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RNA binding proteins (RBPs) containing intrinsically disordered regions (IDRs) are present in diverse molecular complexes where they function as dynamic regulators. Their characteristics promote liquid-liquid phase separation (LLPS) and the formation of membraneless organelles such as stress granules and nucleoli. IDR-RBPs are particularly relevant in the nervous system and their dysfunction is associated with neurodegenerative diseases and brain tumor development. Serpine1 mRNA-binding protein 1 (SERBP1) is a unique member of this group, being mostly disordered and lacking canonical RNA-binding domains. We defined SERBP1's interactome, uncovered novel roles in splicing, cell division and ribosomal biogenesis, and showed its participation in pathological stress granules and Tau aggregates in Alzheimer's brains. SERBP1 preferentially interacts with other G-quadruplex (G4) binders, implicated in different stages of gene expression, suggesting that G4 binding is a critical component of SERBP1 function in different settings. Similarly, we identified important associations between SERBP1 and PARP1/polyADP-ribosylation (PARylation). SERBP1 interacts with PARP1 and its associated factors and influences PARylation. Moreover, protein complexes in which SERBP1 participates contain mostly PARylated proteins and PAR binders. Based on these results, we propose a feedback regulatory model in which SERBP1 influences PARP1 function and PARylation, while PARylation modulates SERBP1 functions and participation in regulatory complexes.

Estradiol (E2) and relaxin (Rln) are steroid and polypeptide hormones, respectively, with important roles in the female reproductive tract, including myometrium. Some actions of Rln, which are mediated by its membrane receptor RXFP1, require or are augmented by E2 signaling through its cognate nuclear steroid receptor, estrogen receptor alpha (ERα). In contrast, other actions of Rln act in opposition to the effects of E2. Here we explored the molecular and genomic mechanisms that underlie the functional interplay between E2 and Rln in the myometrium. We used both ovariectomized female mice and immortalized human myometrial cells expressing wild-type or mutant ERα (hTERT-HM-ERα cells). Our results indicate that Rln modulates the genomic actions and biological effects of estrogen in the myometrium and myometrial cells by reducing phosphorylation of ERα on serine 118 (S118), as well as by reducing the E2-dependent binding of ERα across the genome. These effects were associated with changes in the hormone-regulated transcriptome, including a decrease in the E2-dependent expression of some genes and enhanced expression of others. The inhibitory effects of Rln cotreatment on the E2-dependent phosphorylation of ERα required the nuclear dual-specificity phosphatases DUSP1 and DUSP5. Moreover, the inhibitory effects of Rln were reflected in a concomitant inhibition of the E2-dependent contraction of myometrial cells. Collectively, our results identify a pathway that integrates Rln/RXFP1 and E2/ERα signaling, resulting in a convergence of membrane and nuclear signaling pathways to control genomic and biological outcomes., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

Objectives: Mono(ADP-ribosyl)ation (MARylation), a post translational modification of proteins, is emerging as an important regulator of the biology of cancer cells. PARP7 (TiPARP), a mono (ADP-ribosyl) transferase (MART), MARylates its substrate α-tubulin in ovarian cancer cells, promoting destabilization of microtubules, cell growth, and migration. Recent development of RBN-2397, a potent inhibitor that selectively acts on PARP7, has provided a new tool for exploring the role of PARP7 catalytic activity in biological processes. In this study, we investigated the role of PARP7 catalytic activity in the regulation of ovarian cancer cell biology via MARylation of α-tubulin., Methods: Ovarian cancer cell lines (OVCAR4, OVCAR3) were treated with RBN-2397 and paclitaxel, both separately and in combination. Western blotting and immunoprecipitation confirmed the effects of RBN-2397 on α-tubulin MARylation and stabilization. Cell proliferation and migration were assessed, and α-tubulin stabilization was quantified using immunofluorescent imaging. RNA-sequencing was performed to assess the effects on gene expression changes., Results: RBN-2397 inhibited PARP7 activity, decreasing α-tubulin MARylation, leading to its stabilization, and reducing cancer cell proliferation and migration. The addition of paclitaxel further enhanced these effects, highlighting a synergistic interaction between the two drugs. Mutating the site of PARP7-mediated MARylation on α-tubulin similarly resulted in microtubule stabilization and decreased cell migration in the presence of paclitaxel., Conclusions: This study demonstrates that targeting PARP7 with RBN-2397, particularly in combination with paclitaxel, offers an effective strategy for inhibiting aggressive ovarian cancer cell phenotypes. Our findings underscore the potential of combining PARP7 inhibitors with established chemotherapeutics to enhance treatment efficacy in ovarian cancer.

Mono(ADP-ribosyl)ation (MARylation) is emerging as a critical regulator of ribosome function and translation. Herein, we demonstrate that RACK1, an integral component of the ribosome, is MARylated on three acidic residues by the mono(ADP-ribosyl) transferase (MART) PARP14 in ovarian cancer cells. MARylation of RACK1 is required for stress granule formation and promotes the colocalization of RACK1 in stress granules with G3BP1, eIF3η, and 40S ribosomal proteins. In parallel, we observed reduced translation of a subset of mRNAs, including those encoding key cancer regulators (e.g., AKT). Treatment with a PARP14 inhibitor or mutation of the sites of MARylation on RACK1 blocks these outcomes, as well as the growth of ovarian cancer cells in culture and in vivo. To re-set the system after prolonged stress and recovery, the ADP-ribosyl hydrolase TARG1 deMARylates RACK1, leading to the dissociation of the stress granules and the restoration of translation. Collectively, our results demonstrate a therapeutically targetable pathway that controls stress granule assembly and disassembly in ovarian cancer cells., Competing Interests: Conflict of Interest Statement: W.L.K. is a founder, member of the SAB, member of the BOD, and a stockholder for ARase Therapeutics, Inc. He is also coholder of U.S. Patent 9,599,606 covering the ADP-ribose detection reagents used herein, which has been licensed to and is sold by EMD Millipore.

