Methyl-cytosine Binding Domain Research Articles

MBD2a-NuRD binds to the methylated γ-globin gene promoter and uniquely forms a complex required for silencing of HbF expression.

During human development, there is a switch in the erythroid compartment at birth that results in silencing of expression of fetal hemoglobin (HbF). Reversal of this silencing has been shown to be effective in overcoming the pathophysiologic defect in sickle cell anemia. Among the many transcription factors and epigenetic effectors that are known to mediate HbF silencing, two of the most potent are BCL11A and MBD2-NuRD. In this report, we present direct evidence that MBD2-NuRD occupies the γ-globin gene promoter in adult erythroid cells and positions a nucleosome there that results in a closed chromatin conformation that prevents binding of the transcriptional activator, NF-Y. We show that the specific isoform, MBD2a, is required for the formation and stable occupancy of this repressor complex that includes BCL11A, MBD2a-NuRD, and the arginine methyltransferase, PRMT5. The methyl cytosine binding preference and the arginine-rich (GR) domain of MBD2a are required for high affinity binding to methylated γ-globin gene proximal promoter DNA sequences. Mutation of the methyl cytosine-binding domain(MBD) of MBD2 results in a variable but consistent loss of γ-globin gene silencing, in support of the importance of promoter methylation. The GR domain of MBD2a is also required for recruitment of PRMT5, which in turn results in placement of the repressive chromatin mark H3K8me2s at the promoter. These findings support a unified model that integrates the respective roles of BCL11A, MBD2a-NuRD, PRMT5, and DNA methylation in HbF silencing.

MBD2a-NuRD Binds to the γ-Globin Gene Promoter and Uniquely Forms a Complex Required for Bcl11a Mediated Silencing of Hb F Expression

A mechanistic understanding of γ-globin silencing is of clinical importance because reversal of silencing in adult erythroid cells results in high level expression Hb F which is therapeutic in sickle cell anemia and beta thalassemia. We describe here studies to elucidate the mechanism by which MBD2a-NuRD is an obligate partner of Bcl11a in silencing of HbF. We have previously shown that depletion of MBD2 or disruption of the MBD2-NuRD chromatin remodeling complex, but not MBD3-NuRD complex results in high levels of Hb F despite a concomitant 2-3 fold increase in expression of Bcl11a, a major silencing transcription factor (Yu etal. Haematologica, 2019). At the same time, Bcl11a can interact with both the MBD3 and MBD2 NuRD complexes. To elucidate the specific role of MBD2-NuRD, Chromatin Immunoprecipitation-qPCR(ChIP-qPCR) assays carried out in both adult erythroid phenotype HUDEP-2 and primary progenitor derived adult erythroid cells showed binding at the γ-globin gene promoter. To clarify the role of MBD2-NuRD in establishing closed chromatin configuration at the γ-globin gene loci, we carried out ATAC seq assays which showed that MBD2 knockout resulted in the same pattern of chromatin opening as reported for Bcl11a knockout in HUDEP-2 cells, consistent with our previous data showing ~40-50% Hb F in MBD2 knockout cells. In order to examine the effect of MBD2-NuRD on nucleosome positioning over the γ-globin gene promoter, NOME-SEQ assays were performed in HUDEP 2 cells which showed a nucleosome positioned at -110 to + 36 relative to the transcription start site (TSS) in MBD +/+ cells but not MBD2 KO HUDEP-2 cells, such that the NFY-A transcriptional activator binding site but not the Bcl11a repressor binding site is occluded. MBD2 exists in two isoforms in erythroid cells, resulting from alternative translation initiation sites. MBD2a contains an arginine rich (GR) domain which is absent in MBD2b and MBD3. Addback of MBD2a but not MBD2b in MBD2 KO HUDEP-2 cells resulted in silencing of γ-globin gene expression. Editing of the endogenous MBD2 gene such that predominantly MBD2b was expressed relieved silencing. To understand how the GR domain contributes to silencing we carried out unbiased proteomic analysis of MBD2a vs MBD2b NuRD complexes, which identified only 3 proteins uniquely associated with MBD2a. One of these, PRMT5, has been implicated in silencing of HbF. Knockdown of PRMT5 resulted in a`from <1% to ~15% γ-globin RNA in parental HUDEP-2 cells and a 4-5 fold increase in primary human progenitor derived erythroid cells, but caused no significant further increase in MBD2 KO HUDEP-2 cells. ChIP qPCR assays showed that PRMT5 and its obligate binding partner MEP50 occupy the γ- promoter in parental HUDEP-2 cells and primary erythroid cells but not in MBD2 KO HUDEP-2 cells. Furthermore, knockdown of PRMT5 resulted in frequent displacement of the nucleosome positioned at -110 to + 36 relative to the TSS. In order to determine the role that the GR domain might have in occupancy of MBD2a-NuRD at the γ-promoter, in vitro binding studies where done, which showed a requirement for both CpG methylation and the GR domain for high affinity binding of the methyl cytosine binding domain of MBD2 to the proximal γ-globin gene promoter sequences. We conclude that MBD2a-NuRD is required for silencing of Hb F due to its ability to localize at the methylated γ-globin gene promoter in conjunction with Bcl11a and recruit PRMT5, thereby stably positioning a nucleosome that blocks binding of the transcriptional activator NFY-A. These results reinforce the concept that targeted disruption of this specific complex has therapeutic potential to increase HbF in sickle cell anemia and β thalassemia.

