The nucleosome remodelling and deacetylase (NuRD) complex is essential for the development of complex animals. NuRD has roles in regulating gene expression and repairing damaged DNA. The complex comprises at least six proteins with two or more paralogues of each protein routinely identified when the complex is purified from cell extracts. To understand the structure and function of NuRD, a map of direct subunit interactions is needed. Dozens of published studies have attempted to define direct inter-subunit connectivities. We propose that conclusions reported in many such studies are in fact ambiguous for one of several reasons. First, the expression of many NuRD subunits in bacteria is unlikely to lead to folded, active protein. Second, interaction studies carried out in cells that contain endogenous NuRD complex can lead to false positives through bridging of target proteins by endogenous components. Combining existing information on NuRD structure with a protocol designed to minimize false positives, we report a conservative and robust interaction map for the NuRD complex. We also suggest a 3D model of the complex that brings together the existing data on the complex. The issues and strategies discussed herein are also applicable to the analysis of a wide range of multi-subunit complexes.
The Rett syndrome-associated methyl-CpG binding protein 2 (MeCP2) selectively binds methylated DNA to regulate transcription during the development of mature neurons. Like other members of the methyl-CpG binding domain (MBD) family, MeCP2 functions through the recognition of symmetrical 5-methylcytosines in CpG (mCG) dinucleotides. Advances in base level resolution epigenetic mapping techniques have revealed, however, that MeCP2 can bind asymmetrically methylated and hydroxymethylated CpA (h/mCA) dinucleotides and this alternative binding selectivity modifies gene expression in the developing mammalian brain. The structural determinants of binding to mCA and hydroxymethylated DNA have not been previously investigated. Here, we employ ITC and NMR spectroscopy to characterize MeCP2 binding to methylated and hydroxymethylated mCG and mCA DNA; examine the effects of Rett syndrome-associated missense mutations; and make comparisons to the related and evolutionarily most ancient protein, MBD2. These analyses reveal that MeCP2 binds mCA with high affinity in a strand-specific and orientation-dependent manner. In contrast, MBD2 does not show high affinity or methyl-specific binding to mCA. The Rett-associated missense mutations (T158M, R106W, and P101S) destabilize the MeCP2 MBD and disrupt recognition of mCG and mCA equally. Finally, hydroxymethylation of a high-affinity mCA site does not alter the binding properties; whereas, hemi-hydroxylation of the equivalent cytosine in an mCG site decreases affinity and specificity. Based on these findings, we suggest MeCP2 recognition of methylated/hydroxymethylated CpA dinucleotides functions as an epigenetic switch redistributing MeCP2 among mCG and mCA loci.
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.
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