Chromatin Structure of the Inactive X Chromosome

X-inactivation is the unusual mode of gene regulation by which most genes on one of the two X chromosomes in female mammalian cells are transcriptionally silenced. The underlying mechanism for this widespread transcriptional repression is unknown. This thesis investigates two key aspects of the X-inactivation process. The first aspect is the correlation between chromatin structure and gene expression from the inactive X (Xi). Two features of the Xi chromatin DNA methylation and late replication timing have been shown to correlate with silencing of individual genes. This thesis describes a third feature that correlates with silencing of individual genes on the Xi: promoter-specific hypoacetylation of histone H4. Chromatin immunoprecipitation experiments demonstrated that transcriptionally active genes had elevated levels of H4 acetylation at their promoters on both the active and inactive X. In contrast, promoters of X-inactivated genes were markedly deacetylated, which coincided with the methylation of adjacent CG dinucleotides. This suggests that promoter hypoacetylation may be a key component of an X-inactivation machinery that operates at the level of individual genes. The second focus of this thesis is the nature of the association between XIST RNA and the Xi chromatin. Microscopy studies have shown that the noncoding XIST RNA colocalizes with the Xi. It is unclear, however, if this colocalization is due to physical association of XIST RNA with the Xi chromatin, or if it is a secondary consequence of XIST RNA and the Xi being sequestered to the same nuclear territory. This thesis provides evidence from chromatin immunoprecipitation experiments that XIST RNA is part of the Xi chromatin. First, XIST RNA can be co-precipitated by antisera against macroH2A, a histone H2A variant enriched in the Xi. Second, XIST RNA can be co-precipitated by antisera that recognize unacetylated, but not acetylated, isoforms of histones H3 and H4. As demonstrated in this thesis, hypoacetylated histone H4 is enriched at promoters of X-inactivated genes, whereas hyperacetylated histone H4 is found only at promoters of active genes. The preferential association of XIST RNA with unacetylated histones therefore suggests that the RNA is not uniformly associated with the Xi chromatin. Further evidence for this conclusion comes from fluorescence in situ hybridization in mouse cells, in which Xist RNA is shown to localize with the inactivated Zfx locus, but not with the Sts locus which escapes X-inactivation. These results raise the possibility that association with XIST RNA may be a fourth feature that correlates with silencing of individual genes on the Xi. This physical association between XIST RNA and the Xi chromatin may facilitate X-inactivation. Thesis Supervisor: Dr. Phillip A. Sharp, Title: Salvador E. Luria Professor of Biology