Beyond the Usual Suspects: Examining the Role of Understudied Histone Variants in Breast Cancer.

The incorporation of histone variants has structural ramifications on nucleosome dynamics and stability. Due to their unique sequences, histone variants can alter histone-histone or histone-DNA interactions, impacting the folding of DNA around the histone octamer and the overall higher-order structure of chromatin fibers. These structural modifications alter chromatin compaction and accessibility of DNA by transcription factors and other regulatory proteins to influence gene regulatory processes such as DNA damage and repair, as well as transcriptional activation or repression. Histone variants can also generate a unique interactome composed of histone chaperones and chromatin remodeling complexes. Any of these perturbations can contribute to cellular plasticity and the progression of human diseases. Here, we focus on a frequently overlooked group of histone variants lying within the four human histone gene clusters and their contribution to breast cancer.

A dynamic model of inorganic arsenic-induced carcinogenesis reveals an epigenetic mechanism for epithelial-mesenchymal plasticity.

Inorganic arsenic (iAs) causes cancer by initiating dynamic transitions between epithelial and mesenchymal cell phenotypes. These transitions transform normal cells into cancerous cells, and cancerous cells into metastatic cells. Most in vitro models assume that transitions between states are binary and complete, and do not consider the possibility that intermediate, stable cellular states might exist. In this paper, we describe a new, two-hit in vitro model of iAs-induced carcinogenesis that extends to 28 weeks of iAs exposure. Through week 17, the model faithfully recapitulates known and expected phenotypic, genetic, and epigenetic characteristics of iAs-induced carcinogenesis. By 28 weeks, however, exposed cells exhibit stable, intermediate phenotypes and epigenetic properties, and key transcription factor promoters (SNAI1, ZEB1) enter an epigenetically poised or bivalent state. These data suggest that key epigenetic transitions and cellular states exist during iAs-induced epithelial-to-mesenchymal transition (EMT), and that it is important for our in vitro models to encapsulate all aspects of EMT and the mesenchymal-to-epithelial transition (MET). In so doing, and by understanding the epigenetic systems controlling these transitions, we might find new, unexpected opportunities for developing targeted, cell state-specific therapeutics.

Cooperative nucleic acid binding by Poly ADP-ribose polymerase 1.

Poly (ADP)-ribose polymerase 1 (PARP1) is an abundant nuclear protein well-known for its role in DNA repair yet also participates in DNA replication, transcription, and co-transcriptional splicing, where DNA is undamaged. Thus, binding to undamaged regions in DNA and RNA is likely a part of PARP1's normal repertoire. Here we describe analyses of PARP1 binding to two short single-stranded DNAs, a single-stranded RNA, and a double stranded DNA. The investigations involved comparing the wild-type (WT) full-length enzyme with mutants lacking the catalytic domain (∆CAT) or zinc fingers 1 and 2 (∆Zn1∆Zn2). All three protein types exhibited monomeric characteristics in solution and formed saturated 2:1 complexes with single-stranded T20 and U20 oligonucleotides. These complexes formed without accumulation of 1:1 intermediates, a pattern suggestive of positive binding cooperativity. The retention of binding activities by ∆CAT and ∆Zn1∆Zn2 enzymes suggests that neither the catalytic domain nor zinc fingers 1 and 2 are indispensable for cooperative binding. In contrast, when a double stranded 19mer DNA was tested, WT PARP1 formed a 4:1 complex while the ∆Zn1Zn2 mutant binding saturated at 1:1 stoichiometry. These deviations from the 2:1 pattern observed with T20 and U20 oligonucleotides show that PARP's binding mechanism can be influenced by the secondary structure of the nucleic acid. Our studies show that PARP1:nucleic acid interactions are strongly dependent on the nucleic acid type and properties, perhaps reflecting PARP1's ability to respond differently to different nucleic acid ligands in cells. These findings lay a platform for understanding how the functionally versatile PARP1 recognizes diverse oligonucleotides within the realms of chromatin and RNA biology.

