Regulation Of Chromatin Structre And Transcription By Poly(Adp-Ribose) Polymerase -1
The process of transcription, which is a vital step in the cellular response to physiological and environmental stimuli, is highly regulated at multiple levels. Many proteins are involved in orchestrating transcriptional responses, including proteins that modulate the physiological template for transcription, chromatin. One such protein is the highly abundant nuclear enzyme Poly(ADP-ribose) Polymerase-1 , or PARP-1. Although PARP-1 has classically been studied with relation to its role in the detection and repair of DNA damage, recent work has uncovered physiological functions of PARP-1 in regulating transcription. PARP-1 has been shown to have a range of functions in transcriptional regulating, including acting as a co-activator and as a modulator of chromatin structure. Although there are increasing numbers of studies revealing roles for PARP-1 in many processes, the molecular mechanisms of PARP-1 action in most pathways is largely unknown. In this study, I have investigated transcriptional regulation by PARP-1 in vivo, using both genomic and gene-specific analyses in breast cancer cells and human cardiomyocytes. Using chromatin immunoprecipitation coupled with DNA microarrays (ChIPchip), I show that PARP-1 binds to active promoters in MCF-7 breast cancer cells, and that at genes that are positively-regulated by PARP-1, it acts to exclude the binding of linker histone H1. Further analysis revealed that exclusion of H1 from promoters allows for a favorable chromatin structure which in turn permits the binding of RNA Polymerase II at target genes. This open chromatin conformation also requires methylated histones, which PARP-1 maintains by PARylating and preventing recruitment of the demethylase KDM5B, a pathway which is also utilized by signaldependent transcription. Besides breast cancer, PARP-1 plays a prominent role in other pathologies, one of which is the progression of cardiovascular disease (CVD). I use a human cardiomyocyte cell line to show that TNF-alpha can drastically increase the binding of the transcription factor NF-kappa-B to chromatin, and that this causes changes in the gene expression profile of these cells. PARP-1 is known to cooperate with NF-kappa-B at target genes. I show that in human cardiomyocytes, PARP-1 is required for NF-kappa-B upregulated genes, but not for down-regulated genes, confirming its role as an activator of NF-kappa-B-dependent transcription. Interestingly, I see that the majority of NF-kappa-B binding in the presence of TNF-alpha is not to canonical NF-kappa-B binding sites, suggesting the the majority of the NF-kappa-B response is intricately dependent on other transcription factors, and I show that one of these factors, ATF2, is vital for recruiting NF-kappa-B to promoters and regulating transcription. It will be interesting to further investigate how PARP-1 and ATF2 may be collaborating at target genes. Together, these data demonstrate a conserved binding pattern of PARP-1 on chromatin across cell types, and establish novel connections between PARP-1, signaling pathways, chromatin and gene expression.
dissertation or thesis