ADP-ribosylation (ADPRylation) is a post-translational modification (PTM) of proteins mediated by the activity of a variety of ADP-ribosyltransferase (ART) enzymes, such as the Poly (ADP-ribose) Polymerase (PARP) family of proteins. This PTM is diverse in both form and biological functions, which makes it a highly interesting modification, but difficult to study due to limitations in reagents available to detect the diversity of ADPRylation. Recently we developed a set of recombinant antibody-like ADP-ribose (ADPR) binding proteins using naturally occurring ADPR binding domains (ARBDs), including macrodomains and WWE domains, functionalized by fusion to the constant "Fc" region of rabbit immunoglobulin. Herein, we present an expansion of this biological toolkit, where we have replaced the rabbit Fc sequence with the sequence from two other species, mouse and goat. These new reagents are based on a previously characterized set of naturally occurring ARBDs with known specificity. Characterization of the new reagents demonstrates that they can be detected in a species-dependent manner by secondary immunological tools, recognize specific ADPR moieties, and can be used for simultaneous detection of mono ADPR and poly ADPR at single-cell resolution in various antibody-based assays. The expansion of this toolkit will allow for more multiplexed assessments of the complexity of ADPRylation biology in many biological systems., Competing Interests: Conflict of interest W. L. Kraus is a founder, consultant, and Science Advisory Board member for ARase Therapeutics, Inc. W. L. Kraus is a co-holder of U.S. Patent 9,599,606 covering the ADPR detection reagents described herein. The rabbit ARBD-Fc fusions have been licensed to and are sold by EMD Millipore., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Signal-induced transcriptional programs regulate critical biological processes through the precise spatiotemporal activation of Immediate Early Genes (IEGs); however, the mechanisms of transcription induction remain poorly understood. By combining an acute depletion system with several genomics approaches to interrogate synchronized, temporal transcription, we reveal that KAP1/TRIM28 is a first responder that fulfills the temporal and heightened transcriptional demand of IEGs. Acute KAP1 loss triggers an increase in RNA polymerase II elongation kinetics during early stimulation time points. This elongation defect derails the normal progression through the transcriptional cycle during late stimulation time points, ultimately leading to decreased recruitment of the transcription apparatus for re-initiation thereby dampening IEGs transcriptional output. Collectively, KAP1 plays a counterintuitive role by negatively regulating transcription elongation to support full activation across multiple transcription cycles of genes critical for cell physiology and organismal functions., (© 2024. The Author(s).)

Progesterone (P 4 ), acting via its nuclear receptor (PR), is critical for pregnancy maintenance by suppressing proinflammatory and contraction-associated protein (CAP)/contractile genes in the myometrium. P 4 /PR partially exerts these effects by tethering to NF-κB bound to their promot-ers, thereby decreasing NF-κB transcriptional activity. However, the underlying mechanisms whereby P 4 /PR interaction blocks proinflammatory and CAP gene expression are not fully understood. Herein, we characterized CCR-NOT transcription complex subunit 1 (CNOT1) as a corepressor that also interacts within the same chromatin complex as PR-B. In mouse myome-trium increased expression of CAP genes Oxtr and Cx43 at term coincided with a marked decline in expression and binding of CNOT1 to NF-κB-response elements within the Oxtr and Cx43 promoters. Increased CAP gene expression was accompanied by a pronounced decrease in enrichment of repressive histone marks and increase in enrichment of active histone marks to this genomic region. These changes in histone modification were associated with changes in expression of corresponding histone modifying enzymes. Myometrial tissues from P 4 -treated 18.5 dpc pregnant mice manifested increased Cnot1 expression at 18.5 dpc, compared to vehicle-treated controls. P 4 treatment of PR-expressing hTERT-HM cells enhanced CNOT1 expression and its recruitment to PR bound NF-κB-response elements within the CX43 and OXTR promoters. Furthermore, knockdown of CNOT1 significantly increased expression of contractile genes. These novel findings suggest that decreased expression and DNA-binding of the P 4 /PR-regulated transcriptional corepressor CNOT1 near term and associated changes in histone modifications at the OXTR and CX43 promoters contribute to the induction of myometrial contractility leading to parturition., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cancer cells with DNA repair defects (e.g., BRCA1/2 mutant cells) are vulnerable to PARP inhibitors (PARPi) due to induction of synthetic lethality. However, recent clinical evidence has shown that PARPi can prevent the growth of some cancers irrespective of their BRCA1/2 status, suggesting alternative mechanisms of action. We previously discovered one such mechanism in breast cancer involving DDX21, an RNA helicase that localizes to the nucleoli of cells and is a target of PARP1. We have now extended this observation in endometrial and ovarian cancers and provided links to patient outcomes. When PARP1-mediated ADPRylation of DDX21 is inhibited by niraparib, DDX21 is mislocalized to the nucleoplasm resulting in decreased rDNA transcription, which leads to a reduction in ribosome biogenesis, protein translation, and ultimately endometrial and ovarian cancer cell growth. High PARP1 expression was associated with high nucleolar localization of DDX21 in both cancers. High nucleolar DDX21 negatively correlated with calculated IC50s for niraparib. By studying endometrial cancer patient samples, we were able to show that high DDX21 nucleolar localization was significantly associated with decreased survival. Our study suggests that the use of PARPi as a cancer therapeutic can be expanded to further types of cancers and that DDX21 localization can potentially be used as a prognostic factor and as a biomarker for response to PARPi., Significance: Currently, there are no reliable biomarkers for response to PARPi outside of homologous recombination deficiency. Herein we present a unique potential biomarker, with clear functional understanding of the molecular mechanism by which DDX21 nucleolar localization can predict response to PARPi., (© 2024 The Authors; Published by the American Association for Cancer Research.)

Pyridone-containing adenine dinucleotides, ox-NAD, are formed by overoxidation of nicotinamide adenine dinucleotide (NAD + ) and exist in three distinct isomeric forms. Like the canonical nucleosides, the corresponding pyridone-containing nucleosides (PYR) are chemically stable, biochemically versatile, and easily converted to nucleotides, di- and triphosphates, and dinucleotides. The 4-PYR isomer is often reported with its abundance increasing with the progression of metabolic diseases, age, cancer, and oxidative stress. Yet, the pyridone-derived nucleotides are largely under-represented in the literature. Here, we report the efficient synthesis of the series of ox-NAD and pyridone nucleotides and measure the abundance of ox-NAD in biological specimens using liquid chromatography coupled with mass spectrometry (LC-MS). Overall, we demonstrate that all three forms of PYR and ox-NAD are found in biospecimens at concentrations ranging from nanomolar to midmicromolar and that their presence affects the measurements of NAD(H) concentrations when standard biochemical redox-based assays are applied. Furthermore, we used liver extracts and 1 H NMR spectrometry to demonstrate that each ox-NAD isomer can be metabolized to its respective PYR isomer. Together, these results suggest a need for a better understanding of ox-NAD in the context of human physiology since these species are endogenous mimics of NAD + , the key redox cofactor in metabolism and bioenergetics maintenance.