Exploring the interplay between PARP1 and circRNA biogenesis and function.

PARP1 (poly-ADP-ribose polymerase 1) is a multidomain protein with a flexible and self-folding structure that allows it to interact with a wide range of biomolecules, including nucleic acids and target proteins. PARP1 interacts with its target molecules either covalently via PARylation or non-covalently through its PAR moieties induced by auto-PARylation. These diverse interactions allow PARP1 to participate in complex regulatory circuits and cellular functions. Although the most studied PARP1-mediated functions are associated with DNA repair and cellular stress response, subsequent discoveries have revealed additional biological functions. Based on these findings, PARP1 is now recognized as a major modulator of gene expression. Several discoveries show that this multifunctional protein has been intimately connected to several steps of mRNA biogenesis, from transcription initiation to mRNA splicing, polyadenylation, export, and translation of mRNA to proteins. Nevertheless, our understanding of PARP1's involvement in the biogenesis of both coding and noncoding RNA, notably circular RNA (circRNA), remains restricted. In this review, we outline the possible roles of PARP1 in circRNA biogenesis. A full examination of the regulatory roles of PARP1 in nuclear processes with an emphasis on circRNA may reveal new avenues to control dysregulation implicated in the pathogenesis of several diseases such as neurodegenerative disorders and cancers. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Processing > Splicing Regulation/Alternative Splicing.

Co-Targeting Plk1 and DNMT3a in Advanced Prostate Cancer.

Because there is no effective treatment for late-stage prostate cancer (PCa) at this moment, identifying novel targets for therapy of advanced PCa is urgently needed. A new network-based systems biology approach, XDeath, is developed to detect crosstalk of signaling pathways associated with PCa progression. This unique integrated network merges gene causal regulation networks and protein-protein interactions to identify novel co-targets for PCa treatment. The results show that polo-like kinase 1 (Plk1) and DNA methyltransferase 3A (DNMT3a)-related signaling pathways are robustly enhanced during PCa progression and together they regulate autophagy as a common death mode. Mechanistically, it is shown that Plk1 phosphorylation of DNMT3a leads to its degradation in mitosis and that DNMT3a represses Plk1 transcription to inhibit autophagy in interphase, suggesting a negative feedback loop between these two proteins. Finally, a combination of the DNMT inhibitor 5-Aza-2'-deoxycytidine (5-Aza) with inhibition of Plk1 suppresses PCa synergistically.

Chronic Exposure to Cadmium Induces Differential Methylation in Mice Spermatozoa.

Cadmium exposure is ubiquitous and has been linked to diseases including cancers and reproductive defects. Since cadmium is nonmutagenic, it is thought to exert its gene dysregulatory effects through epigenetic reprogramming. Several studies have implicated germline exposure to cadmium in developmental reprogramming. However, most of these studies have focused on maternal exposure, while the impact on sperm fertility and disease susceptibility has received less attention. In this study, we used reduced representation bisulfite sequencing to comprehensively investigate the impact of chronic cadmium exposure on mouse spermatozoa DNA methylation. Adult male C57BL/J6 mice were provided water with or without cadmium chloride for 9 weeks. Sperm, testes, liver, and kidney tissues were collected at the end of the treatment period. Cadmium exposure was confirmed through gene expression analysis of metallothionein-1 and 2, 2 well-known cadmium-induced genes. Analysis of sperm DNA methylation changes revealed 1788 differentially methylated sites present at regulatory regions in sperm of mice exposed to cadmium compared with vehicle (control) mice. Furthermore, most of these differential methylation changes positively correlated with changes in gene expression at both the transcription initiation stage as well as the splicing levels. Interestingly, the genes targeted by cadmium exposure are involved in several critical developmental processes. Our results present a comprehensive analysis of the sperm methylome in response to chronic cadmium exposure. These data, therefore, highlight a foundational framework to study gene expression patterns that may affect fertility in the exposed individual as well as their offspring, through paternal inheritance.