Early-Life Stress Induced Epigenetic Changes of Corticotropin-Releasing Factor Gene in Anorexic Low Body Weight-Selected Chicks.

The expression of neuropeptide Y (NPY) in the arcuate nucleus (ARC) and corticotropin-releasing factor (CRF) in the paraventricular nucleus (PVN) were increased when low body weight–selected (LWS) line chicks, which are predisposed to anorexia, were subjected to a combination of nutritional and thermal stressors at hatch. We hypothesized that such changes resulted from epigenetic modifications. We determined global DNA methylation, DNA methyltransferase (DNMT) activity, and methylation near the promoter regions of NPY and CRF, in the hypothalamus of LWS chicks on day 5 post-hatch. Stress exposure at hatch induced global hypermethylation and increased DNMT activity in the ARC but not PVN. In the PVN of stressed LWS chicks, there was decreased methylation of a CpG site located at the core binding domain of methyl cytosine binding domain protein 2 (MBD2), near the CRF gene promoter. We then demonstrated that this was associated with disrupted binding of MBD2. There was also reduced utilization of yolk reserves and lean and fat masses in chicks that were stress-exposed. These findings provide novel insights on molecular mechanisms through which stressful events induce or intensify anorexia in predisposed individuals and a novel molecular target for further studies.

CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level.

The methylcytosine-binding domain 2 (MBD2) protein recruits the nucleosome remodeling and deacetylase complex (NuRD) to methylated DNA to modify chromatin and regulate transcription. Importantly, MBD2 functions within CpG islands that contain 100s to 1000s of potential binding sites. Since NuRD physically rearranges nucleosomes, the dynamic mobility of this complex is directly related to function. In these studies, we use NMR and single-molecule atomic force microscopy and fluorescence imaging to study DNA binding dynamics of MBD2. Single-molecule fluorescence tracking on DNA tightropes containing regions with CpG-rich and CpG-free regions reveals that MBD2 carries out unbiased 1D diffusion on CpG-rich DNA but subdiffusion on CpG-free DNA. In contrast, the protein stably and statically binds to methylated CpG (mCpG) regions. The intrinsically disordered region (IDR) on MBD2 both reduces exchange between mCpG sites along the DNA as well as the dissociation from DNA, acting like an anchor that restricts the dynamic mobility of the MBD domain. Unexpectedly, MBD2 binding to methylated CpGs induces DNA bending that is augmented by the IDR region of the protein. These results suggest that MBD2 targets NuRD to unmethylated or methylated CpG islands where its distinct dynamic binding modes help maintain open or closed chromatin, respectively.

