MeCP2 binds to methylated DNA independently of phase separation and heterochromatin organisation

Abstract

Correlative evidence has suggested that DNA methylation promotes the formation of transcriptionally silent heterochromatin. Accordingly, the methyl-CpG binding domain protein MeCP2 is often portrayed as a constituent of heterochromatin. This interpretation has been reinforced by the use of mouse cells as an experimental system for studying the mammalian epigenome, as heterochromatin, DNA methylation and MeCP2 colocalise in prominent foci. The findings presented here revise this view. We show that focal localisation of MeCP2 in mice is independent of heterochromatin, as DNA methylation-dependent MeCP2 foci persist even when the signature heterochromatin histone mark H3K9me3 is absent and heterochromatin protein HP1 is diffuse. Contrary to the proposal that MeCP2 forms condensates at mouse heterochromatic foci via liquid-liquid phase transition, the short methyl-CpG binding domain, which lacks the disordered domains thought to be required for condensation, is sufficient to target foci in mouse cells. Importantly, we find that the formation of MeCP2 foci in mice is highly atypical, as they are indetectable in 14 out of 16 other mammalian species, including humans. Notably, MeCP2 foci are absent in Mus spretus which can interbreed with Mus musculus but lacks its highly methylated pericentric satellite DNA repeats. We conclude that MeCP2 has no intrinsic tendency to form nuclear condensates and its localisation is independent of heterochromatin formation. Instead, the distribution of MeCP2 in the nucleus is primarily determined by global DNA methylation patterns and is typically euchromatic.

HDAC inhibitors rescue MeCP2T158Mspeckles in a high content screen

“…The differential phenotype of MeCP2 and MeCP2 T158M has been studied in murine NIH3T3 12,13 . Wild-type MeCP2 forms clear pericentromeric speckles while MeCP2 T158M displays less speckles due to impaired affinity to chromatin 12 .…”

“…The crucial part of this work was to create a cell model in which the condensate forming capability of MeCP2 and MeCP2 T158M could be discriminated. Initially, we tried to establish the assay in human cell lines however, the difference observed between MeCP2 and MeCP2 T158M in human cell lines was less clear than in mouse cell lines since the speckled pattern is mouse specific as described earlier 13 . The speckled pattern displayed by mouse cells is due to heterochromatin arrangement independently from MeCP2 13 .…”

“…Initially, we tried to establish the assay in human cell lines however, the difference observed between MeCP2 and MeCP2 T158M in human cell lines was less clear than in mouse cell lines since the speckled pattern is mouse specific as described earlier 13 . The speckled pattern displayed by mouse cells is due to heterochromatin arrangement independently from MeCP2 13 . Despite the high identity of human and mouse MeCP2 we engineered NIH3T3 (mouse fibroblasts) to express human MeCP2 to ensure that the identified hits indeed act on the human protein.…”

“…MeCP2E1 expression levels are low during early stages of development but increase at the start of neuronal differentiation 8 . In adults, the protein is expressed at high levels in the cortical regions, hippocampus, cerebellum, and olfactory bulb 13,16 .…”

“…It is known that the chromatin is organized differently in mouse and human cells. In the mouse nucleus, the chromatin forms clear pericentromeric domains were MeCP2 binds while in human cells, these pericentromeric domains are less prominent 13 .…”