The placenta is a dynamic organ that must perform a remarkable variety of functions during its relatively short existence in order to support a developing fetus. These functions include nutrient delivery, gas exchange, waste removal, hormone production, and immune barrier protection. Proper placenta development and function are critical for healthy pregnancy outcomes, but the underlying genomic regulatory events that control this process remain largely unknown. We hypothesized that mapping sites of transcriptional enhancer activity and associated changes in gene expression across gestation in human placenta tissue would identify genomic loci and predicted transcription factor activity related to critical placenta functions. We used a suite of genomic assays [i.e., RNA-sequencing (RNA-seq), Precision run-on-sequencing (PRO-seq), and Chromatin immunoprecipitation-sequencing (ChIP-seq)] and computational pipelines to identify a set of >20 000 enhancers that are active at various time points in gestation. Changes in the activity of these enhancers correlate with changes in gene expression. In addition, some of these enhancers encode risk for adverse pregnancy outcomes. We further show that integrating enhancer activity, transcription factor motif analysis, and transcription factor expression can identify distinct sets of transcription factors predicted to be more active either in early pregnancy or at term. Knockdown of selected identified transcription factors in a trophoblast stem cell culture model altered the expression of key placental marker genes. These observations provide a framework for future mechanistic studies of individual enhancer-transcription factor-target gene interactions and have the potential to inform genetic risk prediction for adverse pregnancy outcomes., (© The Author(s) 2023. Published by Oxford University Press behalf of Society for the Study of Reproduction.)

ADP-ribosylation (ADPRylation) is a reversible posttranslational modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally occurring protein motifs and domains, including WWEs, PBZs (PAR binding zinc fingers), and macrodomains, act as "readers" for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe the preparation and use of poly(ADP-ribose) (PAR) Trackers (PAR-Ts)-optimized dimerization-dependent or split-protein reassembly PAR sensors containing a naturally occurring PAR binding domain fused to both halves of dimerization-dependent GFP (ddGFP) or split nano luciferase (NanoLuc), respectively. We also describe how these tools can be used for the detection and quantification of PAR levels in biochemical assays with extracts and in living cells. These protocols will allow users to explore the broad utility of PAR-Ts for detecting PAR in various experimental and biological systems., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Gene regulation in the nucleus requires precise control of the molecular processes that dictate how, when, and which genes are transcribed. The posttranslational modification (PTM) of histones in chromatin is an effective means to link cellular signaling to gene expression outcomes. The repertoire of histone PTMs includes phosphorylation, acetylation, methylation, ubiquitylation, and ADP-ribosylation (ADPRylation). ADPRylation is a reversible PTM that results in the covalent transfer of ADP-ribose units derived from NAD + to substrate proteins on glutamate, aspartate, serine, and other amino acids. Histones were the first substrate proteins identified for ADPRylation, over five decades ago. Since that time, histone ADPRylation has been shown to be a widespread and critical regulator of chromatin structure and function during transcription, DNA repair, and replication. Here, we describe a set of protocols that allow the user to investigate site-specific histone ADPRylation and its functional consequences in biochemical assays and in cells in a variety of biological systems. With the recent discovery that some cancer-causing histone mutations (i.e., oncohistone mutations) occur at functional sites of regulatory ADPRylation, these protocols may have additional utility in studies of oncology., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD + onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Long noncoding RNAs have been implicated in many of the hallmarks of cancer. Herein, we found that the expression of lncRNA152 (lnc152; a.k.a. DRAIC), which we annotated previously, is highly upregulated in luminal breast cancer (LBC) and downregulated in triple-negative breast cancer (TNBC). Knockdown of lnc152 promotes cell migration and invasion in LBC cell lines. In contrast, ectopic expression of lnc152 inhibits growth, migration, invasion, and angiogenesis in TNBC cell lines. In mice, lnc152 inhibited the growth of TNBC cell xenografts, as well as metastasis of TNBC cells in an intracardiac injection model. Transcriptome analysis of the xenografts indicated that lnc152 downregulates genes controlling angiogenesis. Using pull down assays followed by LC/MS-MS, we identified RBM47, a known tumor suppressor in breast cancer, as a lnc152-interacting protein. The effects of lnc152 in TNBC cells are mediated, in part, by regulating the expression of RBM47. Collectively, our results demonstrate that lnc152 is an angiogenesis-inhibiting tumor suppressor that attenuates the aggressive cancer-related phenotypes found in TNBC., Implications: This study identifies lncRNA152 as an angiogenesis-inhibiting tumor suppressor that attenuates the aggressive cancer-related phenotypes found in TNBC by upregulating the expression of the tumor suppressor RBM47. As such, lncRNA152 may serve as a biomarker to track aggressiveness of breast cancer, as well as therapeutic target for treating TNBC., (©2022 American Association for Cancer Research.)

Cervical cancer continues to be a significant cause of cancer-related deaths in women. The most common treatment for cervical cancer involves the use of the drug cisplatin in conjunction with other therapeutics. However, the development of cisplatin resistance in patients can hinder the efficacy of these treatments, so alternatives are needed. In this study, we found that PARP inhibitors (PARPi) could attenuate the growth of cells representing cervical adenocarcinoma and cervical squamous cell carcinoma. Moreover, a combination of PARPi with cisplatin increased cisplatin-mediated cytotoxicity in cervical cancer cells. This was accompanied by a dramatic alteration of the transcriptome. The FOS gene, which encodes the transcription factor Fos, was one of the most highly upregulated genes in the dual treatment condition, leading to increased Fos protein levels, greater Fos binding to chromatin, and the subsequent induction of Fos target genes. Increased expression of Fos was sufficient to hinder cervical cancer growth, as shown by ectopic expression of Fos in cervical cancer cells. Conversely, Fos knockdown enhanced cell growth. Collectively, these results indicate that by inducing FOS expression, PARPi treatment in combination with cisplatin leads to inhibition of cervical cancer proliferation, likely through a Fos-specific gene expression program., Implications: Our observations, which link the gene regulatory effects of PARPi + cisplatin to the growth inhibitory effects of FOS expression in cervical cancer cells, strengthen the rationale for using PARPi with cisplatin as a therapy for cervical cancer., (©2022 American Association for Cancer Research.)

