Chemical Genetic and Genomic Discovery of PARP-1-Dependent Mechansims of Transcriptional Regulation




Gibson, Bryan Andrew

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Poly(ADP-ribose) polymerases, or PARPs, are a family of enzymes that modulate diverse biological processes through covalent transfer of ADP-ribose from NAD+ onto target proteins. These targets of post-translational modification as well as the genomic targets, or binding sites, to which the nuclear PARP family members localize reflects the molecular biology and cellular function of an individual PARP. Given that PARP proteins are implicated in the most devastating of human disease, including cancer, heart disease, stroke, and neuropathology, a deeper understanding of their proteomic and genomic targets may guide effective therapeutic intervention within this family of enzymes. Here, I report the development of new methodologies in an effort to identify the targets of PARPs. To identify the sites of ADP- ribsoylation, I've developed a simple and robust analog-sensitive approach for PARPs, which allows PARP-specific clickable ADP-ribosylation. Using this approach, I have mapped hundreds of protein targets and sites of ADP-ribosylation for PARPs 1, 2, and 3. I found that PARP-1 ADP-ribosylates and inhibits RNA-binding by NELF, a protein complex that regulates promoter-proximal pausing by RNA polymerase II. I have used this analog-sensitive approach to discover genomic sites of PARP-1-mediated ADP-ribosylation and their relationship to paused RNA Polymerase II. Furthermore, I have found that knockdown of PARP-1 resulted in the accumulation of paused RNA Polymerase II, implicating PARP-1 in RNA Polymerase II elongation through ADP-ribosylation and inhibition of NELF. PARP-1 is activated by both DNA lesions and nucleosomally wrapped DNA, the latter of which is a likely substrate for PARP-1 in its role as a regulator of transcription. In order to identify the nucleosomes that PARP-1 binds, I've developed an MNase ChIP-seq method using crosslinked cells called XL- MNase ChIP-seq. This technical advance has revealed that PARP-1 binds a nuclease-sensitive nucleosome that spans the "nucleosome free" region of regulatory elements of the genome, implicating PARP-1 as a modulator of genomic access at regulatory regions across the genome. The utility of these new methodologies is evident in their use in the discovery of new biological roles for PARP-1 in transcriptional regulation.

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