Poly (ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme involved in DNA repair, chromatin remodeling and gene expression. PARP1 interactions with chromatin architectural multi-protein complexes (i.e. nucleosomes) alter chromatin structure resulting in changes in gene expression. Chromatin structure impacts gene regulatory processes including transcription, splicing, DNA repair, replication and recombination. It is important to delineate whether PARP1 randomly associates with nucleosomes or is present at specific nucleosome regions throughout the cell genome. We performed genome-wide association studies in breast cancer cell lines to address these questions. Our studies show that PARP1 associates with epigenetic regulatory elements genome-wide, such as active histone marks, CTCF and DNase hypersensitive sites. Additionally, the binding of PARP1 to chromatin genome-wide is mutually exclusive with DNA methylation pattern suggesting a functional interplay between PARP1 and DNA methylation. Indeed, inhibition of PARylation results in genome-wide changes in DNA methylation patterns. Our results suggest that PARP1 controls the fidelity of gene transcription and marks actively transcribed gene regions by selectively binding to transcriptionally active chromatin. These studies provide a platform for developing our understanding of PARP1’s role in gene regulation.

Background: Histone wrapping of DNA into nucleosomes almost certainly evolved in the Archaea, and predates Eukaryotes. In Eukaryotes, nucleosome positioning plays a central role in regulating gene expression and is directed by primary sequence motifs that together form a nucleosome positioning code. The experiments reported were undertaken to determine if archaeal histone assembly conforms to the nucleosome positioning code. Results: Eukaryotic nucleosome positioning is favored and directed by phased helical repeats of AA/TT/AT/TA and CC/GG/CG/GC dinucleotides, and disfavored by longer AT-rich oligonucleotides. Deep sequencing of genomic DNA protected from micrococcal nuclease digestion by assembly into archaeal nucleosomes has established that archaeal nucleosome assembly is also directed and positioned by these sequence motifs, both in vivo in Methanothermobacter thermautotrophicus and Thermococcus kodakarensis and in vitro in reaction mixtures containing only one purified archaeal histone and genomic DNA. Archaeal nucleosomes assembled at the same locations in vivo and in vitro, with much reduced assembly immediately upstream of open reading frames and throughout the ribosomal rDNA operons. Providing further support for a common positioning code, archaeal histones assembled into nucleosomes on eukaryotic DNA and eukaryotic histones into nucleosomes on archaeal DNA at the same locations. T. kodakarensis has two histones, designated HTkA and HTkB, and strains with either but not both histones deleted grow normally but do exhibit transcriptome differences. Comparisons of the archaeal nucleosome profiles in the intergenic regions immediately upstream of genes that exhibited increased or decreased transcription in the absence of HTkA or HTkB revealed substantial differences but no consistent pattern of changes that would correlate directly with archaeal nucleosome positioning inhibiting or stimulating transcription. Conclusions: The results obtained establish that an archaeal histone and a genome sequence together are sufficient to determine where archaeal nucleosomes preferentially assemble and where they avoid assembly. We confirm that the same nucleosome positioning code operates in Archaea as in Eukaryotes and presumably therefore evolved with the histone-fold mechanism of DNA binding and compaction early in the archaeal lineage, before the divergence of Eukaryotes.

Specialized chromatin structures such as nucleosomes with specific histone modifications decorate exons in eukaryotic genomes, suggesting a functional connection between chromatin organization and the regulation of pre-mRNA splicing. Through profiling the functional location of Poly (ADP) ribose polymerase, we observed that it is associated with the nucleosomes at exon/intron boundaries of specific genes, suggestive of a role for this enzyme in alternative splicing. Poly (ADP) ribose polymerase has previously been implicated in the PARylation of splicing factors as well as regulation of the histone modification H3K4me3, a mark critical for co-transcriptional splicing. In light of these studies, we hypothesized that interaction of the chromatin-modifying factor, Poly (ADP) ribose polymerase with nucleosomal structures at exon–intron boundaries, might regulate pre-mRNA splicing. Using genome-wide approaches validated by gene-specific assays, we show that depletion of PARP1 or inhibition of its PARylation activity results in changes in alternative splicing of a specific subset of genes. Furthermore, we observed that PARP1 bound to RNA, splicing factors and chromatin, suggesting that Poly (ADP) ribose polymerase serves as a gene regulatory hub to facilitate co-transcriptional splicing. These studies add another function to the multi-functional protein, Poly (ADP) ribose polymerase, and provide a platform for further investigation of this protein’s function in organizing chromatin during gene regulatory processes.

Recent studies implicate Poly (ADP-ribose) polymerase 1 (PARP1) in alternative splicing regulation, and PARP1 may be an RNA-binding protein. However, detailed knowledge of RNA targets and the RNA-binding region for PARP1 are unknown. Here we report the first global study of PARP1–RNA interactions using PAR–CLIP in HeLa cells. We identified a largely overlapping set of 22 142 PARP1–RNA-binding peaks mapping to mRNAs, with 20 484 sites located in intronic regions. PARP1 preferentially bound RNA containing GC-rich sequences. Using a Bayesian model, we determined positional effects of PARP1 on regulated exon-skipping events: PARP1 binding upstream and downstream of the skipped exons generally promotes exon inclusion, whereas binding within the exon of interest and intronic regions closer to the skipped exon promotes exon skipping. Using truncation mutants, we show that removal of the Zn1Zn2 domain switches PARP1 from a DNA binder to an RNA binder. This study represents a first step into understanding the role of PARP1–RNA interaction. Continued identification and characterization of the functional interplay between PARPs and RNA may provide important insights into the role of PARPs in RNA regulation.

Arsenic (As) exposure is a significant worldwide environmental health concern. Low dose, chronic arsenic exposure has been associated with a higher than normal risk of skin, lung, and bladder cancer, as well as cardiovascular disease and diabetes. While arsenic-induced biological changes play a role in disease pathology, little is known about the dynamic cellular changes resulting from arsenic exposure and withdrawal. In these studies, we sought to understand the molecular mechanisms behind the biological changes induced by arsenic exposure. A comprehensive global approach was employed to determine genome-wide changes to chromatin structure, transcriptome patterns and splicing patterns in response to chronic low dose arsenic and its subsequent withdrawal. Our results show that cells exposed to chronic low doses of sodium arsenite have distinct temporal and coordinated chromatin, gene expression, and miRNA changes consistent with differentiation and activation of multiple biochemical pathways. Most of these temporal patterns in gene expression are reversed when arsenic is withdrawn. However, some gene expression patterns remained altered, plausibly as a result of an adaptive response by cells. Additionally, the correlation of changes to gene expression and chromatin structure solidify the role of chromatin structure in gene regulatory changes due to arsenite exposure. Lastly, we show that arsenite exposure influences gene regulation both at the initiation of transcription as well as at the level of splicing. Our results show that adaptation of cells to iAs-mediated EMT is coupled to changes in chromatin structure effecting differential transcriptional and splicing patterns of genes. These studies provide new insights into the mechanism of iAs-mediated pathology, which includes epigenetic chromatin changes coupled with changes to the transcriptome and splicing patterns of key genes.