Neuronal non-CG methylation is an essential target for MeCP2 function

Rebekah Tillotson,Justyna Cholewa-Waclaw,Kashyap Chhatbar,John C Connelly,Sophie A Kirschner,Shaun Webb,Martha V Koerner,Jim Selfridge,David A Kelly,Dina De Sousa,Kyla Brown,Matthew J Lyst,Skirmantas Kriaucionis,Adrian Bird

doi:10.1016/j.molcel.2021.01.011

Rebekah Tillotson, Justyna Cholewa-Waclaw + Show 12 more

Open Access

https://doi.org/10.1016/j.molcel.2021.01.011

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Abstract

SummaryDNA methylation is implicated in neuronal biology via the protein MeCP2, the mutation of which causes Rett syndrome. MeCP2 recruits the NCOR1/2 co-repressor complexes to methylated cytosine in the CG dinucleotide, but also to sites of non-CG methylation, which are abundant in neurons. To test the biological significance of the dual-binding specificity of MeCP2, we replaced its DNA binding domain with an orthologous domain from MBD2, which can only bind mCG motifs. Knockin mice expressing the domain-swap protein displayed severe Rett-syndrome-like phenotypes, indicating that normal brain function requires the interaction of MeCP2 with sites of non-CG methylation, specifically mCAC. The results support the notion that the delayed onset of Rett syndrome is due to the simultaneous post-natal accumulation of mCAC and its reader MeCP2. Intriguingly, genes dysregulated in both Mecp2 null and domain-swap mice are implicated in other neurological disorders, potentially highlighting targets of relevance to the Rett syndrome phenotype.

Highlights

Heterozygous loss-of-function mutations in the X-linked MECP2 gene result in Rett syndrome (RTT), a neurological disorder affecting $1 in 10,000 live female births (Amir et al, 1999)
We used Bio-Layer Interferometry (BLI) to quantify the interactions between the methyl-CpG binding domain (MBD) of MeCP2 and DNA probes containing each of three methylated motifs (Figure 1A)
To visualize a MeCP2 binding footprint at mCAC sites with this method, we focused on the subset of sites with >75% methylation (Figures 1E and S1B)

Summary

Introduction

Heterozygous loss-of-function mutations in the X-linked MECP2 gene result in Rett syndrome (RTT), a neurological disorder affecting $1 in 10,000 live female births (Amir et al, 1999). In the absence of functional MeCP2, indirect mechanisms lead to the downregulation of many genes, perhaps connected with a global reduction in total RNA levels (Kinde et al, 2016; Lagger et al, 2017; Li et al, 2013; Yazdani et al, 2012) This multitude of subtle changes to neuronal gene expression is thought to underlie RTT. Post-mitotic neurons are unique among mammalian somatic cell types in that they accumulate high levels of CH methylation, most prevalently in CAC trinucleotides (Guo et al, 2014; Varley et al, 2013; Xie et al, 2012).

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