Recent studies have identified cancer-associated mutations in histone genes that lead to the expression of mutant versions of core histones called oncohistones. Many oncohistone mutations occur at Asp and Glu residues, two amino acids known to be ADP-ribosylated (ADPRylated) by PARP1. We screened 25 Glu or Asp oncohistone mutants for their effects on cell growth in breast and ovarian cancer cells. Ectopic expression of six mutants of three different core histones (H2B, H3, and H4) altered cell growth in at least two different cell lines. Two of these sites, H2B-D51 and H4-D68, were indeed sites of ADPRylation in wild-type (unmutated) histones, and mutation of these sites inhibited ADPRylation. Mutation of H2B-D51 dramatically altered chromatin accessibility at enhancers and promoters, as well as gene expression outcomes, whereas mutation of H4-D68 did not. Additional biochemical, cellular, proteomic, and genomic analyses demonstrated that ADPRylation of H2B-D51 inhibits p300-mediated acetylation of H2B at many Lys residues. In breast cancer cell xenografts in mice, H2B-D51A promoted tumor growth, but did not confer resistance to the cytotoxic effects of PARP inhibition. Collectively, these results demonstrate that functional Asp and Glu ADPRylation sites on histones are mutated in cancers, allowing cancer cells to escape the growth-regulating effects of post-translational modifications via distinct mechanisms., Significance: This study identifies cancer-driving mutations in histones as sites of PARP1-mediated ADP-ribosylation in breast and ovarian cancers, providing a molecular pathway by which cancers may subvert the growth-regulating effects of PARP1., (©2022 American Association for Cancer Research.)

ADP-ribosylation (ADPRylation) is a post-translational modification of proteins catalyzed by ADP-ribosyl transferase (ART) enzymes, including nuclear PARPs (e.g., PARP1 and PARP2). Historically, studies of ADPRylation and PARPs have focused on DNA damage responses in cancers, but more recent studies elucidate diverse roles in a broader array of biological processes. Here, we summarize the expanding array of molecular mechanisms underlying the biological functions of nuclear PARPs with a focus on PARP1, the founding member of the family. This includes roles in DNA repair, chromatin regulation, gene expression, ribosome biogenesis, and RNA biology. We also present new concepts in PARP1-dependent regulation, including PAR-dependent post-translational modifications, "ADPR spray," and PAR-mediated biomolecular condensate formation. Moreover, we review advances in the therapeutic mechanisms of PARP inhibitors (PARPi) as well as the progress on the mechanisms of PARPi resistance. Collectively, the recent progress in the field has yielded new insights into the expanding universe of PARP1-mediated molecular and therapeutic mechanisms in a variety of biological processes., Competing Interests: Declaration of interests W.L.K. is a founder of Ribon Therapeutics and a founder, consultant, and SAB member of ARase Therapeutics. He is also a co-holder of U.S. Patent 9,599,606 covering a set of ADP-ribose detection reagents, which have been licensed to and is sold by EMD Millipore. D.H. declares no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

To better understand the functions of non-coding enhancer RNAs (eRNAs), we annotated the estrogen-regulated eRNA transcriptome in estrogen receptor α (ERα)-positive breast cancer cells using PRO-cap and RNA sequencing. We then cloned a subset of the eRNAs identified, fused them to single guide RNAs, and targeted them to their ERα enhancers of origin using CRISPR/dCas9. Some of the eRNAs tested modulated the expression of cognate, but not heterologous, target genes after estrogen treatment by increasing ERα recruitment and stimulating p300-catalyzed H3K27 acetylation at the enhancer. We identified a ∼40 nucleotide functional eRNA regulatory motif (FERM) present in many eRNAs that was necessary and sufficient to modulate gene expression, but not the specificity of activation, after estrogen treatment. The FERM interacted with BCAS2, an RNA-binding protein amplified in breast cancers. The ectopic expression of a targeted eRNA controlling the expression of an oncogene resulted in increased cell proliferation, demonstrating the regulatory potential of eRNAs in breast cancer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The differentiation of embryonic stem cells (ESCs) into a lineage-committed state is a dynamic process involving changes in cellular metabolism, epigenetic modifications, post-translational modifications, gene expression, and RNA processing. Here we integrated data from metabolomic, proteomic, and transcriptomic assays to characterize how alterations in NAD + metabolism during the differentiation of mouse ESCs lead to alteration of the PARP1-mediated ADP-ribosylated (ADPRylated) proteome and mRNA isoform specialization. Our metabolomic analyses indicate that mESCs use distinct NAD + biosynthetic pathways in different cell states: the de novo pathway in the pluripotent state, and the salvage and Preiss-Handler pathways as differentiation progresses. We observed a dramatic induction of PARP1 catalytic activity driven by enhanced nuclear NAD + biosynthesis during the early stages of mESC differentiation (e.g., within 12 h of LIF removal). PARP1-modified proteins in mESCs are enriched for biological processes related to stem cell maintenance, transcriptional regulation, and RNA processing. The PARP1 substrates include core spliceosome components, such as U2AF35 and U2AF65, whose splicing functions are modulated by PARP1-mediated site-specific ADP-ribosylation. Finally, we observed that splicing is dysregulated genome-wide in Parp1 knockout mESCs. Together, these results demonstrate a role for the NAD + -PARP1 axis in the maintenance of mESC state, specifically in the splicing program during differentiation., (© 2022 Jones and Kraus; Published by Cold Spring Harbor Laboratory Press.)

ADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as 'readers' for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts)-optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems., Competing Interests: SC, KR K.W.R., S.C., and W.L.K. have a patent pending for the PAR-T sensors described herein, AW, JA, CC No competing interests declared, WK W.L.K. is a founder and consultant for Ribon Therapeutics, Inc and ARase Therapeutics, Inc He is also coholder of U.S. Patent 9,599,606 covering the ADP-ribose detection reagent used herein, which has been licensed to and is sold by EMD Millipore, (© 2022, Challa et al.)

