MBD3L2 Interacts with MBD3 and Components of the NuRD Complex and Can Oppose MBD2-MeCP1-mediated Methylation Silencing

Seung-Gi Jin,Chun-Ling Jiang,Tibor Rauch,Hongwei Li,Gerd P Pfeifer

doi:10.1074/jbc.m413492200

Abstract

MBD2 and MBD3 are two proteins that contain methyl-CpG binding domains and have a transcriptional repression function. Both proteins are components of a large CpG-methylated DNA binding complex named MeCP1, which consists of the nucleosome remodeling and histone deacetylase complex Mi2-NuRD and MBD2. MBD3L2 (methyl-CpG-binding protein 3-like 2) is a protein with substantial homology to MBD2 and MBD3, but it lacks the methyl-CpG-binding domain. Unlike MBD3L1, which is specifically expressed in haploid male germ cells, MBD3L2 expression is more widespread. MBD3L2 interacts with MBD3 in vitro and in vivo, co-localizes with MBD3 but not MBD2, and does not localize to methyl-CpG-rich regions in the nucleus. In glutathione S-transferase pull-down assays, MBD3L2 is found associated with several known components of the Mi2-NuRD complex, including HDAC1, HDAC2, MTA1, MBD3, p66, RbAp46, and RbAp48. Gel shift experiments with nuclear extracts and a CpG-methylated DNA probe indicate that recombinant MBD3L2 can displace a form of the MeCP1 complex from methylated DNA. MBD3L2 acts as a transcriptional repressor when tethered to a GAL4-DNA binding domain. Repression by GAL4-MBD3L2 is relieved by MBD2 and vice versa, and repression by MBD2 from a methylated promoter is relieved by MBD3L2. The data are consistent with a role of MBD3L2 as a transcriptional modulator that can interchange with MBD2 as an MBD3-interacting component of the NuRD complex. Thus, MBD3L2 has the potential to recruit the MeCP1 complex away from methylated DNA and reactivate transcription.

Highlights

DNA methylation has the capacity to inhibit gene expression through proteins that can bind to methylated CpG sequences in critical regulatory sequences [1,2,3]
We previously reported the identification of two mammalian homologues of MBD2 and MBD3, which were named MBD3L1 and MBD3L2 [30]
We show that the MBD3L2 protein can act as a transcriptional repressor when tethered to a promoter and is associated with components of the NuRD complex

Summary

Introduction

DNA methylation has the capacity to inhibit gene expression through proteins that can bind to methylated CpG sequences in critical regulatory sequences [1,2,3]. MeCP2 is physically associated with the transcriptional corepressor Sin and histone deacetylases in both mammalian cells and Xenopus oocytes [6, 7] This suggests that MeCP2 may play a role in the assembly of a specialized chromatin structure at methylated CpG sequences. In 1998, four additional genes, which encode methyl-CpG binding domain (MBD) proteins, were identified [8]. MBD3 proteins can bind to methylated DNA, presumably due to an amino acid difference in the MBD [1]. Both zebrafish [22] and Xenopus [23] lack the global demethylation event characteristic of early mammalian development [24]

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Conclusion