Scott Holmes; Robert Lane; Amy MacQueen; Don Oliver
Delicate DNA threads need to be both organized and maintain their accessibility to fit in the small dimension of the nucleus and allow biological processes to occur. The basic unit of DNA compaction is the nucleosome, which consists of a histone octamer (two copies of each histone H2A, H2B, H3 and H4), 147 base pairs of DNA that wrap around the octamer, and a linker histone protein H1, which sits outside the core nucleosome and binds to linker DNA at the entry and exit site.
Early in vitro studies suggested that linker histone H1 would be an essential protein since it facilitates higher-order chromatin structure formation, and functions as a transcription repressor. In vivo studies in budding yeast, however, showed that linker histone H1 is not essential for viability, but that H1 is important for chromatin structure and compaction, and its role in transcriptional regulation has been shown in a gene-specific manner.
Despite efforts to reveal the function of linker histone H1, a clear description of the role of H1 has yet to be described. The minor phenotypes observed when HHO1 is deleted suggest that Hho1 might function redundantly with nucleosome proteins. To investigate the role of H1, my research project explores the interactions between H1 and the core nucleosome proteins H3 and H4, as well as the histone variant H2A.Z. To assess the interactions between Hho1 and H3/H4 and the interaction between Hho1 and H2A.Z, I performed silencing assays at telomere and rDNA loci in budding yeast containing viable H3/H4 mutants and their HHO1 deletion counterpart, and examined changes in silencing in H3/H4 mutant strains upon deletion of HHO1. In addition, I also performed Chromatin Immunoprecipitation (ChIP) to look at whether H2A.Z affects Hho1 association at variant loci in the genome, or vice versa.
We found that deletion of HHO1 increased or decreased silencing dependent on the specific H3/H4 mutant allele, suggesting unique interactions between Hho1 and H3/H4, and the influence of Hho1 and H3/H4 interactions on silencing at telomere and rDNA regions are not correlated. Hho1-ChIP results showed that Hho1 association with chromatin was decreased at all loci examined in Δhtz1 (cells missing H2A.Z) strains. However, partially decreased Hho1 association with chromatin at some loci was observed in a Δswr1 strain, while similar to wild type level of Hho1 association was observed in some loci in Δswr1Δhtz1 cells. Our observations suggest that Hho1 association might correlate with the presence of H2A.Z, Hho1 might serve as a docking site, via an interaction with Arp4 (a component of SWR1 remodeling complex), for Swr1 to deposit H2A.Z onto the nucleosomes. Our hypothesis might help us to define the role of Hho1 and provide a basis for future experiments.
Huang, Junning, "Investigating the Role of Linker Histone in Saccharomyces Cerevisiae" (2017). Masters Theses. 143.
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