In this issue of Cell Chemical Biology, Palve et al. (2022) identified PARP16 as a non-canonical therapeutic target of the PARP1 inhibitor talazoparib, which synergizes with the WEE1 inhibitor adavosertib to enhance its efficacy. The dual targeting of PARP1 and PARP16 may explain the greater efficacy of talazoparib in some cancers., Competing Interests: Declaration of interests W.L.K. is a founder for Ribon Therapeutics, Inc.; a founder, consultant, and SAB member for ARase Therapeutics, Inc.; and a consultant and SAB member for Alphina Therapeutics, Inc. He is also a co-holder of U.S. Patent 9,599,606 covering a set of ADP-ribose detection reagents, which have been licensed to and are sold by EMD Millipore., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

PARPs and tankyrases (TNKS) represent a family of 17 proteins. PARPs and tankyrases were originally identified as DNA repair factors, nevertheless, recent advances have shed light on their role in lipid metabolism. To date, PARP1, PARP2, PARP3, tankyrases, PARP9, PARP10, PARP14 were reported to have multi-pronged connections to lipid metabolism. The activity of PARP enzymes is fine-tuned by a set of cholesterol-based compounds as oxidized cholesterol derivatives, steroid hormones or bile acids. In turn, PARPs modulate several key processes of lipid homeostasis (lipotoxicity, fatty acid and steroid biosynthesis, lipoprotein homeostasis, fatty acid oxidation, etc.). PARPs are also cofactors of lipid-responsive nuclear receptors and transcription factors through which PARPs regulate lipid metabolism and lipid homeostasis. PARP activation often represents a disruptive signal to (lipid) metabolism, and PARP-dependent changes to lipid metabolism have pathophysiological role in the development of hyperlipidemia, obesity, alcoholic and non-alcoholic fatty liver disease, type II diabetes and its complications, atherosclerosis, cardiovascular aging and skin pathologies, just to name a few. In this synopsis we will review the evidence supporting the beneficial effects of pharmacological PARP inhibitors in these diseases/pathologies and propose repurposing PARP inhibitors already available for the treatment of various malignancies., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Miscarriage is a common complication of pregnancy for which there are few clinical interventions. Deficiency in endometrial stromal cell decidualization is considered a major contributing factor to pregnancy loss; however, our understanding of the underlying mechanisms of decidual deficiency are incomplete. ADP ribosylation by PARP-1 and PARP-2 has been linked to physiological processes essential to successful pregnancy outcomes. Here, we report that the catalytic inhibition or genetic ablation of PARP-1 and PARP-2 in the uterus lead to pregnancy loss in mice. Notably, the absence of PARP-1 and PARP-2 resulted in increased p53 signaling and an increased population of senescent decidual cells. Molecular and histological analysis revealed that embryo attachment and the removal of the luminal epithelium are not altered in uterine Parp1 , Parp2 knockout mice, but subsequent decidualization failure results in pregnancy loss. These findings provide evidence for a previously unknown function of PARP-1 and PARP-2 in mediating decidualization for successful pregnancy establishment., Competing Interests: Competing interest statement: W.L.K. is a founder, consultant, and Scientific Advisory Board member for Ribon Therapeutics, Inc. and ARase Therapeutics, Inc. He is also coholder of US Patent 9,599,606 covering the ADP-ribose detection reagent used herein, which has been licensed to and is sold by EMD Millipore.

Poly(ADP-ribose) polymerase-1 (PARP-1) has gained considerable attention as a target for therapeutic inhibitors in breast cancers. Previously we showed that PARP-1 localizes to active gene promoters to regulate histone methylation and RNA polymerase II activity (Pol II), altering the expression of various tumor-related genes. Here we report a role for PARP-1 in estrogen-dependent transcription in estrogen receptor alpha (ERα)-positive (ER + ) breast cancers. Global nuclear run-on and sequencing analyses functionally linked PARP-1 to the direct control of estrogen-regulated gene expression in ER + MCF-7 breast cancer cells by promoting transcriptional elongation by Pol II. Furthermore, chromatin immunoprecipitation sequencing analyses revealed that PARP-1 regulates the estrogen-dependent binding of ERα and FoxA1 to a subset of genomic ERα binding sites, promoting active enhancer formation. Moreover, we found that the expression levels of the PARP-1- and estrogen-coregulated gene set are enriched in the luminal subtype of breast cancer, and high PARP-1 expression in ER + cases correlates with poor survival. Finally, treatment with a PARP inhibitor or a transcriptional elongation inhibitor attenuated estrogen-dependent growth of multiple ER + breast cancer cell lines. Taken together, our results show that PARP-1 regulates critical molecular pathways that control the estrogen-dependent gene expression program underlying the proliferation of ER + breast cancer cells. IMPLICATIONS: PARP-1 regulates the estrogen-dependent genomic binding of ERα and FoxA1 to regulate critical gene expression programs by RNA Pol II that underlie the proliferation of ER + breast cancers, providing a potential therapeutic opportunity for PARP inhibitors in estrogen-responsive breast cancers., (©2021 American Association for Cancer Research.)

Complementary papers by Nguyen et al. (2021) and Baek et al. (2021) track the assembly of the pre-initiation complexes at gene promoters using single-molecule microscopy, revealing dynamic spatiotemporal regulation of transcription initiation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Defects in translation lead to changes in the expression of proteins that can serve as drivers of cancer formation. Here, we show that cytosolic NAD + synthesis plays an essential role in ovarian cancer by regulating translation and maintaining protein homeostasis. Expression of NMNAT-2, a cytosolic NAD + synthase, is highly upregulated in ovarian cancers. NMNAT-2 supports the catalytic activity of the mono(ADP-ribosyl) transferase (MART) PARP-16, which mono(ADP-ribosyl)ates (MARylates) ribosomal proteins. Depletion of NMNAT-2 or PARP-16 leads to inhibition of MARylation, increased polysome association and enhanced translation of specific mRNAs, aggregation of their translated protein products, and reduced growth of ovarian cancer cells. Furthermore, MARylation of the ribosomal proteins, such as RPL24 and RPS6, inhibits polysome assembly by stabilizing eIF6 binding to ribosomes. Collectively, our results demonstrate that ribosome MARylation promotes protein homeostasis in cancers by fine-tuning the levels of protein synthesis and preventing toxic protein aggregation., Competing Interests: Declaration of interests W.L.K. is a founder and consultant for Ribon Therapeutics, Inc. and ARase Therapeutics, Inc. He is also coholder of U.S. Patent 9,599,606 covering the ADP-ribose detection reagent used herein, which has been licensed to and is sold by EMD Millipore., (Copyright © 2021 Elsevier Inc. All rights reserved.)