The multifaceted role of PARP1 in RNA biogenesis.

Poly(ADP-ribose) polymerases (PARPs) are abundant nuclear proteins that synthesize ADP ribose polymers (pADPr) and catalyze the addition of (p)ADPr to target biomolecules. PARP1, the most abundant and well-studied PARP, is a multifunctional enzyme that participates in numerous critical cellular processes. A considerable amount of PARP research has focused on PARP1's role in DNA damage. However, an increasing body of evidence outlines more routine roles for PARP and PARylation in nearly every step of RNA biogenesis and metabolism. PARP1's involvement in these RNA processes is pleiotropic and has been ascribed to PARP1's unique flexible domain structures. PARP1 domains are modular self-arranged enabling it to recognize structurally diverse substrates and to act simultaneously through multiple discrete mechanisms. These mechanisms include direct PARP1-protein binding, PARP1-nucleic acid binding, covalent PARylation of target molecules, covalent autoPARylation, and induction of noncovalent interactions with PAR molecules. A combination of these mechanisms has been implicated in PARP1's context-specific regulation of RNA biogenesis and metabolism. We examine the mechanisms of PARP1 regulation in transcription initiation, elongation and termination, co-transcriptional splicing, RNA export, and post-transcriptional RNA processing. Finally, we consider promising new investigative avenues for PARP1 involvement in these processes with an emphasis on PARP1 regulation of subcellular condensates. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing.

Exacerbated obesogenic response in female mice exposed to early life stress is linked to fat depot-specific upregulation of leptin protein expression.

Early life stress (ELS) is an independent risk factor for increased BMI and cardiometabolic disease risk later in life. We have previously shown that a mouse model of ELS, maternal separation and early weaning (MSEW), exacerbates high-fat diet (HF)-induced obesity only in adult female mice. Therefore, the aim of this study was to investigate 1) whether the short- and long-term effects of HF on leptin expression are influenced by MSEW in a sex-specific manner and 2) the potential epigenetic mechanisms underlying the MSEW-induced changes in leptin expression. After 1 wk of HF, both MSEW male and female mice displayed increased fat mass compared with controls (P < 0.05). However, only MSEW female mice showed elevated leptin mRNA expression in gonadal white adipose tissue (gWAT; P < 0.05). After 12 wk of HF, fat mass remained increased only in female mice (P < 0.05). Moreover, plasma leptin and both leptin mRNA and protein expression in gWAT were augmented in MSEW female mice compered to controls (P < 0.05), but not in MSEW male mice. This association was not present in subcutaneous WAT. Furthermore, among 16 CpG sites in the leptin promoter, we identified three hypomethylated sites in tissue from HF-fed MSEW female mice compared with controls (3, 15, and 16, P < 0.05). These hypomethylated sites showed greater binding of key adipogenic factors such as PPARγ (P < 0.05). Taken together, our study reveals that MSEW superimposed to HF increases leptin protein expression in a sex- and fat depot-specific fashion. Our data suggest that the mechanism by which MSEW increases leptin expression could be epigenetic.

Latexin regulation by HMGB2 is required for hematopoietic stem cell maintenance.

