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P66Alpha-MBD2 coiled-coil interaction and recruitment of Mi-2 are critical for globin gene silencing by the MBD2-NuRD complex.

ABSTRACT: Nucleosome remodeling complexes comprise several large families of chromatin modifiers that integrate multiple epigenetic control signals to play key roles in cell type-specific transcription regulation. We previously isolated a methyl-binding domain protein 2 (MBD2)-containing nucleosome remodeling and deacetylation (NuRD) complex from primary erythroid cells and showed that MBD2 contributes to DNA methylation-dependent embryonic and fetal ?-type globin gene silencing during development in vivo. Here we present structural and biophysical details of the coiled-coil interaction between MBD2 and p66?, a critical component of the MBD2-NuRD complex. We show that enforced expression of the isolated p66? coiled-coil domain relieves MBD2-mediated globin gene silencing and that the expressed peptide interacts only with a subset of components of the MBD2-NuRD complex that does not include native p66? or Mi-2. These results demonstrate the central importance of the coiled-coil interaction and suggest that MBD2-dependent DNA methylation-driven gene silencing can be disrupted by selectively targeting this coiled-coil complex.

SUBMITTER: Gnanapragasam MN

PROVIDER: S-EPMC3088629 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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p66Alpha-MBD2 coiled-coil interaction and recruitment of Mi-2 are critical for globin gene silencing by the MBD2-NuRD complex.

Gnanapragasam Merlin Nithya MN Scarsdale J Neel JN Amaya Maria L ML Webb Heather D HD Desai Megha A MA Walavalkar Ninad M NM Wang Shou Zhen SZ Zu Zhu Sheng S Ginder Gordon D GD Williams David C DC

Proceedings of the National Academy of Sciences of the United States of America 20110413 18

Nucleosome remodeling complexes comprise several large families of chromatin modifiers that integrate multiple epigenetic control signals to play key roles in cell type-specific transcription regulation. We previously isolated a methyl-binding domain protein 2 (MBD2)-containing nucleosome remodeling and deacetylation (NuRD) complex from primary erythroid cells and showed that MBD2 contributes to DNA methylation-dependent embryonic and fetal β-type globin gene silencing during development in vivo ...[more]

PMID: 21490301

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Project description: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.

Dataset Information

P66Alpha-MBD2 coiled-coil interaction and recruitment of Mi-2 are critical for globin gene silencing by the MBD2-NuRD complex.

Publications

p66Alpha-MBD2 coiled-coil interaction and recruitment of Mi-2 are critical for globin gene silencing by the MBD2-NuRD complex.

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