Complete Profiling of Methyl-CpG-Binding Domains for Combinations of Cytosine Modifications at CpG Dinucleotides Reveals Differential Read-out in Normal and Rett-Associated States

Benjamin C Buchmuller,Brinja Kosel,Daniel Summerer

doi:10.1038/s41598-020-61030-1

Benjamin C Buchmuller, Brinja Kosel + Show 1 more

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https://doi.org/10.1038/s41598-020-61030-1

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Journal: Scientific Reports	Publication Date: Mar 4, 2020
Citations: 18	License type: open-access

Affiliation: TU Dortmund University

Abstract

5-Methylcytosine (mC) exists in CpG dinucleotides of mammalian DNA and plays key roles in chromatin regulation during development and disease. As a main regulatory pathway, fully methylated CpG are recognized by methyl-CpG-binding domain (MBD) proteins that act in concert with chromatin remodelers, histone deacetylases and methyltransferases to trigger transcriptional downregulation. In turn, MBD mutations can alter CpG binding, and in case of the MBD protein MeCP2 can cause the neurological disorder Rett syndrome (RTT). An additional layer of complexity in CpG recognition is added by ten-eleven-translocation (TET) dioxygenases that oxidize mC to 5-hydroxymethyl-, 5-formyl- and 5-carboxylcytosine, giving rise to fifteen possible combinations of cytosine modifications in the two CpG strands. We report a comprehensive, comparative interaction analysis of the human MBD proteins MeCP2, MBD1, MBD2, MBD3, and MBD4 with all CpG combinations and observe individual preferences of each MBD for distinct combinations. In addition, we profile four MeCP2 RTT mutants and reveal that although interactions to methylated CpGs are similarly affected by the mutations, interactions to oxidized mC combinations are differentially affected. These findings argue for a complex interplay between local TET activity/processivity and CpG recognition by MBDs, with potential consequences for the transcriptional landscape in normal and RTT states.

Highlights

Proteins containing a methyl-CpG-binding domain (MBD) are the central readers of methylated CpG dinucleotides and interact with both DNA strands[20]
A deeper understanding of the direct interactions of MBDs, including MeCP2 Rett syndrome (RTT) mutants, with oxidized mC combinations at CpGs is of particular interest, since they may translate into altered genomic distributions of MBD occupancy and transcriptional activity in normal and RTT-associated states
We recombinantly expressed the MBDs of the five human MBD family proteins hMBD1–4 and hMeCP2 (Fig. 2a), which all adopt a highly similar three-dimensional fold and share 50–60% of polypeptide sequence identity, especially at the residues interacting with the DNA double-strand (Fig. 2b)[20]

Summary

Introduction

Proteins containing a methyl-CpG-binding domain (MBD) are the central readers of methylated CpG dinucleotides and interact with both DNA strands[20]. These studies delivered highly valuable insights into the interplay of individual MBDs and particular combinations of cytosine modifications, they are incomplete in view of the tested MBD-CpG combinations and allow only limited comparisons The latter is due to the use of either full-length or isolated MBDs from different organisms, different expression construct designs, as well as different binding conditions and target DNA sequences. We profile four MeCP2 RTT mutants and report that though binding to methylated CpGs is reduced by the mutations, their interaction to frequent oxidized mC combinations are differentially affected These findings argue for a more complex interplay between TET activity and MBD than previously thought that may translate into altered genomic landscapes of MBD occupancy and transcriptional activity

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