Hematopoietic stem cells provide life-long production of blood cells and undergo self-renewal division in order to sustain the stem cell pool. Homeostatic maintenance of hematopoietic stem cell pool and blood cell production is vital for the organism to survive. We previously reported that latexin is a negative regulator of hematopoietic stem cells in mice. Its natural variation in the expression is inversely correlated with hematopoietic stem cell number. However, the molecular mechanisms regulating latexin transcription remain largely unknown, and the genetic factors contributing to its natural variation are not clearly defined. Here we discovered a chromatin protein, high-mobility group protein B2, as a novel transcriptional suppressor of latexin by using DNA pull-down and mass spectrometry. High-mobility group protein B2 knockdown increases latexin expression at transcript and protein levels, and decreases hematopoietic stem cell number and regeneration capacity in vivo Concomitant blockage of latexin activation significantly reverses these phenotypic changes, suggesting that latexin is one of the downstream targets and functional mediators of high-mobility group protein B2. We further identified a functional single nucleotide polymorphism, rs31528793, in the latexin promoter that binds to high-mobility group protein B2 and affects the promoter activity. G allelic variation in rs31528793 associates with the higher latexin expression and lower hematopoietic stem cell number, whereas C allele indicates the lower latexin expression and higher stem cell number. This study reveals for the first time that latexin transcription is regulated by both transacting (high-mobility group protein B2) and cis-acting (single nucleotide polymorphism rs31528793) factors. It uncovers the functional role of naturally occurring genetic variants, in combination with epigenetic regulator, in determining differential gene expression and phenotypic diversity in the hematopoietic stem cell population.

Transcriptional Regulation Factors of the Human Mitochondrial Aspartate/Glutamate Carrier Gene, Isoform 2 (SLC25A13): USF1 as Basal Factor and FOXA2 as Activator in Liver Cells.

Mitochondrial carriers catalyse the translocation of numerous metabolites across the inner mitochondrial membrane, playing a key role in different cell functions. For this reason, mitochondrial carrier gene expression needs tight regulation. The human SLC25A13 gene, encoding for the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), catalyses the electrogenic exchange of aspartate for glutamate plus a proton, thus taking part in many metabolic processes including the malate-aspartate shuttle. By the luciferase (LUC) activity of promoter deletion constructs we identified the putative promoter region, comprising the proximal promoter (-442 bp/-19 bp), as well as an enhancer region (-968 bp/-768 bp). Furthermore, with different approaches, such as in silico promoter analysis, gene silencing and chromatin immunoprecipitation, we identified two transcription factors responsible for SLC25A13 transcriptional regulation: FOXA2 and USF1. USF1 acts as a positive transcription factor which binds to the basal promoter thus ensuring SLC25A13 gene expression in a wide range of tissues. The role of FOXA2 is different, working as an activator in hepatic cells. As a tumour suppressor, FOXA2 could be responsible for SLC25A13 high expression levels in liver and its downregulation in hepatocellular carcinoma (HCC).

PARP1 is a versatile factor in the regulation of mRNA stability and decay.

PARP1 is an abundant nuclear protein with many pleiotropic functions involved in epigenetic and transcriptional controls. Abundance of mRNA depends on the balance between synthesis and decay of a particular transcript. PARP1 binds RNA and its depletion results in increased expression of genes involved in nonsense-mediated decay, suggesting that PARP1 might be involved in mRNA stability. This is of interest considering RNA binding proteins play key roles in post-transcriptional processes in all eukaryotes. We tested the direct impact of PARP1 and PARylation on mRNA stability and decay. By measuring the half-lives of two PARP1-mRNA targets we found that the half-lives were significantly decreased in PARP1-depleted cells. PARP1 depletion impacted both the synthesis of nascent mRNA and the stability of mature mRNAs. PARylation impacted the production of nascent mRNA and the stability of mature mRNA, albeit to a lesser extent than PARP1 KD. PARylation enhanced the impact of PARP1 depletion. These studies provide the first direct comparative role of PARP1 and PARylation in RNA stability and decay, adding a new dimension as to how PARP1 regulates gene expression. These studies present a platform to begin to tease out the influence of PARP1 at each step of RNA biogenesis and decay to fine-tune gene expression.

Coupling of PARP1-mediated chromatin structural changes to transcriptional RNA polymerase II elongation and cotranscriptional splicing.

