Abstract
Methyl-CpG-binding protein 2 (MeCP2) is generally considered to act as a transcriptional repressor, whereas recent studies suggest that MeCP2 is also involved in transcription activation. To gain insight into this dual function of MeCP2, we assessed the impact of MeCP2 on higher-order chromatin structure in living cells using mammalian cell systems harbouring a lactose operator and reporter gene-containing chromosomal domain to assess the effect of lactose repressor-tagged MeCP2 (and separate MeCP2 domains) binding in living cells. Our data reveal that targeted binding of MeCP2 elicits extensive chromatin unfolding. MeCP2-induced chromatin unfolding is triggered independently of the methyl-cytosine-binding domain. Interestingly, MeCP2 binding triggers the loss of HP1γ at the chromosomal domain and an increased HP1γ mobility, which is not observed for HP1α and HP1β. Surprisingly, MeCP2-induced chromatin unfolding is not associated with transcriptional activation. Our study suggests a novel role for MeCP2 in reorganizing chromatin to facilitate a switch in gene activity.
Highlights
Gene activity is governed by the interplay between various proteins that modulate the epigenetic composition of chromatin (e.g. DNA methylation, histone modifications) [1]
methyl-CpG-binding protein 2 (MeCP2) targeting causes local chromatin unfolding To investigate the effect of MeCP2 targeting on chromatin folding we used cell lines that enable targeting of (EGFP-lacRtagged) MeCP2 and separate MeCP2 domains to an integrated lac operator (lacO) genomic region, that is present in a highly amplified chromosomal domain in hamster cells or as a multicopy genomic integration in human cells
Fluorescent immunolabeling using MeCP2 specific antibodies that recognize both exogenous and endogenous MeCP2 showed that mCherry-lac repressor (lacR) tagged MeCP2 contained a,25% higher MeCP2 level compared to the endogenous MeCP2 level in non-transfected cells (Figure 1B, C)
Summary
Gene activity is governed by the interplay between various proteins that modulate the epigenetic composition of chromatin (e.g. DNA methylation, histone modifications) [1]. Histone modifications and DNA methylation are linked by CpG-binding proteins such as methyl-CpG-binding protein 2 (MeCP2) [2] through, for instance, cross-talk between MeCP2 and heterochromatin protein 1 (HP1) isoforms [3]. HP1 is a chromatin-binding protein that bridges H3K9methylated histones with other chromatin-associated proteins thereby advancing the ‘spreading’ of heterochromatin [8,9]. Both the clustering of pericentromeric heterochromatin domains and the relocalization of HP1 (in particular HP1c) occur during myogenic differentiation when the level of methyl-CpG-binding proteins is up-regulated [3,10]
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