STAT1α is a key transcription factor driving pro-inflammatory responses in macrophages. We found that the interferon gamma (IFNγ)-regulated transcriptional program in macrophages is controlled by ADP-ribosylation (ADPRylation) of STAT1α, a post-translational modification resulting in the site-specific covalent attachment of ADP-ribose moieties. PARP-1, the major nuclear poly(ADP-ribose) polymerase (PARP), supports IFNγ-stimulated enhancer formation by regulating the genome-wide binding and IFNγ-dependent transcriptional activation of STAT1α. It does so by ADPRylating STAT1α on specific residues in its DNA-binding domain (DBD) and transcription activation (TA) domain. ADPRylation of the DBD controls STAT1α binding to its cognate DNA elements, whereas ADPRylation of the TA domain regulates enhancer activation by modulating STAT1α phosphorylation and p300 acetyltransferase activity. Loss of ADPRylation at either site leads to diminished IFNγ-dependent transcription and downstream pro-inflammatory responses. We conclude that PARP-1-mediated ADPRylation of STAT1α drives distinct enhancer activation mechanisms and is a critical regulator of inflammatory responses in macrophages.

Mono(ADP-ribosyl)ation (MARylation) is a regulatory post-translational modification of proteins that controls their functions through a variety of mechanisms. MARylation is catalyzed by mono(ADP-ribosyl) transferase (MART) enzymes, a subclass of the poly(ADP-ribosyl) polymerase (PARP) family of enzymes. Although the role of PARPs and poly(ADP-ribosyl)ation (PARylation) in cellular pathways, such as DNA repair and transcription, is well studied, the role of MARylation and MARTs (i.e., the PARP 'monoenzymes') are not well understood. Moreover, compared to PARPs, the development of MART-targeted therapeutics is in its infancy. Recent studies are beginning to shed light on the structural features, catalytic targets, and biological functions of MARTs. The development of new technologies to study MARTs have uncovered essential roles for these enzymes in the regulation of cellular processes, such as RNA metabolism, cellular transport, focal adhesion, and stress responses. These insights have increased our understanding of the biological functions of MARTs in cancers, neuronal development, and immune responses. Furthermore, several novel inhibitors of MARTs have been developed and are nearing clinical utility. In this review, we summarize the biological functions and molecular mechanisms of MARTs and MARylation, as well as recent advances in technology that have enabled detection and inhibition of their activity. We emphasize PARP-7, which is at the forefront of the MART subfamily with respect to understanding its biological roles and the development of therapeutically useful inhibitors. Collectively, the available studies reveal a growing understanding of the biochemistry, chemical biology, physiology, and pathology of MARTs., Competing Interests: W.L.K. is a founder and consultant for Ribon Therapeutics, Inc. He is a coholder of U.S. Patent 9,599,606 covering the set of ADP-ribose detection reagents described herein, which have been licensed to and are sold by EMD Millipore. He is also a coholder of U.S. Patent 9,926,340 covering the clickable NAD+ analogs and analog sensitive PARP mutants described herein. S.C. and M.S.S. declare no conflict of interest.

Responses to developmental and environmental cues depend on precise spatiotemporal control of gene transcription. Enhancers, which comprise DNA elements bound by regulatory proteins, can activate target genes in response to these external signals. Recent studies have shown that enhancers are transcribed to produce enhancer RNAs (eRNAs). Do eRNAs play a functional role in activating gene expression or are they non-functional byproducts of nearby transcription machinery? The unstable nature of eRNAs and over-reliance on knockdown approaches have made elucidating the possible functions of eRNAs challenging. We focus here on studies using cloned eRNAs to study their function as transcripts, revealing roles for eRNAs in enhancer-promoter looping, recruiting transcriptional machinery, and facilitating RNA polymerase pause-release to regulate gene expression., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

PARP-7 (TiPARP) is a mono(ADP-ribosyl) transferase whose protein substrates and biological activities are poorly understood. We observed that PARP7 mRNA levels are lower in ovarian cancer patient samples compared to non-cancerous tissue, but PARP-7 protein nonetheless contributes to several cancer-related biological endpoints in ovarian cancer cells (e.g. growth, migration). Global gene expression analyses in ovarian cancer cells subjected to PARP-7 depletion indicate biological roles for PARP-7 in cell-cell adhesion and gene regulation. To identify the MARylated substrates of PARP-7 in ovarian cancer cells, we developed an NAD + analog-sensitive approach, which we coupled with mass spectrometry to identify the PARP-7 ADP-ribosylated proteome in ovarian cancer cells, including cell-cell adhesion and cytoskeletal proteins. Specifically, we found that PARP-7 MARylates α-tubulin to promote microtubule instability, which may regulate ovarian cancer cell growth and motility. In sum, we identified an extensive PARP-7 ADP-ribosylated proteome with important roles in cancer-related cellular phenotypes., Competing Interests: LP, SC, TN, MS, DH, JL No competing interests declared, BG holds the patents on the anti-MAR binding reagent (United States Patent No. 9,599,606) and the asPARP technology (United States Patent No. 9,926,340) described herein. UT Southwestern Medical Center has licensed the anti-MAR binding reagent to EMD Millipore, which markets it for research purposes. BIOLOG Life Science Institute, a coholder of United States Patent No. 9,926,340, sells the NAD+ analog 8-Bu(3-yne)T-NAD+. WK is a founder consultant for Ribon (Therapeutics, Inc). Holds the patents on the anti-MAR binding reagent (United States Patent No. 9,599,606) and the asPARP technology (United States Patent No. 9,926,340) described herein. UT Southwestern Medical Center has licensed the anti-MAR binding reagent to EMD Millipore, which markets it for research purposes. BIOLOG Life Science Institute, a coholder of United States Patent No. 9,926,340, sells the NAD+ analog 8-Bu(3-yne)T-NAD+., (© 2021, Palavalli Parsons et al.)

Homologous recombination (HR) repair deficiency impairs the proper maintenance of genomic stability, thus rendering cancer cells vulnerable to loss or inhibition of DNA repair proteins, such as poly(ADP-ribose) polymerase-1 (PARP-1). Inhibitors of nuclear PARPs are effective therapeutics for a number of different types of cancers. Here we review key concepts and current progress on the therapeutic use of PARP inhibitors (PARPi). PARPi selectively induce synthetic lethality in cancer cells with homologous recombination deficiencies (HRDs), the most notable being cancer cells harboring mutations in the BRCA1 and BRCA2 genes. Recent clinical evidence, however, shows that PARPi can be effective as cancer therapeutics regardless of BRCA1/2 or HRD status, suggesting that a broader population of patients might benefit from PARPi therapy. Currently, four PARPi have been approved by the Food and Drug Administration (FDA) for the treatment of advanced ovarian and breast cancer with deleterious BRCA mutations. Although PARPi have been shown to improve progression-free survival, cancer cells inevitably develop resistance, which poses a significant obstacle to the prolonged use of PARP inhibitors. For example, somatic BRCA1/2 reversion mutations are often identified in patients with BRCA1/2-mutated cancers after treatment with platinum-based therapy, causing restoration of HR capacity and thus conferring PARPi resistance. Accordingly, PARPi have been studied in combination with other targeted therapies to overcome PARPi resistance, enhance PARPi efficacy, and sensitize tumors to PARP inhibition. Moreover, multiple clinical trials are now actively underway to evaluate novel combinations of PARPi with other anticancer therapies for the treatment of PARPi-resistant cancer. In this review, we highlight the mechanisms of action of PARP inhibitors with or without BRCA1/2 defects and provide an overview of the ongoing clinical trials of PARPi. We also review the current progress on PARPi-based combination strategies and PARP inhibitor resistance.

