Date of Award
histone H3 variants, chromatin, embryonic stem cells, H3.3 deposition, Hira
The eukaryotic genome is composed of chromatin, a complex polymer of genomic DNA, RNA, and closely associated proteins. Histone proteins form the core of the nucleosome, the fundamental repeating unit of chromatin. Variant histone proteins play critical roles in the epigenetic regulation of gene expression and in the development of multicellular organisms. In this thesis, I describe the first genome-wide profiles of histone H3 variants in pluripotent mammalian embryonic stem (ES) cells, and I establish the dependence and independence of these patterns on the histone chaperone Hira. To distinguish H3 variants, I use designed zinc finger nucleases (ZFNs) to rapidly knock epitope tags into a single allele of the endogenous histone H3.3B gene in mouse ES cells. Genome-wide analysis reveals that H3.3 is enriched in specific patterns at active and repressed genes with high CpG content promoters, including developmentally repressed bivalent H3K4me3 / H3K27me3 genes in ES cells, in addition to transcribed non-coding regions, telomeres, ribosomal DNA (rDNA), and genic and intergenic transcription factor binding sites (TFBS). Transcriptional termination sites of highly transcribed genes are marked by peaks of H3.3 and phosphorylated RNA polymerase II. Differentiation of ES cells into neural precursor cells (NPCs) leads to specific changes in H3.3 localization, demonstrating that the localization of H3.3 is dependent on cellular state. Targeted gene editing of H3.3B to H3.2 or H3.1 using ZFNs demonstrates that these patterns are dependent on the amino acid sequence of endogenous H3.3. Using wild-type and Hira -/- ES cells, I show that the H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for deposition of H3.3 at rDNA, telomeres, and specific TFBS. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Using Atrxflox and Atrxnull mouse ES cells, I find that Atrx is specifically required for Hira-independent enrichment of H3.3 at telomeres and rDNA, and for repression of telomeric and ribosomal RNA. Overall, the data in this thesis demonstrate that multiple and distinct pathways are responsible for H3.3 deposition at specific genomic locations in mammalian cells.
Goldberg, Aaron David, "Genome-Wide Localization And Novel Deposition Pathways Of Histone Variant H3.3 In Embryonic Stem And Neuronal Precursor Cells" (2010). Student Theses and Dissertations. 98.