Novel Functional Residues in the Core Domain of Histone H2B Regulate Yeast Gene Expression and Silencing and Affect the Response to DNA Damage

Abstract

Previous studies have identified novel modifications in the core fold domain of histone H2B, but relatively little is known about the function of these putative histone modification sites. We have mutated core modifiable residues that are conserved in Saccharomyces cerevisiae histone H2B and characterized the effects of the mutants on yeast silencing, gene expression, and the DNA damage response. We identified three histone H2B core modifiable residues as functionally important. We find that mutating H2B K49 in yeast confers a UV sensitivity phenotype, and we confirm that the homologous residue in human histone H2B is acetylated and methylated in human cells. Our results also indicate that mutating H2B K111 impairs the response to methyl methanesulfonate (MMS)-induced DNA lesions and disrupts telomeric silencing and Sir4 binding. In contrast, mutating H2B R102 enhances silencing at yeast telomeres and the HML silent mating loci and increases Sir4 binding to these regions. The H2B R102A mutant also represses the expression of endogenous genes adjacent to yeast telomeres, which is likely due to the ectopic spreading of the Sir complex in this mutant strain. We propose a structural model by which H2B R102 and K111 regulate the binding of the Sir complex to the nucleosome.

We thank Robert Morris and Brandon Kyriss for helpful comments on the manuscript. We are grateful to Kasey Vargo for assistance in constructing a few of the histone H2B mutants, and we thank Stacy Hathcox and Katie Bergstrom for their assistance with the deletion of the endogenous H2A and H2B genes in the HML silencing tester strain. We also thank Stacy Hathcox for her help in testing the anti-H2B K111me2 antibodies, and we thank Erna Agmata for her assistance with the peptide dot blots. We are grateful to Derek Pouchnik for technical assistance with DNA microarray experiments. We thank Ray Reeves for the MCF-7 cells and Julie Stanton and Li Mao for preparing the MCF-7 cell protein extracts. We thank Daniel Gottschling for the gift of the yeast silencing strains and David Allis for the gift of yeast strain JHY205 and plasmid pQQ18.

We gratefully acknowledge support from the Washington State University College of Sciences Undergraduate Minigrant program (Kasey Vargo and J.A.S.), from a NIH Biotechnology Graduate Training grant (M.N.M.K and I.J.G.), and from the Washington State Fraternal Order of Eagles.