MeCP2 interacts with HP1 and modulates its heterochromatin association during myogenic differentiation

Abstract

There is increasing evidence of crosstalk between epigenetic modifications such as histone and DNA methylation, recognized by HP1 and methyl CpG-binding proteins, respectively. We have previously shown that the level of methyl CpG-binding proteins increased dramatically during myogenesis leading to large-scale heterochromatin reorganization. In this work, we show that the level of HP1 isoforms did not change significantly throughout myogenic differentiation but their localization did. In particular, HP1gamma relocalization to heterochromatin correlated with MeCP2 presence. Using co-immunoprecipitation assays, we found that these heterochromatic factors interact in vivo via the chromo shadow domain of HP1 and the first 55 amino acids of MeCP2. We propose that this dynamic interaction of HP1 and MeCP2 increases their concentration at heterochromatin linking two major gene silencing pathways to stabilize transcriptional repression during differentiation.

Figure 1.

Level of HP1 proteins during differentiation. (A) Schematic representation of myogenesis. (B) Western blot analysis of the level of HP1 isoforms and of HP1-binding site on chromatin (H3K9Me3) in MB versus MC/MT. Lamin B and histone H3 are taken as controls for equal nuclear protein amounts and for total histone H3, respectively. (C) Quantitative analysis of western blots. Error bars indicate SDs.

Figure 2.

Pericentric heterochromatin association of HP1γ increases during differentiation and correlates with the presence of MeCP2 and MBD1 proteins. (A) Cells were stained with HP1γ and MeCP2-specific antibodies and DNA counterstained with DAPI, highlighting the chromocenters. In the upper panels, overview images and below them representative magnified MB cells are shown, of which only the MeCP2 positive cell has HP1γ accumulated at chromocenters. The lower panels show an overview of a differentiated culture, with most nuclei having HP1γ at chromocenters. Scale bar: 20 µm. (B) Percentage of cells with HP1γ at pericentric heterochromatin and correlation with MeCP2 and MBD1 proteins. Error bars indicate SD.

Figure 3.

MeCP2 interacts with HP1 in vivo. (A) Schematic representation of the fusion proteins. Numbers represent amino acid coordinates. (B and C) HEK293-EBNA cells were transfected with the plasmids indicated and extracts prepared the next day. Immunoprecipitations were done using either anti-GFP (B) or anti-mRFP (C) antibody. (D) Extracts from MB and MT were subjected to immunoprecipitation using the antibodies, as indicated. Input (I) and bound (B) fractions were loaded in the percentages mentioned and analyzed by western blotting using anti-HP1γ (B, D) or anti-GFP (C).

Figure 4.

MeCP2 interacts via its N-terminal domain with the CSD domain of HP1. Schematic representation of the fusion proteins. Numbers represent amino acid coordinates. HEK293-EBNA cells were transfected with the plasmids indicated. Immunoprecipitations were done using either anti-mRFP or anti-GFP antibody. Input (I) and bound (B) fractions were loaded in the percentages mentioned and analyzed by western blotting using anti-GFP or anti-HP1γ (shown here is the endogenous HP1γ).