Molecular Biology and Biochemistry
Chromatin is responsible for processes ranging from gene expression to
chromosome dynamics during cell division. The fundamental organizing structure of chromatin is composed of a histone octamer (two copies each of H2A, H2B, H3, and H4) that packages 147 bp of DNA into a unit called the nucleosome. Histone H1 is a fifth histone that resides outside of the nucleosome and interacts at the DNA entrance and exit site of the nucleosome and the linker DNA that connects adjacent nucleosomes.
The function of histone H1 is poorly understood. Early in vitro studies
suggested that histone H1 facilitated chromatin condensation. As a result of its role in DNA packaging, it was thought that histone H1 might serve as a transcriptional repressor. However, initial in vivo studies in Saccharomyces cerevisiae have shown that Hho1 (histone H one) was not essential for cell viability, and its deletion yielded no detectable changes in chromatin structure, growth rates, or transcriptional silencing at the constitutively silenced loci.
In order to clarify the role of Hho1 and its mechanism of function, my
research project aims to explore the interaction between Hho1 and the histone
octamer proteins H3 and H4. The characterization of this relationship will help us determine the nature of Hho1’s influence on transcriptional silencing.
To examine the Hho1 and nucleosome interaction, we eliminated the HHO1
gene in strains containing particular histone H3 or histone H4 mutations and looked for the suppression or exacerbation of silencing phenotypes at the three constitutively silenced loci in Saccharomyces cerevisiae: the HM loci, the telomeres, and the rDNA repeats. We selected nucleosomal mutants that were most likely to impact transcriptional silencing or interact with Hho1. These regions of these mutations included histone tails, the DNA entrance and exit site, the LRS (loss of rDNA silencing) region, and modifiable residues.
We observe significant changes in transcriptional silencing in nucleosomal
mutants upon the deletion of HHO1, indicating a functional interaction between Hho1 and the nucleosome. This interaction is complicated, as the deletion of HHO1 may decrease or increase transcriptional silencing depending on the mutant allele. Interestingly, the influences of Hho1 on transcriptional silencing at the telomere and rDNA do not correlate. We took advantage of these differences to identify mutants that significantly contribute to global or locus-specific changes in silencing. Strikingly, using a Monte Carlo method bootstrapping without replacement approach, we determined that histone H3 tail mutants significantly contribute to a decrease in silencing and that lysine mutants significantly contribute to an increase in silencing at both the telomere and rDNA upon the deletion of HHO1. This suggests that these mutants impact a global Hho1-dependent transcriptional silencing effect. In addition, histone H4 mutants are significantly overrepresented in strains that exhibit a decrease in telomeric and an increase in rDNA silencing upon the deletion of HHO1. These mutants, therefore, impact a locus-specific Hho1-dependent transcriptional silencing effect. Our observations lead to hypotheses about the role of Hho1 that provide a basis for future experimentation.
Schilit, Samantha Linn Price, "The Role of Histone H1 and Its Interaction with the Nucleosome in Saccharomyces cerevisiae" (2011). Masters Theses. 13.
© Copyright is owned by author of this document