The numerous biological roles of NAD + are organized and coordinated via its compartmentalization within cells. The spatial and temporal partitioning of this intermediary metabolite is intrinsic to understanding the impact of NAD + on cellular signaling and metabolism. We review evidence supporting the compartmentalization of steady-state NAD + levels in cells, as well as how the modulation of NAD + synthesis dynamically regulates signaling by controlling subcellular NAD + concentrations. We further discuss potential benefits to the cell of compartmentalizing NAD + , and methods for measuring subcellular NAD + levels., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Although ADP-ribosylation of histones by PARP-1 has been linked to genotoxic stress responses, its role in physiological processes and gene expression has remained elusive. We found that NAD + -dependent ADP-ribosylation of histone H2B-Glu35 by small nucleolar RNA (snoRNA)-activated PARP-1 inhibits AMP kinase-mediated phosphorylation of adjacent H2B-Ser36, which is required for the proadipogenic gene expression program. The activity of PARP-1 on H2B requires NMNAT-1, a nuclear NAD + synthase, which directs PARP-1 catalytic activity to Glu and Asp residues. ADP-ribosylation of Glu35 and the subsequent reduction of H2B-Ser36 phosphorylation inhibits the differentiation of adipocyte precursors in cultured cells. Parp1 knockout in preadipocytes in a mouse lineage-tracing genetic model increases adipogenesis, leading to obesity. Collectively, our results demonstrate a functional interplay between H2B-Glu35 ADP-ribosylation and H2B-Ser36 phosphorylation that controls adipogenesis., Competing Interests: Declaration of Interests W.L.K. is a founder and consultant for Ribon Therapeutics. He is also a co-holder of U.S. Patent 9,599,606 covering the ADP-ribose detection reagents used herein, which have been licensed to and are sold by EMD Millipore. All other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The differentiation of embryonic stem cells into various lineages is highly dependent on the chromatin state of the genome and patterns of gene expression. To identify lineage-specific enhancers driving the differentiation of progenitors into pancreatic cells, we used a previously described computational framework called Total Functional Score of Enhancer Elements (TFSEE), which integrates multiple genomic assays that probe both transcriptional and epigenomic states. First, we evaluated and compared TFSEE as an enhancer-calling algorithm with enhancers called using GRO-seq-defined enhancer transcripts (method 1) versus enhancers called using histone modification ChIP-seq data (method 2). Second, we used TFSEE to define the enhancer landscape and identify transcription factors (TFs) that maintain the multipotency of a subpopulation of endodermal stem cells during differentiation into pancreatic lineages. Collectively, our results demonstrate that TFSEE is a robust enhancer-calling algorithm that can be used to perform multilayer genomic data integration to uncover cell type-specific TFs that control lineage-specific enhancers., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2020.)

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant and ubiquitous nuclear enzyme that catalyzes the transfer of ADP-ribose from donor NAD + molecules to specific amino acids on substrate proteins. The catalytic activity of PARP-1 has long been known to be allosterically stimulated by the free ends of DNA, such as those found at double-strand breaks in the genome. A number of studies have also shown that the catalytic activity of PARP-1 can also be stimulated by various types of RNA. A recent study by Nakamoto et al., however, has contradicted these results, concluding that the apparent stimulatory activity of the RNAs was due to contaminating DNA in the RNA preparations used in the biochemical assays. Here we show using a carefully controlled set of biochemical assays that DNA-free, in vitro-transcribed, PARP-1-interacting snoRNAs can stimulate PARP-1 catalytic activity. We confirmed the activation of PARP-1 by snoRNAs using a chemically synthesized snoRNA, as well as CRISPR/Cas9-mediated knockout of snoRNAs in cells. Finally, we provide a set of considerations and experimental conditions for the careful evaluation of RNA-stimulated PARP-1 catalytic activity that will help researchers avoid artifacts.

Estrogen-responsive breast cancer cells exhibit both basal and estrogen-regulated transcriptional programs, which lead to the transcription of many different transcription units (i.e., genes), including those that produce coding and non-coding sense (e.g., mRNA, lncRNA) and antisense (i.e., asRNA) transcripts. We have previously characterized the global basal and estrogen-regulated transcriptomes in estrogen receptor alpha (ERα)-positive MCF-7 breast cancer cells. Herein, we have mined genomic data to define three classes of antisense transcription in MCF-7 cells based on where their antisense transcription termination sites reside relative to their cognate sense mRNA and lncRNA genes. These three classes differ in their response to estrogen treatment, the enrichment of a number of genomic features associated with active promoters (H3K4me3, RNA polymerase II, open chromatin architecture), and the biological functions of their cognate sense genes as analyzed by DAVID gene ontology. We further characterized two estrogen-regulated antisense transcripts arising from the MYC gene in MCF-7 cells, showing that these antisense transcripts are 5'-capped, 3'-polyadenylated, and localized to different compartments of the cell. Together, our analyses have revealed distinct classes of antisense transcription correlated to different biological processes and response to estrogen stimulation, uncovering another layer of hormone-regulated gene regulation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

ADP-ribosylation (ADPRylation) is a posttranslational modification of proteins discovered nearly six decades ago, but many important questions remain regarding its molecular functions and biological roles, as well as the activity of the ADP-ribose (ADPR) transferase enzymes (PARP family members) that catalyze it. Growing evidence indicates that PARP-mediated ADPRylation events are key regulators of the protein biosynthetic pathway, leading from rDNA transcription and ribosome biogenesis to mRNA synthesis, processing, and translation. In this review we describe the role of PARP proteins and ADPRylation in all facets of this pathway. PARP-1 and its enzymatic activity are key regulators of rDNA transcription, which is a critical step in ribosome biogenesis. An emerging role of PARPs in alternative splicing of mRNAs, as well as direct ADPRylation of mRNAs, highlight the role of PARP members in RNA processing. Furthermore, PARP activity, stimulated by cellular stresses, such as viral infections and ER stress, leads to the regulation of mRNA stability and protein synthesis through posttranscriptional mechanisms. Dysregulation of PARP activity in these processes can promote disease states. Collectively, these results highlight the importance of PARP family members and ADPRylation in gene regulation, mRNA processing, and protein abundance. Future studies in these areas will yield new insights into the fundamental mechanisms and a broader utility for PARP-targeted therapeutic agents., (© 2020 Kim et al.; Published by Cold Spring Harbor Laboratory Press.)