Methylation specific targeting of a chromatin remodeling complex from sponges to humans

DNA cytosine methylation and methyl-cytosine binding domain (MBD) containing proteins are found throughout all vertebrate species studied to date. However, both the presence of DNA methylation and pattern of methylation varies among invertebrate species. Invertebrates generally have only a single MBD protein, MBD2/3, that does not always contain appropriate residues for selectively binding methylated DNA. Therefore, we sought to determine whether sponges, one of the most ancient extant metazoan lineages, possess an MBD2/3 capable of recognizing methylated DNA and recruiting the associated nucleosome remodeling and deacetylase (NuRD) complex. We find that Ephydatia muelleri has genes for each of the NuRD core components including an EmMBD2/3 that selectively binds methylated DNA. NMR analyses reveal a remarkably conserved binding mode, showing almost identical chemical shift changes between binding to methylated and unmethylated CpG dinucleotides. In addition, we find that EmMBD2/3 and EmGATAD2A/B proteins form a coiled-coil interaction known to be critical for the formation of NuRD. Finally, we show that knockdown of EmMBD2/3 expression disrupts normal cellular architecture and development of E. muelleri. These data support a model in which the MBD2/3 methylation-dependent functional role emerged with the earliest multicellular organisms and has been maintained to varying degrees across animal evolution.

CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level

CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level

Effects of atrazine and chlorpyrifos on DNA methylation in the brain and gonad of the common carp

DNA methylation is known to play an important role in the regulation of gene expression in animal. The purpose of the present study was to examine the effect of atrazine (ATR), chlorpyrifos (CPF) and combined ATR/CPF exposure on DNA methylation in the brain and gonad of common carp (Cyprinus carpio L.). The carp were sampled after a 40-d exposure to CPF and ATR, individually or in combination, followed by a 40-d recovery to measure the levels of global DNA methylation and the expression of methylation enzymes (DNA methyltransferases (DNMTs) and methylcytosine binding domain 2 (MBD2)) in the brain and gonad tissues. The results revealed that a significant global DNA hypomethylation in the common carp exposed to ATR, CPF and their mixture was observed compared to the control fish. The MBD2 mRNA expression was up-regulated in the brain and gonad of the common carp exposed to ATR, CPF and their mixture, in contrast, the DNMTs mRNA expression was down-regulated. The information regarding the effects of ATR and CPF on DNA methylation status generated in this study is important for pesticides toxicology evaluation. However, the effect of ATR and CPF on the methylation status of specific genes, as well as its detailed mechanism requires further investigation.

Solution structure of MBD4 methyl-cytosine binding domain bound to methylated DNA

Solution structure of MBD4 methyl-cytosine binding domain bound to methylated DNA

Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches.

Unlike other members of the methyl-cytosine binding domain (MBD) family, MBD4 serves as a potent DNA glycosylase in DNA mismatch repair specifically targeting mCpG/TpG mismatches arising from spontaneous deamination of methyl-cytosine. The protein contains an N-terminal MBD (MBD4MBD) and a C-terminal glycosylase domain (MBD4GD) separated by a long linker. This arrangement suggests that the MBD4MBD either directly augments enzymatic catalysis by the MBD4GD or targets the protein to regions enriched for mCpG/TpG mismatches. Here we present structural and dynamic studies of MBD4MBD bound to dsDNA. We show that MBD4MBD binds with a modest preference formCpG as compared to mismatch, unmethylated and hydroxymethylated DNA. We find that while MBD4MBD exhibits slow exchange between molecules of DNA (intermolecular exchange), the domain exhibits fast exchange between two sites in the same molecule of dsDNA (intramolecular exchange). Introducing a single-strand defect between binding sites does not greatly reduce the intramolecular exchange rate, consistent with a local hopping mechanism for moving along the DNA. These results support a model in which the MBD4MBD4 targets the intact protein to mCpG islands and promotes scanning by rapidly exchanging between successive mCpG sites which facilitates repair of nearby mCpG/TpG mismatches by the glycosylase domain.

