| The maintenance of dynamic chromatin structures is critical for the proper regulation of cellular activities. The plasticity of chromatin structures can be mediated in several ways, two of which include the incorporation of histone variants and the activities of trans-acting factors. In this dissertation, biochemical methods were used to determine the effects of the histone variant H2A.Z or the methyl-CpG binding protein 2 (MeCP2) on the structural composition of native chromatin.;MeCP2 is a chromatin binding protein best known for its ability to repress transcription. While its roles in neuron development have been well-studied, little is known of its interactions within native chromatin. Shortly after MeCP2 was discovered, it was postulated that MeCP2 would behave as a global repressor. However, recent findings have contested this idea. If MeCP2 does act as a universal silencer, it was hypothesized that changes to global chromatin modifications would affect the distribution of MeCP2 within chromatin. HeLa S3 cultures were chemically treated with 3-aminobenzamide or butyrate to induce either DNA hypermethylation or histone hyperacetylation. Neither treated culture resulted in a redistribution of MeCP2 within chromatin. Moreover, the majority of MeCP2 was present within nuclease-accessible, active chromatin. Interestingly, the butyrate treatment resulted in proportional losses of MeCP2 within fractionated chromatin that were not due to changes in MeCP2 transcription. MeCP2 was also observed to bind to mononucleosomes containing DNA that was >146 bp - ∼160 bp. These results suggested that MeCP2 does not act as an indiscriminate silencer, but more likely as a specific transcriptional regulator.;Most studies of MeCP2 interactions with chromatin were performed using reconstituted chromatin templates in vitro. However, it is not known if MeCP2 interacts with chromatin in a tissue-specific manner. In addition, as MeCP2 has a broad distribution throughout all chromatin types, it is not known if histone variants or PTMs influence MeCP2 deposition. Therefore, the tissue specificity of MeCP2 binding and the influence of nucleosomal components were investigated. MeCP2 has a differential v distribution throughout chromatin extracted from rat brain, liver, and testis. The brain has significantly more MeCP2 than the liver or testis and this was reflected in the MECP2 mRNA amounts. Using native co-immunoprecipitations, MeCP2 was shown to interact with mononucleosomes containing specific histone variants and PTMs: H2AX, H3K27me3, and H3K9me2. These novel interactions may further specialize the MeCP2-bound chromatin regions.;Finally, two novel hypotheses regarding the regulation of MeCP2 are proposed. In the first, the regulation of MeCP2 turnover is proposed to occur through the poly-ubiquitination of the two MeCP2 PEST domains, followed by proteolytic degradation. The second hypothesis proposes that the use of histone deacetylase inhibitors could be used to control the levels of MeCP2 expression, in conjunction with gene therapies, for the treatment of Rett syndrome.;Early, independent biophysical studies of the stability of reconstituted H2A.Z chromatin structures yielded contradictory results. As these studies used H2A.Z expressed as a recombinant protein, it was possible that the absence of any essential folding or post-translational modifications (PTMs) may have been responsible for the diametric findings. To resolve this issue, the stability of various native chromatin structures containing H2A.Z was determined. Using gel filtration chromatography, sucrose gradient sedimentation, and hydroxyapatite chromatography, the partitioning of H2A.Z within dissociated octamers, mononucleosomes, and chromatin fibres were respectively assessed. Within all three structures, H2A.Z associated with stabilized forms. However, the salt-dependent thermal analysis of H2A.Z-H2B dimers by circular dichroism showed that the variant dimer was largely unstructured. The deposition of H2A.Z also occurred independently of linker histones. |
