UC Berkeley

The size constraints of the nucleus necessitate condensation of eukaryotic DNA into chromatin. The fundamental subunit of chromatin is the nucleosome, ~147 bp of DNA wound about the histone octamer. Each octamer typically contains two copies each of the canonical histones H2A, H2B, H3 and H4. However, packaging DNA limits its availability to enzymes necessary for the maintenance and expression of our heritable material. More precisely, all chromatin-dependent processes--transcription, replication, recombination, and repair--are affected by the position and occupancy of nucleosomes. Given the transcriptional challenges inherent to DNA packaging, this dissertation documents studies aimed at addressing this fundamental question: How does a cell modify chromatin to achieve proper gene expression? To this end, I pursued functional studies in S. cerevisiae of a potential chromatin modifier, Yta7, and a novel chromatin modification, H2A.Z acetylation.

My studies on Yta7, a conserved bromodomain-containing protein with AAA-ATPase homology, identified this protein as a novel regulator of histone H3 eviction or degradation. Cells lacking Yta7 exhibited both increased levels of chromatin-incorporated histone H3 and decreased nucleosome spacing. Importantly, this modulation of H3 levels occurred post-transcriptionally. The yta7Δ mutant's transcriptional defects were partially suppressed by decreased dosage of histones H3 and H4, indicating the transcriptional impact of this increased nucleosome density. Additionally, Yta7 associated with inducible genes only upon transcriptional activation, with prominent enrichment within open reading frames. Yta7 and its ATPase function were required for the proper induction of these genes. Further, loss of local Yta7 activity resulted in a 5' to 3' gradient of H3 accumulation within a large open reading frame upon transcriptional activation, indicating a direct requirement for Yta7's regulation of H3 levels at that gene. In support of a direct mechanism of histone eviction or degradation by Yta7, Yta7 directly interacts with histone H3 in vitro. Further, over-expressing Yta7 resulted in a ~65% decrease in levels of chromatin-bound H3, as assayed by chromatin immunoprecipitation. Taken together, my studies support a model in which Yta7 utilizes the energy released upon ATP hydrolysis to evict and/or facilitate the degradation of histone H3. As bulk chromatin from cells without Yta7 exhibited increased nucleosome density and decreased dosage of either H3 or H4 suppresses the growth defect of the yta7Δ mutant, Yta7 presumably evicts or degrades an H3/H4 dimer or tetramer. Thus, these studies identified a protein that limited the extent of DNA packaging, thereby facilitating RNA polymerase activity upon transcription.

Restricting nucleosome density represents one mechanism for enabling transcriptional activation. Another possible mechanism is modifying the nucleosomes themselves, by covalently modifying the incorporated histones or changing which histones are incorporated. Although nucleosomes typically contain two copies of each canonical histone, histone variants, such as H2A.Z and H3.3, can be substituted at specific genomic locations for their cognate canonical histone. The histone H2A variant H2A.Z is conserved and essential in all multicellular eukaryotes assayed. Yeast cells lacking H2A.Z exhibit a broad range of chromatin-based phenotypes, including defective gene induction, genomic instability, and spreading of the Sir-silencing complex from heterochromatin into euchromatic domains. However, the importance of its N-terminal tail acetylations to these functions remained unclear. Therefore, I undertook studies to determine the genome-wide requirements for H2A.Z acetylation, assess the role of individual acetylation sites and identify which proteins might interpret these modifications.

My work on H2A.Z acetylation indicated that the transcriptome of cells lacking H2A.Z acetylation exhibited fewer expression defects than cells lacking H2A.Z. In contrast to proposed roles in transcriptional activation, cells lacking H2A.Z acetylation exhibited a bias toward up-regulation of genes. Genes that were down-regulated in these cells, however, were highly enriched for telomere-adjacent genes, consistent with Sir silencing antagonism or altered telomeric structure. In keeping with more recent work, my data supported a model of acetylation-site equivalence and additive activity of H2A.Z acetylation. Additionally, this work identified the double bromodomain-containing TFIID-associated Bdf1 as interacting with H2A.Z in an acetylation-dependent manner in vivo. As Bdf1 is required to inhibit Sir-complex spreading from the telomeres, this work provides insight into the potential mechanism of Bdf1's Sir complex antagonism. Further work will have to be performed to determine if the down-regulation of telomere-adjacent genes in cells that cannot acetylate H2A.Z is Sir-dependent and whether these genes' requirement for acetylated H2A.Z is a direct one. However, these studies on H2A.Z acetylation are consistent with a model in which H2A.Z acetylation prevents chromatin condensation by the Sir proteins, an alternative mechanism for maintaining proper gene expression.