Mechanisms Of Transcriptional Regulation By Proteins In The Nad+ Metabolic Pathway

Abstract

Poly(ADP-ribosyl)ation (PARylation) is an enzymatic reaction whereby ADPribose units from donor NAD+ molecules are covalently attached onto target proteins. The regulation of this reaction is overseen by two nuclear enzymes, Poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG), that modify target proteins in the nucleus by the addition and removal, respectively, of ADP-ribose polymers. While PARP-1 has generally been studied with respect to its role in DNA damage repair and cell death pathways, recent studies have revealed a role for PARP-1 in transcriptional regulation. The role of PARG in transcriptional regulation, however, is less characterized. In this study, I have investigated the coordinate patterns of gene regulation by PARP-1 and PARG in vivo using genomic and gene-specific analyses. Specifically, I show that PARP-1 and PARG coordinately regulate global patterns of gene expression by affecting genes in the same direction and with similar magnitudes. Further analysis revealed that PARP-1 and PARG localized to the promoters of both positively and negatively regulated target genes in parallel binding patterns. I also show that PARP-1 and PARG enzymatic activities are required for some, but not all, target genes. My results indicate that PARP-1 and PARG, two nuclear enzymes with opposing enzymatic activities, localize to target promoters and act in a similar, rather than antagonistic, manner to regulate gene expression. In a follow-up study, I have used a novel method known as Global Run-on Sequencing (GRO-seq) to define the role of PARP-1 on the estrogen-regulated transcriptome at the level of the nascent transcript, rather than steady-state mRNA levels. I have produced libraries from MCF-7 cells treated with vehicle or 17[beta]estradiol (E2) under three conditions: (i) a control knockdown; (ii) a control knockdown plus a PARP inhibitor, PJ34; and (iii) a PARP-1 knockdown. I have determined that the estrogen response is highly maintained under PARP-1 knockdown or inhibition. Accordingly, upon estrogen treatment, PARP-1 localization patterns are largely unaffected. However, deeper analyses reveal a small number of genes where PARP-1 knockdown or inhibition reduces the estrogen response at the transcription level (GRO-seq) and at the steady state mRNA level (RT-qPCR). The NAD+ metabolite generated from the PARP-1/PARG reaction, ADPribose (ADPR), is a small molecule ligand that is used by macro domain-containing proteins. The histone variant macroH2A1 is one such protein that has generally been studied with respect to its role in transcriptional repression on the inactive X chromosome. However, recent studies have begun to explore a role for macroH2A1 in autosomal gene regulation, as a transcriptional repressor and a transcriptional activator. Recent results from the Kraus lab have shown that the transcriptional coactivator Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) interacts with the macro domain of macroH2A1 in a ligand-independent manner and shows a similar genomic localization pattern. I have followed up these observations by investigating the mechanisms of gene regulation by macroH2A1 and its interacting proteins using genespecific analyses. Specifically, I have shown that macroH2A1 recruits PELP1 to macroH2A1 target gene promoters, but PELP1 is dispensable for the nucleosomal deposition of macroH2A1 at these loci. Together, macroH2A1 and PELP1 cooperatively regulate the expression of a subset of macroH2A1 target genes. Collectively, my studies expand our understanding of the cooperative actions of proteins in the NAD+ metabolic pathway in regulating transcription.