Probing the Dynamic Distribution of Bound States for Methylcytosine-binding Domains on DNA

Although highly homologous to other methylcytosine-binding domain (MBD) proteins, MBD3 does not selectively bind methylated DNA, and thus the functional role of MBD3 remains in question. To explore the structural basis of its binding properties and potential function, we characterized the solution structure and binding distribution of the MBD3 MBD on hydroxymethylated, methylated, and unmethylated DNA. The overall fold of this domain is very similar to other MBDs, yet a key loop involved in DNA binding is more disordered than previously observed. Specific recognition of methylated DNA constrains the structure of this loop and results in large chemical shift changes in NMR spectra. Based on these spectral changes, we show that MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated cytosine-guanosine dinucleotides (CpGs), yet does not distinguish between hydroxymethylated and unmethylated sites. Measuring residual dipolar couplings for the different bound states clearly shows that the MBD3 structure does not change between methylation-specific and nonspecific binding modes. Furthermore, residual dipolar couplings measured for MBD3 bound to methylated DNA can be described by a linear combination of those for the methylation and nonspecific binding modes, confirming the preferential localization to methylated sites. The highly homologous MBD2 protein shows similar but much stronger localization to methylated as well as unmethylated CpGs. Together, these data establish the structural basis for the relative distribution of MBD2 and MBD3 on genomic DNA and their observed occupancy at active and inactive CpG-rich promoters.

Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches

Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches

Unique Features of the Anti-parallel, Heterodimeric Coiled-coil Interaction between Methyl-cytosine Binding Domain 2 (MBD2) Homologues and GATA Zinc Finger Domain Containing 2A (GATAD2A/p66α)

The methyl-cytosine binding domain 2 (MBD2)-nucleosome remodeling and deacetylase (NuRD) complex recognizes methylated DNA and silences expression of associated genes through histone deacetylase and nucleosome remodeling functions. Our previous structural work demonstrated that a coiled-coil interaction between MBD2 and GATA zinc finger domain containing 2A (GATAD2A/p66α) proteins recruits the chromodomain helicase DNA-binding protein (CHD4/Mi2β) to the NuRD complex and is necessary for MBD2-mediated DNA methylation-dependent gene silencing in vivo (Gnanapragasam, M. N., Scarsdale, J. N., Amaya, M. L., Webb, H. D., Desai, M. A., Walavalkar, N. M., Wang, S. Z., Zu Zhu, S., Ginder, G. D., and Williams, D. C., Jr. (2011) p66α-MBD2 coiled-coil interaction and recruitment of Mi-2 are critical for globin gene silencing by the MBD2-NuRD complex. Proc. Natl. Acad. Sci. U.S.A. 108, 7487-7492). The p66α-MBD2 interaction differs from most coiled-coils studied to date by forming an anti-parallel heterodimeric complex between two peptides that are largely monomeric in isolation. To further characterize unique features of this complex that drive heterodimeric specificity and high affinity binding, we carried out biophysical analyses of MBD2 and the related homologues MBD3, MBD3-like protein 1 (MBD3L1), and MBD3-like protein 2 (MBD3L2) as well as specific mutations that modify charge-charge interactions and helical propensity of the coiled-coil domains. Analytical ultracentrifugation analyses show that the individual peptides remain monomeric in isolation even at 300 μM in concentration for MBD2. Circular dichroism analyses demonstrate a direct correlation between helical content of the coiled-coil domains in isolation and binding affinity for p66α. Furthermore, complementary electrostatic surface potentials and inherent helical content of each peptide are necessary to maintain high-affinity association. These factors lead to a binding affinity hierarchy of p66α for the different MBD2 homologues (MBD2 ≈ MBD3 > MBD3L1 ≈ MBD3L2) and suggest a hierarchical regulatory model in tissue and life cycle stage-specific silencing by NuRD complexes.

