The CHD chromatin remodeling factors in schizosaccharomyces pombe

Sammanfattning: Regulation of chromatin structure is essential in a wide variety of processes including transcriptional regulation, recombination, replication, chromosome segregation, development and differentiation. The enzymes that are central in regulating chromatin structure can be classified into two major groups. The first group of proteins consists of the histone modifying enzymes that catalyse the addition or removal of posttranslational modifications of histones. The second group of proteins is the highly conserved ATP-dependent remodeling factors that modify the nucleosome structure. Evidence is emerging that these two groups of proteins are intimately linked in chromatin function. This thesis describes the roles of the S. pombe Hrp1 and Hrp3 CHD remodeling factors in chromatin regulation, which have been shown to be important in centromere function and transcriptional regulation. The Hrp remodeling factors are functionally linked to the histone chaperone Nap1 as well as acetylation and methylation activities. We have demonstrated that Hrp1 has both independent and overlapping roles with Hrp3 in regulating centromere assembly and function. Both hrp1 and hrp3 deficient cells are disrupted in centromere silencing and display various chromosome segregation defects indicative of functions at both the outer repeats and the central core of the centromere. These phenotypes are likely to originate from the requirement of Hrp1 in keeping the centromeres hypoacetylated and for maintaining the histone H3 variant CENP-A at the central core of the centromere. Genetic interactions combined with chromatin immunoprecipitation and fluorescent in situ hybridisation indicate that Hrp1 stimulates CENP-A assembly during DNA replication. In addition to their centromere functions, the Hrp remodeling factors contribute to transcriptional regulation by promoting histone removal. Biochemical purifications identified a physical interaction between Hrp1 and Hrp3 and with the histone chaperone Nap1. Consistent with the physical interaction data, genome wide analysis showed that the CHD remodeling factors together with Nap1 have a common function in removing histones particularly at promoter regions. Interestingly, we found that histone disassembly in coding regions by both Hrp1 and Hrp3 promote transcriptional activation. Cell synchronisation studies revealed that the Hrp1 dependent histone disassembly occurs in a DNA replication independent manner. A functional interaction between acetylation and remodeling activity was established based on the high degree of overlap between the Hrp ATPases, regions affected by Nap1 histone density, and corresponding histone deacetylase and histone acetylase targets. Finally, we discovered that regions with upregulated genes and altered levels of histone modifications in the HDAC clr6-1 mutant were significantly similar to equivalent lists for the histone demethyl transferase swm1 mutant. In addition, the same regions with upregulated genes and effects on histone modification levels in the swm1 and clr6 mutant overlapped with Hrp1 and Hrp3 binding targets. Thus, it is likely that Swm1 act in concert with Clr6 and Hrp1 to mediate transcriptional silencing. Thus, HDACs, HATs, and HMTs are intimately linked in vivo to CHD nucleosome remodeling factors as well as histone chaperones in centromere assembly and transcriptional regulation.

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