Methyl-CpG-binding protein 2 (MeCP2) is a chromatin-binding protein that mediates transcriptional regulation, and is highly abundant in brain. The nature of its binding to reconstituted templates has been well characterized in vitro. However, its interactions with native chromatin are less understood. Here we show that MeCP2 displays a distinct distribution within fractionated chromatin from various tissues and cell types. Artificially induced global changes in DNA methylation by 3-aminobenzamide or 5-aza-2′-deoxycytidine, do not significantly affect the distribution or amount of MeCP2 in HeLa S3 or 3T3 cells. Most MeCP2 in brain is chromatin-bound and localized within highly nuclease-accessible regions. We also show that, while in most tissues and cell lines, MeCP2 forms stable complexes with nucleosome, in brain, a fraction of it is loosely bound to chromatin, likely to nucleosome-depleted regions. Finally, we provide evidence for novel associations of MeCP2 with mononucleosomes containing histone H2A.X, H3K9me2 and H3K27me3 in different chromatin fractions from brain cortex and in vitro. We postulate that the functional compartmentalization and tissue-specific distribution of MeCP2 within different chromatin types may be directed by its association with nucleosomes containing specific histone variants, and post-translational modifications.

The functional and structural chromatin roles of H2A.Z are still controversial. This work represents a further attempt to resolve the current functional and structural dichotomy by characterizing chromatin structures containing native H2A.Z. We have analyzed the role of this variant in mediating the stability of the histone octamer in solution using gel-filtration chromatography at different pH. It was found that decreasing the pH from neutral to acidic conditions destabilized the histone complex. Furthermore, it was shown that the H2A.Z-H2B dimer had a reduced stability. Sedimentation velocity analysis of nucleosome core particles (NCPs) reconstituted from native H2A.Z-containing octamers indicated that these particles exhibit a very similar behavior to that of native NCPs consisting of canonical H2A. Sucrose gradient fractionation of native NCPs under different ionic strengths indicated that H2A.Z had a subtle tendency to fractionate with more stabilized populations. An extensive analysis of the salt-dependent dissociation of histones from hydroxyapatite-adsorbed chromatin revealed that, whereas H2A.Z co-elutes with H3-H4, hyperacetylation of histones (by treatment of chicken MSB cells with sodium butyrate) resulted in a significant fraction of this variant eluting with the canonical H2A. These studies also showed that the late elution of this variant (correlated to enhanced binding stability) was independent of the chromatin size and of the presence or absence of linker histones.

Despite the identification of H2A.Bbd as a new vertebrate-specific replacement histone variant several years ago, and despite the many in vitro structural characterizations using reconstituted chromatin complexes consisting of this variant, the existence of H2A.Bbd in the cell and its location has remained elusive. Here, we report that the native form of this variant is present in highly advanced spermiogenic fractions of mammalian testis at the time when histones are highly acetylated and being replaced by protamines. It is also present in the nucleosomal chromatin fraction of mature human sperm. The ectopically expressed non-tagged version of the protein is associated with micrococcal nuclease-refractory insoluble fractions of chromatin and in mouse (20T1/2) cell line, H2A.Bbd is enriched at the periphery of chromocenters. The exceedingly rapid evolution of this unique X-chromosome-linked histone variant is shared with other reproductive proteins including those associated with chromatin in the mature sperm (protamines) of many vertebrates. This common rate of evolution provides further support for the functional and structural involvement of this protein in male gametogenesis in mammals.