Chromatin Remodeling Around Nucleosome Free Regions Represses Non-Coding RNA By DNA Looping and Transcription Factor Dependent Targeting of Isw2
Yadon, Adam Nicholas
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The efficient three-dimensional packaging of DNA into eukaryotic nuclei is accomplished through spatially organizing and compacting DNA into chromatin. Maintaining proper access to DNA, by modulation of either the location of DNA within the nucleus or the positions of nucleosomes, is essential for regulating DNA-dependent processes. The work in this dissertation focuses on elucidating the functions and molecular mechanisms by which the three-dimensional packaging of eukaryotic DNA affects DNA-dependent processes. Here I show that the ATP-dependent chromatin remodeling enzyme Isw2 is a global repressor of non-coding RNA (ncRNA) transcription that initiates from the edges of nucleosome free regions (NFRs) genome-wide. Isw2-dependent chromatin remodeling activity is required to reduce accessibility to DNA by sliding nucleosomes toward NFRs and occluding transcription start sites. This work establishes Isw2 as the first factor that functions to reduce the size of NFRs in vivo. My evidence also suggests that proper repression of ncRNA by Isw2 prevents transcriptional interference of mRNA, providing an important biological role for Isw2-dependent chromatin remodeling. Analysis of the targeting mechanisms of Isw2 to NFRs uncovered the sequence-specific transcription factors (TFs) Ume6, Nrg1, Cin5, and Sok2 as globally required for Isw2 recruitment to many target loci genome-wide. This establishes the first comprehensive genome-wide map for TF-dependent targeting of a chromatin remodeling enzyme. The observation that Isw2 is targeted in a TF-dependent fashion to a large number of loci not containing an annotated TF binding site led to the discovery that Isw2 can also be targeted to specific loci via Ume6- and TFIIB-dependent DNA looping. Both Ume6 and TFIIB-dependent DNA looping are required to maintain transcriptional repression at target loci. I have thus identified DNA looping as a previously unknown mechanism to target a chromatin remodeling enzyme and uncovered a novel physiological role for DNA looping. My work has led to a better understanding of how the three dimensional packaging of DNA into eukaryotic nuclei affects a DNA-dependent process in which transcriptional repression is facilitated by DNA-looping mediated TF-dependent targeting of a chromatin remodeling enzyme.