Absence of MeCP2 binding to non-methylated GT-rich sequences in vivo

Verdiana Steccanella,Bartlomiej Waclaw,John C Connelly,Justyna Cholewa-Waclaw,Adrian Bird,Shaun Webb

doi:10.1093/nar/gkaa102

Verdiana Steccanella, Bartlomiej Waclaw + Show 4 more

Open Access

https://doi.org/10.1093/nar/gkaa102

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Abstract

MeCP2 is a nuclear protein that binds to sites of cytosine methylation in the genome. While most evidence confirms this epigenetic mark as the primary determinant of DNA binding, MeCP2 is also reported to have an affinity for non-methylated DNA sequences. Here we investigated the molecular basis and in vivo significance of its reported affinity for non-methylated GT-rich sequences. We confirmed this interaction with isolated domains of MeCP2 in vitro and defined a minimal target DNA sequence. Binding depends on pyrimidine 5′ methyl groups provided by thymine and requires adjacent guanines and a correctly orientated A/T-rich flanking sequence. Unexpectedly, full-length MeCP2 protein failed to bind GT-rich sequences in vitro. To test for MeCP2 binding to these motifs in vivo, we analysed human neuronal cells using ChIP-seq and ATAC-seq technologies. While both methods robustly detected DNA methylation-dependent binding of MeCP2 to mCG and mCAC, neither showed evidence of MeCP2 binding to GT-rich motifs. The data suggest that GT binding is an in vitro phenomenon without in vivo relevance. Our findings argue that MeCP2 does not read unadorned DNA sequence and therefore support the notion that its primary role is to interpret epigenetic modifications of DNA.

Highlights

The DNA base cytosine can exist in a variety of modified forms of which 5-methylcytosine is the most abundant in vertebrates (1)
The 5-methylcytosine binding protein MeCP2 is present at high levels in neuronal cells (5) where it interacts with both methyl-CG and methyl-CAC (6–8)
To investigate the molecular basis of MeCP2 binding to non-methylated DNA in vitro we performed electrophoretic mobility shift assays (EMSAs) using a recombinant Nterminal fragment of MeCP2 comprising amino-acids 1– 205

Summary

Introduction

The DNA base cytosine can exist in a variety of modified forms of which 5-methylcytosine (mC) is the most abundant in vertebrates (1). The 5-methylcytosine binding protein MeCP2 is present at high levels in neuronal cells (5) where it interacts with both methyl-CG (mCG) and methyl-CAC (mCAC) (6–8). A primary function of MeCP2 is to recruit the NCoR1/2 corepressor complex to these methylated sites and thereby restrain neuronal transcription (6,8–10). Discrete protein domains responsible for methyl-CpG/mCAC binding (the methyl-binding domain: MBD) and NCoR1/2 interaction domain (NID) have been defined by deletion analysis and X-ray crystallography of the protein–DNA and protein–protein complexes (9,12–14). These two domains alone are sufficient to rescue survival of MeCP2-deficient mice (15)

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