Direct Homo- and Hetero-Interactions of MeCP2 and MBD2

Epigenetic marks like methylation of cytosines at CpG dinucleotides are essential for mammalian development and play a major role in the regulation of gene expression and chromatin architecture. The methyl-cytosine binding domain (MBD) protein family recognizes and translates this methylation mark. We have recently shown that the level of MeCP2 and MBD2, two members of the MBD family, increased during differentiation and their ectopic expression induced heterochromatin clustering in vivo. As oligomerization of these MBD proteins could constitute a factor contributing to the chromatin clustering effect, we addressed potential associations among the MBD family performing a series of different interaction assays in vitro as well as in vivo. Using recombinant purified MBDs we found that MeCP2 and MBD2 showed the stronger self and cross association as compared to the other family members. Besides demonstrating that these homo- and hetero-interactions occur in the absence of DNA, we could confirm them in mammalian cells using co-immunoprecipitation analysis. Employing a modified form of the fluorescent two-hybrid protein-protein interaction assay, we could clearly visualize these associations in single cells in vivo. Deletion analysis indicated that the region of MeCP2 comprising amino acids 163–309 as well the first 152 amino acids of MBD2 are the domains responsible for MeCP2 and MBD2 associations. Our results strengthen the possibility that MeCP2 and MBD2 direct interactions could crosslink chromatin fibers and therefore give novel insight into the molecular mechanism of MBD mediated global heterochromatin architecture.

Solution structure and dynamic analysis of chicken MBD2 methyl binding domain bound to a target-methylated DNA sequence

The epigenetic code of DNA methylation is interpreted chiefly by methyl cytosine binding domain (MBD) proteins which in turn recruit multiprotein co-repressor complexes. We previously isolated one such complex, MBD2-NuRD, from primary erythroid cells and have shown it contributes to embryonic/fetal β-type globin gene silencing during development. This complex has been implicated in silencing tumor suppressor genes in a variety of human tumor cell types. Here we present structural details of chicken MBD2 bound to a methylated DNA sequence from the ρ-globin promoter to which it binds in vivo and mediates developmental transcriptional silencing in normal erythroid cells. While previous studies have failed to show sequence specificity for MBD2 outside of the symmetric mCpG, we find that this domain binds in a single orientation on the ρ-globin target DNA sequence. Further, we show that the orientation and affinity depends on guanine immediately following the mCpG dinucleotide. Dynamic analyses show that DNA binding stabilizes the central β-sheet, while the N- and C-terminal regions of the protein maintain mobility. Taken together, these data lead to a model in which DNA binding stabilizes the MBD2 structure and that binding orientation and affinity is influenced by the DNA sequence surrounding the central mCpG.

Abstract 116: Methylcytosine binding domain protein 2(MBD2) dependent proliferation and survival of breast cancer cells

Abstract Methyl Cytosine Binding Domain Protein 2 (MBD2) functions as an interpreter of DNA methylation by binding promoter sequences with high methylated CpG content and recruiting a Nuclear Remodeling and Deacetylase (NuRD) co-repressor complex. MBD2 has been shown to bind to and mediate repression of methylated tumor suppressor genes in cancer cells where re-patterning of CpG methylation and associated gene silencing is common. We have investigated the role of MBD2 in breast cancer cell growth and tumor suppressor gene expression. We show that stable shRNA mediated knockdown of MBD2 leads to significant growth suppression of cultured human mammary epithelial cancer lines, SK-BR-3 (92%); MDA-MB-231 (77%); MDA-MB-435 (94%); with the peak anti-proliferative effect occurring only after sustained, stable knockdown and lasting over 4 weeks. MBD2 knockdown also leads to markedly decreased propensity for these aggressive breast cancer cell lines to form in vivo xenograft tumors in BALB/C nu/nu mice. The growth effects of MBD2 knockdown are accompanied by de-repression of specific tumor suppressor genes including DAPK1 and KLK10. Chromatin immunoprecipitation assays and bisulfite sequencing demonstrate MBD2 binding directly to the hyper-methylated CpG-rich promoters of both DAPK1 and KLK10. Expression of an shRNA-resistant MBD2 protein results in restoration of growth and re-silencing of the MBD2 dependent tumor suppressor genes. Our data suggest that uncoupling CpG-methylation from repressive chromatin remodeling and histone modifications by removing MBD2 is sufficient to initiate and maintain tumor suppressor gene transcription and suppress neoplastic cell growth. These results demonstrate a role for MBD2 in aggressive breast cancer cell growth and provide support for the prospect of targeting MBD2 therapeutically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 116. doi:10.1158/1538-7445.AM2011-116