BACKGROUND: Recently, we showed that PARP1 is involved in cotranscriptional splicing, possibly by bridging chromatin to RNA and recruiting splicing factors. It also can influence alternative splicing decisions through the regulation of RNAPII elongation. In this study, we investigated the effect of PARP1-mediated chromatin changes on RNAPII movement, during transcription and alternative splicing. RESULTS: We show that RNAPII pauses at PARP1-chromatin structures within the gene body. Knockdown of PARP1 abolishes this RNAPII pausing, suggesting that PARP1 may regulate RNAPII elongation. Additionally, PARP1 alters nucleosome deposition and histone post-translational modifications at specific exon-intron boundaries, thereby affecting RNAPII movement. Lastly, genome-wide analyses confirmed that PARP1 influences changes in RNAPII elongation by either reducing or increasing the rate of RNAPII elongation depending on the chromatin context. CONCLUSIONS: These studies suggest a context-specific effect of PARP1-chromatin binding on RNA polymerase movement and provide a platform to delineate PARP1's role in RNA biogenesis and processing.

A comparison of nucleosome organization in Drosophila cell lines.

Changes in the distribution of nucleosomes along the genome influence chromatin structure and impact gene expression by modulating the accessibility of DNA to transcriptional machinery. However, the role of genome-wide nucleosome positioning in gene expression and in maintaining differentiated cell states remains poorly understood. Drosophila melanogaster cell lines represent distinct tissue types and exhibit cell-type specific gene expression profiles. They thus could provide a useful tool for investigating cell-type specific nucleosome organization of an organism's genome. To evaluate this possibility, we compared genome-wide nucleosome positioning and occupancy in five different Drosophila tissue-specific cell lines, and in reconstituted chromatin, and then tested for correlations between nucleosome positioning, transcription factor binding motifs, and gene expression. Nucleosomes in all cell lines were positioned in accordance with previously known DNA-nucleosome interactions, with helically repeating A/T di-nucleotide pairs arranged within nucleosomal DNAs and AT-rich pentamers generally excluded from nucleosomal DNA. Nucleosome organization in all cell lines differed markedly from in vitro reconstituted chromatin, with highly expressed genes showing strong nucleosome organization around transcriptional start sites. Importantly, comparative analysis identified genomic regions that exhibited cell line-specific nucleosome enrichment or depletion. Further analysis of these regions identified 91 out of 16,384 possible heptamer sequences that showed differential nucleosomal occupation between cell lines, and 49 of the heptamers matched one or more known transcription factor binding sites. These results demonstrate that there is differential nucleosome positioning between these Drosophila cell lines and therefore identify a system that could be used to investigate the functional significance of differential nucleosomal positioning in cell type specification.

Microarray dataset of transient and permanent DNA methylation changes in HeLa cells undergoing inorganic arsenic-mediated epithelial-to-mesenchymal transition.

The novel dataset presented here represents the results of the changing pattern of DNA methylation profiles in HeLa cells exposed to chronic low dose (0.5 µM) sodium arsenite, resulting in epithelial-to-mesenchymal transition, as well as DNA methylation patterns in cells where inorganic arsenic has been removed. Inorganic arsenic is a known carcinogen, though not mutagenic. Several mechanisms have been proposed as to how inorganic arsenic drives carcinogenesis such as regulation of the cell׳s redox potential and/or epigenetics. In fact, there are gene specific studies and limited genome-wide studies that have implicated epigenetic factors such as DNA methylation in inorganic arsenic-mediated epithelial-to-mesenchymal transition (EMT). However, genome-wide studies about the impact of 1) chronic, low-dose inorganic arsenic exposure on DNA methylation patterns during inorganic arsenic-induced epithelial-to-mesenchymal transition, and 2) the removal inorganic arsenic (reversal) on DNA methylation patterns, is lacking. For this dataset, two replicates were performed with each of the samples - non-treated, inorganic arsenic-treated, and reverse-treated cells. We provide normalized and processed data, and log2 fold change in DNA methylation. The raw microarray data are available through NCBI GEO, accession number GSE95232 and a related research paper has been accepted for published in Toxicology and Applied Pharmacology (Eckstein et al., 2017) [1].

Transient and permanent changes in DNA methylation patterns in inorganic arsenic-mediated epithelial-to-mesenchymal transition.