The ovarian hormones estrogen and progesterone orchestrate the transcriptional programs required to direct functions of the uterus for initiation and maintenance of pregnancy. Estrogen, acting via estrogen receptor alpha, regulates gene expression by activating and repressing distinct genes involved in signaling pathways that regulate cellular and physiological responses including cell division, water influx, and immune cell recruitment. Historically, these transcriptional responses have been postulated to reflect a biphasic physiological response. In this study, we explored the transcriptional responses of the ovariectomized mouse uterus to 17β-estradiol (E2) by RNA-seq to obtain global expression profiles of protein-coding transcripts (mRNAs) and long noncoding RNAs (lncRNAs) following 0.5, 1, 2, and 6 hours of treatment. The E2-regulated mRNA and lncRNA expression profiles in the mouse uterus indicate an association between lncRNAs and mRNAs that regulate E2-driven pathways and reproductive phenotypes in the mouse. The transient E2-regulated transcriptome is reflected in the time-dependent shifting of biological processes regulated in the uterus in response to E2. Moreover, high expression of some conserved lncRNAs that are E2 regulated in the mouse uterus are predictive of low overall survival in endometrial carcinoma patients (e.g., H19, KCNQ1OT1, MIR17HG, and FTX). Collectively, this study (1) describes a genomic approach for identifying E2-regulated lncRNAs that may serve critical function in the uterus and (2) provides new insights into our understanding of the regulation of hormone-regulated transcriptional responses with implications in pregnancy and endometrial pathologies., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Inhibitors of nuclear PARP enzymes (e.g., PARP-1) have improved clinical outcomes in ovarian cancer, especially in patients with BRCA1/2 gene mutations or additional homologous recombination (HR) DNA repair pathway deficiencies. These defects serve as biomarkers for response to PARP inhibitors (PARPi). We sought to identify an additional biomarker that could predict responses to both conventional chemotherapy and PARPi in ovarian cancers. We focused on cellular ADP-ribosylation (ADPRylation), which is catalyzed by PARP enzymes and detected by detection reagents we developed previously. We determined molecular phenotypes of 34 high-grade serous ovarian cancers and associated them with clinical outcomes. We used the levels and patterns of ADPRylation and PARP-1 to distribute ovarian cancers into distinct molecular phenotypes, which exhibit dramatically different gene expression profiles. In addition, the levels and patterns of ADPRylation, PARP-1 protein, and gene expression correlated with clinical outcomes in response to platinum-based chemotherapy, with cancers exhibiting the highest levels of ADPRylation having the best outcomes independent of BRCA1/2 status. Finally, in cell culture-based assays using patient-derived ovarian cancer cell lines, ADPRylation levels correlated with sensitivity to the PARPi, Olaparib, with cell lines exhibiting high levels of ADPRylation having greater sensitivity to Olaparib. Collectively, our study demonstrates that ovarian cancers exhibit a wide range of ADPRylation levels, which correlate with therapeutic responses and clinical outcomes. These results suggest ADPRylation may be a useful biomarker for PARPi sensitivity in ovarian cancers, independent of BRCA1/2 or homologous recombination deficiency status., (©2019 American Association for Cancer Research.)

The bromodomain family member proteins (BRD; BET proteins) are key coregulators for estrogen receptor alpha (ERα)-mediated transcriptional enhancers. The use of BRD-selective inhibitors has gained much attention as a potential treatment for various solid tumors, including ER-positive breast cancers. However, the roles of individual BET family members have largely remained unexplored. Here, we describe the role of BRDs in estrogen (E2)-dependent gene expression in ERα-positive breast cancer cells. We observed that chemical inhibition of BET family proteins with JQ1 impairs E2-regulated gene expression and growth in breast cancer cells. In addition, RNAi-mediated depletion of each BET family member (BRDs 2, 3, and 4) revealed partially redundant roles at ERα enhancers and for target gene transcription. Furthermore, we found a unique role of BRD3 as a molecular sensor of total BET family protein levels and activity through compensatory control of its own protein levels. Finally, we observed that BRD3 is recruited to a subset of ERα-binding sites (ERBS) that are enriched for active enhancer features, located in clusters of ERBSs likely functioning as "super enhancers," and associated with highly E2-responsive genes. Collectively, our results illustrate a critical and specific role for BET family members in ERα-dependent gene transcription. IMPLICATIONS: BRD3 is recruited to and controls the activity of a subset ERα transcriptional enhancers, providing a therapeutic opportunity to target BRD3 with BET inhibitors in ERα-positive breast cancers., (©2019 American Association for Cancer Research.)

PARP inhibitors (PARPi) prevent cancer cell growth by inducing synthetic lethality with DNA repair defects (e.g., in BRCA1/2 mutant cells). We have identified an alternative pathway for PARPi-mediated growth control in BRCA1/2-intact breast cancer cells involving rDNA transcription and ribosome biogenesis. PARP-1 binds to snoRNAs, which stimulate PARP-1 catalytic activity in the nucleolus independent of DNA damage. Activated PARP-1 ADP-ribosylates DDX21, an RNA helicase that localizes to nucleoli and promotes rDNA transcription when ADP-ribosylated. Treatment with PARPi or mutation of the ADP-ribosylation sites reduces DDX21 nucleolar localization, rDNA transcription, ribosome biogenesis, protein translation, and cell growth. The salient features of this pathway are evident in xenografts in mice and human breast cancer patient samples. Elevated levels of PARP-1 and nucleolar DDX21 are associated with cancer-related outcomes. Our studies provide a mechanistic rationale for efficacy of PARPi in cancer cells lacking defects in DNA repair whose growth is inhibited by PARPi., (Copyright © 2019 Elsevier Inc. All rights reserved.)