Decreased expression of MBD2 and MBD4 gene and genomic-wide hypomethylation in patients with primary immune thrombocytopenia

Genome-wide hypomethylation has been confirmed in patients with primary immune thrombocytopenia (ITP). Proteins containing methylcytosine-binding domain (MBD) are involved in promoter methylation as transcriptional repressors and promote the gene-silencing effect of DNA methylation. The purpose of this study was to investigate the methylation pattern of T cells and the relationship between genomic methylation and the expression of MBD2 and MBD4 in ITP patients. DNA deoxymethylcytosine content of CD4 + cells from peripheral blood mononuclear cells was measured by enzyme-linked immunoassay. Real-time polymerase chain reaction was performed to quantify the transcription levels of MBD2 and MBD4 in peripheral blood mononuclear cells and CD4 + cells. DNA dmC content in CD4 + cells of ITP patients was significantly lower than in the controls ( p = 0.001). The mRNA level of MBD2 and MBD4 in CD4 + cells of ITP patients was statistically lower than those of the controls ( p < 0.001). Positive correlations between methylation indexes and expression of each enzyme were observed in the control group ( r 2 = 0.718, p = 0.004 for MBD2; r 2 = 0.608, p = 0.015 for MBD4). However, inverse correlations were found in ITP patients ( r 2 = 0.604, p = 0.008 for MBD2; r 2 = 0.498, p = 0.027 for MBD4). Our results indicate that decreased expression of MBD2 and MBD4 might involve in the pathogenesis of ITP.

MBD2 contributes to developmental silencing of the human ε-globin gene

During erythroid development, the embryonic ε-globin gene becomes silenced as erythropoiesis shifts from the yolk sac to the fetal liver where γ-globin gene expression predominates. Previous studies have shown that the ε-globin gene is autonomously silenced through promoter proximal cis-acting sequences in adult erythroid cells. We have shown a role for the methylcytosine binding domain protein 2 (MBD2) in the developmental silencing of the avian embryonic ρ-globin and human fetal γ-globin genes. To determine the roles of MBD2 and DNA methylation in human ε-globin gene silencing, transgenic mice containing all sequences extending from the 5′ hypersensitive site 5 (HS5) of the β-globin locus LCR to the human γ-globin gene promoter were generated. These mice show correct developmental expression and autonomous silencing of the transgene. Either the absence of MBD2 or treatment with the DNA methyltransferase inhibitor 5-azacytidine increases ε-globin transgene expression by 15–20 fold in adult mice. Adult mice containing the entire human β-globin locus also show an increase in expression of both the ε-globin gene transgene and endogenous εY and βH1 genes in the absence of MBD2. These results indicate that the human ε-globin gene is subject to multilayered silencing mediated in part by MBD2.

Targeting a Methyl Cytosine-Binding Protein Complex to Augment Fetal/Embryonic Globin Expression.

