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UBC Theses and Dissertations

Regulation of Chromatin by FACT and NuA3 Chruscicki, Adam Tadeusz


The presence of nucleosomes over vast regions of genome negatively influences transcription creating a need for temporal and structural regul ation of chromatin. The default transcription-repressive state can be countered by addition of post-translational modifications by ch romatin modifiers or chromatin alteration by histone chaperones. Chaperones alter chromatin structure before the RNAPII passage and restore it afterwards. How modifiers and chaperones function within chromatin is an area of intense research. Here we show how two complexes, the yeast FACT and NuA3 contribute to chromatin function. Yeast Facilitates Chromatin Transactions (yFACT) is a histone chaperone that maintains chromatin structure. The model of yFACT function in vivo is a subject of much debate. We provide evidence that yFACT acts by stably binding and altering nucleosomes. We also present the EM structure of yFACT associated with nucleosomes. We find that yFACT-associated nucleosomes are hyper-acetylated and show evidence for it being an effect of a direct interaction between yFACT and NuA3. At the same time, acetylation of the H3K56 residue by the histone acetyltransferase Rtt109, acts to recruit yFACT to chromatin through a nucleosome-dependent mechanism. To determine the distribution of yFACT- associated nucleosomes we constructed a map of yFACT-nucleosome localization at single-nucleosome resolution. We show that while yFACT-bound nucleosomes are distributed thought the genome they are also positioned over the canonical Nucleosome Depleted Regions (NDR). The yFACT- bound nucleosomes are positioned around TATA-elements and Nhp6 - target sequences genome-wide. Deletion of NHP6A/B leads to loss of chromatin at these loci. Our work suggests the first ever sequence-dependent mechanism of histone chaperone action in Saccharomyces cerevisiae. We also examined NuA3 recruitment to chromatin and showed that Yng1, a subunit of NuA3 with a known affinity for H3K4me3 is a bivalent protein. While as previously shown, the C-terminal PHD finer of Yng1 binds to H3K4me3, the N-terminus of Yng1 can also bind to unmodified chromatin. Although these motifs can bind independently, together they increase the apparent association of Yng1 with chromatin. Yng1 binding to chromatin is regulated by the HDA1 complex.

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