Chronic low dose inorganic arsenic exposure causes cells to take on an epithelial-to-mesenchymal phenotype, which is a crucial process in carcinogenesis. Inorganic arsenic is not a mutagen and thus epigenetic alterations have been implicated in this process. Indeed, during the epithelial-to-mesenchymal transition, morphologic changes to cells correlate with changes in chromatin structure and gene expression, ultimately driving this process. However, studies on the effects of inorganic arsenic exposure/withdrawal on the epithelial-to-mesenchymal transition and the impact of epigenetic alterations in this process are limited. In this study we used high-resolution microarray analysis to measure the changes in DNA methylation in cells undergoing inorganic arsenic-induced epithelial-to-mesenchymal transition, and on the reversal of this process, after removal of the inorganic arsenic exposure. We found that cells exposed to chronic, low-dose inorganic arsenic exposure showed 30,530 sites were differentially methylated, and with inorganic arsenic withdrawal several differential methylated sites were reversed, albeit not completely. Furthermore, these changes in DNA methylation mainly correlated with changes in gene expression at most sites tested but not at all. This study suggests that DNA methylation changes on gene expression are not clear-cut and provide a platform to begin to uncover the relationship between DNA methylation and gene expression, specifically within the context of inorganic arsenic treatment.

Genome-wide DNA methylation reprogramming in response to inorganic arsenic links inhibition of CTCF binding, DNMT expression and cellular transformation.

Chronic low dose inorganic arsenic (iAs) exposure leads to changes in gene expression and epithelial-to-mesenchymal transformation. During this transformation, cells adopt a fibroblast-like phenotype accompanied by profound gene expression changes. While many mechanisms have been implicated in this transformation, studies that focus on the role of epigenetic alterations in this process are just emerging. DNA methylation controls gene expression in physiologic and pathologic states. Several studies show alterations in DNA methylation patterns in iAs-mediated pathogenesis, but these studies focused on single genes. We present a comprehensive genome-wide DNA methylation analysis using methyl-sequencing to measure changes between normal and iAs-transformed cells. Additionally, these differential methylation changes correlated positively with changes in gene expression and alternative splicing. Interestingly, most of these differentially methylated genes function in cell adhesion and communication pathways. To gain insight into how genomic DNA methylation patterns are regulated during iAs-mediated carcinogenesis, we show that iAs probably targets CTCF binding at the promoter of DNA methyltransferases, regulating their expression. These findings reveal how CTCF binding regulates DNA methyltransferase to reprogram the methylome in response to an environmental toxin.

Transcriptome-wide identification of the RNA-binding landscape of the chromatin-associated protein PARP1 reveals functions in RNA biogenesis.

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.

Comparative analysis of linker histone H1, MeCP2, and HMGD1 on nucleosome stability and target site accessibility.

Chromatin architectural proteins (CAPs) bind the entry/exit DNA of nucleosomes and linker DNA to form higher order chromatin structures with distinct transcriptional outcomes. How CAPs mediate nucleosome dynamics is not well understood. We hypothesize that CAPs regulate DNA target site accessibility through alteration of the rate of spontaneous dissociation of DNA from nucleosomes. We investigated the effects of histone H1, high mobility group D1 (HMGD1), and methyl CpG binding protein 2 (MeCP2), on the biophysical properties of nucleosomes and chromatin. We show that MeCP2, like the repressive histone H1, traps the nucleosome in a more compact mononucleosome structure. Furthermore, histone H1 and MeCP2 hinder model transcription factor Gal4 from binding to its cognate DNA site within the nucleosomal DNA. These results demonstrate that MeCP2 behaves like a repressor even in the absence of methylation. Additionally, MeCP2 behaves similarly to histone H1 and HMGD1 in creating a higher-order chromatin structure, which is susceptible to chromatin remodeling by ISWI. Overall, we show that CAP binding results in unique changes to nucleosome structure and dynamics.

Involvement of PARP1 in the regulation of alternative splicing.

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.