing complexes. MBD2 is a component of the MeCP1 complex containing several HDACs (17). In vitro interaction of MBD2 with a chromatin remodeling and HDAC containing complex (Mi-2/NuRD) has also been shown (16). If transcriptional repression is mediated by direct or indirect binding of MBD2 to HDACs, a minimal trans-repression domain should exist conferring both repression and binding to HDACs. Here we show that a short region of MBD2b is able to mediate repression. This TRD (amino acids 27-82) overlaps with the MBD such that the C-terminal end of the MBD contributes to full repression. TRDs identified within other MBD containing proteins are found at quite distinct locations. MBD1 contains CXXC domains, which are also found in the DNA methyltransferase Dnmt1 (37, 38). The C-terminal CXXC domain coincides with an unspecific repression function of MBD1 (35), whereas the very C-terminal part of MBD1 exhibits repression dependent on DNA binding. Because repression by MBD1 is trichostatin A-sensitive, direct or indirect binding of the TRD to HDACs is likely but has not been shown. For MeCP2, a C-terminal TRD has been identified that binds Sin3A and recruits HDACs (15, 39, 40). Here we find that the minimal TRD of MBD2b also binds Sin3A. However, the TRDs of MeCP2 and MBD2 are quite distinct in that the MeCP2 TRD is well separated from the MBD, whereas the MBD2b TRD encompasses part of the MBD. In addition to Sin3A binding, association of the MeCP2 TRD with the transcription factor TFIIB has also been found (15), which is supported by a trichostatin A-resistant repression (14).

Because MBD1 and MeCP2 are stably associated with chromosomes, even during mitosis, whereas MBD2 can be released from chromatin by low salt (17, 35), a regulatory role for MBD2 could be envisaged. Such a role remains to be determined in the future.

	ACKNOWLEDGEMENTS

We thank Aria Baniahmad, Martin. L. Privalsky, and Moshe Szyf for plasmids and Helga Wahn for excellent technical assistance. We are grateful to Les Burke for critically reading the manuscript.

	FOOTNOTES

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB397 and by the Fond der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-641-99-35460; Fax: 49-641-99-35469; E-mail: Rainer.Renkawitz@gen.bio. uni-giessen.de.

Published, JBC Papers in Press, August 18, 2000, DOI 10.1074/jbc.M005929200

	ABBREVIATIONS

The abbreviations used are: MBD, methyl-CpG-binding domain; MeCP, methyl-CpG-binding protein; HDAC, histone deacetylase; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; HS, hypersensitive site; tk, thymidine kinase; TRD, transcriptional repression domain; aa, amino acid(s); 6xs, 6 times sense orientation.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				B. E. Reese, K. E. Bachman, S. B. Baylin, and M. R. Rountree The Methyl-CpG Binding Protein MBD1 Interacts with the p150 Subunit of Chromatin Assembly Factor 1 Mol. Cell. Biol., May 1, 2003; 23(9): 3226 - 3236. [Abstract] [Full Text] [PDF]

				F. Lembo, R. Pero, T. Angrisano, C. Vitiello, R. Iuliano, C. B. Bruni, and L. Chiariotti MBDin, a Novel MBD2-Interacting Protein, Relieves MBD2 Repression Potential and Reactivates Transcription from Methylated Promoters Mol. Cell. Biol., March 1, 2003; 23(5): 1656 - 1665. [Abstract] [Full Text] [PDF]

				S. Koizume, K. Tachibana, T. Sekiya, S. Hirohashi, and M. Shiraishi Heterogeneity in the modification and involvement of chromatin components of the CpG island of the silenced human CDH1 gene in cancer cells Nucleic Acids Res., November 1, 2002; 30(21): 4770 - 4780. [Abstract] [Full Text] [PDF]

				M. Brackertz, J. Boeke, R. Zhang, and R. Renkawitz Two Highly Related p66 Proteins Comprise a New Family of Potent Transcriptional Repressors Interacting with MBD2 and MBD3 J. Biol. Chem., October 18, 2002; 277(43): 40958 - 40966. [Abstract] [Full Text] [PDF]

				N. Detich, J. Theberge, and M. Szyf Promoter-specific Activation and Demethylation by MBD2/Demethylase J. Biol. Chem., September 20, 2002; 277(39): 35791 - 35794. [Abstract] [Full Text] [PDF]