Direct Homo- and Hetero-Interactions of MeCP2 and MBD2

Abstract

Epigenetic marks like methylation of cytosines at CpG dinucleotides are essential for mammalian development and play a major role in the regulation of gene expression and chromatin architecture. The methyl-cytosine binding domain (MBD) protein family recognizes and translates this methylation mark. We have recently shown that the level of MeCP2 and MBD2, two members of the MBD family, increased during differentiation and their ectopic expression induced heterochromatin clustering in vivo. As oligomerization of these MBD proteins could constitute a factor contributing to the chromatin clustering effect, we addressed potential associations among the MBD family performing a series of different interaction assays in vitro as well as in vivo. Using recombinant purified MBDs we found that MeCP2 and MBD2 showed the stronger self and cross association as compared to the other family members. Besides demonstrating that these homo- and hetero-interactions occur in the absence of DNA, we could confirm them in mammalian cells using co-immunoprecipitation analysis. Employing a modified form of the fluorescent two-hybrid protein-protein interaction assay, we could clearly visualize these associations in single cells in vivo. Deletion analysis indicated that the region of MeCP2 comprising amino acids 163–309 as well the first 152 amino acids of MBD2 are the domains responsible for MeCP2 and MBD2 associations. Our results strengthen the possibility that MeCP2 and MBD2 direct interactions could crosslink chromatin fibers and therefore give novel insight into the molecular mechanism of MBD mediated global heterochromatin architecture.

Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure

“…MeCP2G.16–18 were generated using site-directed mutagenesis as described previously in detail ( 17 , 18 ). For expression in Sf9 insect cells, the Bac-to-Bac baculovirus expression system (Invitrogen) was used, employing a rat MeCP2G construct and a GFP construct described previously ( 16 , 19 ).…”

“…The nuclear pellet was dissolved in 0.2% Triton X-100 in 1× PBS supplemented with protease inhibitors as described in Ref. 19 , incubated for 10 min on ice and afterwards washed three times with 1× PBS. 300 μl of binding buffer (20 m m imidazole and 0.5 m NaCl in 1× PBS) was added, and sonification was performed twice for 20 s at 70% intensity followed by centrifugation (14,000 rpm, 10 min, 4 °C).…”

MicroRNAs Contribute to Induced Pluripotent Stem Cell Somatic Donor Memory

“…Namely MeCP2 is a member of the methylcytosine-binding domain family and acts as transcriptional regulator imposing local repressive chromatin structures through recruitment of histone-modifying enzymatic activities. It is highly expressed in HPCs as compared with more differentiated hematopoietic cells, because of its role as a global heterochromatin architecture organizer (37,38). Herein, overexpression of miR-155 resulted in a clear reduction in MeCP2 protein levels at day 14 of differentiation as assessed by Western blotting in Tra 1-85ϩ cells (Fig.…”

Astrocyte-specific regulation of hMeCP2 expression in Drosophila

“…In our studies, human MeCP2e2 and RTT R106W transcript levels significantly decrease in astrocyte subsets, while expression of the MeCP2 Δ166 allele is not altered. The region absent in MeCP2 Δ166 contains the MBD domain, a nuclear localizing signal, and a region designated HMG1, due to the amino acid composition similarity to high mobility group (HMG) proteins ( Adams et al, 2007 ; Adkins and Georgel, 2011 ; Becker et al, 2013 ; Ghosh et al, 2010 ). What types of evolutionarily conserved cis -regulatory elements are located in this area of the hMeCP2-coding region?…”