Abstract 974The vertebrate β-type globin genes were among the first genes shown to be regulated, at least in part, by DNA methylation. The mechanism of transcriptional repression by DNA methylation is chiefly through binding of methyl cytosine binding domain (MBD) proteins and their associated co-repressor complexes. The chicken homolog to an MBD2 containing NuRD co-repressor complex (MeCPC) has previously been purified from primary erythroid cells and characterized as binding to the methylated ρ-globin promoter in erythroid cells of adult chickens in which the gene is silent [Kransdorf et al. Blood 2006; 108:2836-45]. Knockdown of MBD2 by siRNA in MEL cells stably transfected with a methylated ρ-globin gene construct leads to a greater than 10-fold increase in ρ-globin gene expression. Likewise, knockout of MBD2 results in a ∼20 fold upregulation of the human gamma globin gene in adult erythroid cells of βYAC transgenic mice [Rupon et al. PNAS 2006; 103:6617-22]. These observations suggest that disruption of the interaction of MBD2 with its co-repressor complex in adult erythropoiesis would increase fetal hemoglobin expression; a therapeutically beneficial effect for both sickle cell anemia and β-thalassemia. This possibility is further supported by the observation that DNA methylation inhibitors such as 5-azacitidine can increase the expression of γ-globin in patients. Based on these studies, we have pursued structural analysis of the interaction between MBD2 and other components from the MeCPC. We have shown that the individual coiled coil regions from MBD2 and a subunit of the NuRD complex, p66α, form a stable heterodimeric complex. Solving the structure of this coiled coil complex by NMR reveals that the interaction involves a combination of hydrophobic and ionic interactions typical of coiled coil complexes as well as a unique charge interaction involving a pair of highly conserved glutamates residues from p66α and arginine residues from MBD2. The key residues involved in binding are conserved across species, between p66α and p66β homologs, as well as between MBD2, MBD3, and the MBD3L1-L5 homologs. We have shown that the p66α coiled coil can stably bind to MBD3 in solution, indicating that similar tertiary interactions are involved in forming both MBD2 and MBD3 containing NuRD complexes. In order to explore this interaction as a potential therapeutic target, we hypothesized that over-expressing the p66α coiled coil region in tissue culture would disrupt the formation of a normal MeCPC and thereby block the function of MBD2. As predicted, expressing this region in both avian (MEL-ρ) and human (CID-βYAC) tissue culture models of globin gene regulation in adult erythroid cells induces embryonic and fetal β-type globin gene expression, respectively. Furthermore, knock-down of p66α induces fetal/embryonic globin gene expression to a similar degree as knock-down of MBD2. These studies suggest a model in which the p66α coiled peptide can bind MBD2 and block recruitment of native p66α to the NuRD complex, thereby acting in a dominant-negative manner to disrupt MBD2 function. We propose that a peptidomimetic of the p66α coiled coil region could be used therapeutically to augment fetal hemoglobin expression. Disclosures:No relevant conflicts of interest to declare.

Multimegabase Silencing in Nucleolar Dominance Involves siRNA-Directed DNA Methylation and Specific Methylcytosine-Binding Proteins

In genetic hybrids, the silencing of nucleolar rRNA genes inherited from one progenitor is the epigenetic phenomenon known as nucleolar dominance. An RNAi knockdown screen identified the Arabidopsis de novo cytosine methyltransferase, DRM2, and the methylcytosine binding domain proteins, MBD6 and MBD10, as activities required for nucleolar dominance. MBD10 localizes throughout the nucleus, but MBD6 preferentially associates with silenced rRNA genes and does so in a DRM2-dependent manner. DRM2 methylation is thought to be guided by siRNAs whose biogenesis requires RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Consistent with this hypothesis, knockdown of DCL3 or RDR2 disrupts nucleolar dominance. Collectively, these results indicate that in addition to directing the silencing of retrotransposons and noncoding repeats, siRNAs specify de novo cytosine methylation patterns that are recognized by MBD6 and MBD10 in the large-scale silencing of rRNA gene loci.

Methyl-CpG binding proteins in the nervous system

Classical methyl-CpG binding proteins contain the conserved DNA binding motif methyl-cytosine binding domain (MBD), which preferentially binds to methylated CpG dinucleotides. These proteins serve as transcriptional repressors, mediating gene silencing via DNA cytosine methylation. Mutations in methyl-CpG binding protein 2 (MeCP2) have been linked to the human mental retardation disorder Rett syndrome, suggesting an important role for methyl-CpG binding proteins in brain development and function. This mini-review summarizes the recent advances in studying the diverse functions of MeCP2 as a prototype for other methyl-CpG binding proteins in the development and function of the vertebrate nervous system.