David C. Williams, Jr, MD, PhD
My laboratory uses structural and biophysical techniques to probe protein-protein and protein-DNA interactions critical to the formation of macromolecular complexes. Over the past several years, we have been studying the Nucleosome Remodeling and Deacetylase (NuRD) complex responsible for recognizing methylated DNA and silencing expression of the associated genes. Our focus has been to understand how a key component of this complex, the methyl-cytosine binding domain protein 2 (MBD2), binds DNA and recruits the rest of NuRD.
DNA methylation represents a key epigenetic signal involved in both normal developmental silencing of genes as well as aberrant silencing of tumor suppressor genes in cancer. The methylation mark is recognized by a family of proteins that contain a ~60 amino acid methyl-cytosine binding domain (MBD) including MeCP2 and MBD1-4. The primary focus of our studies, MBD2, binds methylated promoters and recruits at least five proteins (p66α/β, MTA2, RbAp46/48, HDAC1/2, and Mi2α/β) that alter the chromatin structure and silence transcription of the associated gene. The structural and biophysical details of any of the specific intermolecular interactions forming MBD2-NuRD are an active area of investigation. We recently determined the structure of the coiled-coil complex formed between MBD2 and the p66α protein and showed that disrupting this interaction restores expression of methylated genes.1 Since knockout of MBD2 in mice leads to only mild phenotypic consequences, inhibition of MBD2 represents a potential biological target to restore expression of tumor suppressor genes silenced by DNA methylation. Importantly, work by our collaborators and others have shown that disrupting the function of MBD2 can block tumor cell growth in tissue culture and in vivo.2
Currently we are studying different domains of MBD2 using structural analyses by NMR complimented by a variety of biophysical techniques. The N-terminal methyl-cytosine binding domain adopts a well-folded structure once bound to DNA3 but shows evidence of rapid translocation between methylated sites. We are comparing DNA binding and dynamics between different members of the MBD family to better understand functional differences.4-5 In contrast, the coiled-coil domain in MBD2 forms a very stable and high affinity interaction with p66α of NuRD. We are characterizing the minimal requirements for high affinity coiled-coil complex formation6 with the goal of developing a method to inhibit this interaction. Finally, we are studying a largely unstructured region from MBD2, which is necessary and sufficient for binding and recruiting other components of the NuRD complex.7 Ultimately the goal of these studies is to understand the structural mechanism of methylation dependent gene silencing by MBD2 and to develop inhibitors of complex formation for therapeutic benefit.
- Gnanapragasam, M. N. et al. 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 (2011).
- Mian, O. Y., Wang, S.Z., Zhu, S.Z., Gnanapragasam, M.N., Graham, L., Bear, H.D., & Ginder, G.D. Methyl-binding domain protein 2-dependent proliferation and survival of breast cancer cells. Mol. Cancer. Res. 9, 1152-1162 (2011).
- Scarsdale, J. N., Webb, H. D., Ginder, G. D. & Williams, D. C.,Jr. Solution structure and dynamic analysis of chicken MBD2 methyl binding domain bound to a target-methylated DNA sequence. Nucl. Acids Res. 39, 6741-6752 (2011).
- Walavalkar, N.M., Cramer, J.M., Buckwald, W.A., Scarsdale, J.N., & Williams, D.C., Jr. Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches. Nucl. Acids Res. 42, 11218-11232 (2014).
- Cramer, J.M., Scarsdale, J.N., Walavalkar, N.M., Buckwald, W.A., Ginder, G.D., & Williams, D.C., Jr. Probing the dynamic distribution of bounds states for methylcytosine-binding domains on DNA. J. Biol. Chem. 289, 1294-1302 (2014).
- Walavalkar, N. M., Gordon, N. & Williams, D. C.,Jr. 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). J. Biol. Chem. 288, 3419-3427 (2013).
- Desai, M.A., Webb, H.D., Sinanan, L.M., Scarsdale, J.N., Walavalkar, N.M., Ginder, G.D., & Williams, D.C. Jr. An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex. Nucl. Acids Res. 43, 3100-3113 (2015).