Ribosome ADP-ribosylation inhibits translation and maintains proteostasis in cancers.

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.

PARP Inhibitors for Cancer Therapy.

Rucaparib is an inhibitor of nuclear poly (ADP-ribose) polymerases (inhibition of PARP-1 > PARP-2 > PARP-3), following a similar drug, Olaparib. It disrupts DNA repair and replication pathways (and possibly transcription), leading to selective killing of cancer cells with BRCA1/2 mutations. Rucaparib is approved for recurrent ovarian cancers with germline or somatic mutations in BRCA1/2.

No driver behind the wheel? Targeting transcription in cancer.

Exploiting the dependence of cancer cells on transcription can be used as an effective strategy for targeting aggressive and therapeutically recalcitrant tumors. Wang et al. show that inhibiting transcription using THZ1, a small-molecule inhibitor of cyclin-dependent kinase CDK7, induces apoptotic cell death in triple-negative breast cancers.

The expanding universe of PARP1-mediated molecular and therapeutic mechanisms.

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.

Multiomics analysis of the NAD+-PARP1 axis reveals a role for site-specific ADP-ribosylation in splicing in embryonic stem cells.

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.

Come one, come all? Re-evaluating RNA polymerase II pre-initiation complex assembly using single-molecule microscopy.

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.

Functional Interplay between Histone H2B ADP-Ribosylation and Phosphorylation Controls Adipogenesis.

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.

PARPs and ADP-ribosylation: 60 years on.

Work on PARPs-a family of enzymes that catalyze ADP-ribosylation, a posttranslational modification of proteins-has resulted in major advances and reached important milestones. The past decade has seen new discoveries in areas well beyond the historical focus on DNA repair, which are having impacts on the understanding and treatment of human disease. This special focus section of Genes & Development includes seven reviews that highlight these discoveries and point the way forward for future advances in the field.

PARPs and ADP-ribosylation in RNA biology: from RNA expression and processing to protein translation and proteostasis.

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.

Activation of PARP-1 by snoRNAs Controls Ribosome Biogenesis and Cell Growth via the RNA Helicase DDX21.

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.

A rapid, extensive, and transient transcriptional response to estrogen signaling in breast cancer cells.

We report the immediate effects of estrogen signaling on the transcriptome of breast cancer cells using global run-on and sequencing (GRO-seq). The data were analyzed using a new bioinformatic approach that allowed us to identify transcripts directly from the GRO-seq data. We found that estrogen signaling directly regulates a strikingly large fraction of the transcriptome in a rapid, robust, and unexpectedly transient manner. In addition to protein-coding genes, estrogen regulates the distribution and activity of all three RNA polymerases and virtually every class of noncoding RNA that has been described to date. We also identified a large number of previously undetected estrogen-regulated intergenic transcripts, many of which are found proximal to estrogen receptor binding sites. Collectively, our results provide the most comprehensive measurement of the primary and immediate estrogen effects to date and a resource for understanding rapid signal-dependent transcription in other systems.

Spirits in the Material World: Enhancer RNAs in Transcriptional Regulation.

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.

Decline in Corepressor CNOT1 in the Pregnant Myometrium Near Term Impairs Progesterone Receptor Function and Increases Contractile Gene Expression.

Progesterone (P4), acting via its nuclear receptor (PR), is critical for pregnancy maintenance by suppressing proinflammatory and contraction-associated protein (CAP)/contractile genes in the myometrium. P4/PR partially exerts these effects by tethering to NF-κB bound to their promoters, thereby decreasing NF-κB transcriptional activity. However, the underlying mechanisms whereby P4/PR interaction blocks proinflammatory and CAP gene expression are not fully understood. Herein, we characterized CCR-NOT transcription complex subunit 1 (CNOT1) as a P4-induced corepressor that also interacts within the same chromatin complex as PR-B. In mouse myometrium increased expression of CAP genes Oxtr and Cx43 at term coincided with a marked decline in expression and binding of endogenous CNOT1 to NF-κB-response elements within the Oxtr and Cx43 promoters. Increased CAP gene expression was accompanied by a pronounced decrease in the enrichment of repressive histone marks and an increase in the enrichment of active histone marks to this genomic region. These changes in histone modification were associated with changes in the expression of corresponding histone-modifying enzymes. Myometrial tissues from P4-treated 18.5 dpc pregnant mice manifested increased Cnot1 expression at 18.5 dpc, compared to vehicle-treated controls. In hTERT-HM cells, P4 treatment enhanced CNOT1 expression and its recruitment to NF-κB-response elements within the CX43 and OXTR promoter regions. Furthermore, knockdown of CNOT1 significantly increased the expression of contractile genes. These novel findings suggest that decreased expression and binding of the transcriptional corepressor CNOT1 at the chromatin level near term and associated changes in histone modifications at the OXTR and CX43 promoters contribute to the induction of myometrial contractility leading to parturition.

Catalytic-Independent Functions of PARP-1 Determine Sox2 Pioneer Activity at Intractable Genomic Loci.

Pioneer transcription factors (TFs) function as genomic first responders, binding to inaccessible regions of chromatin to promote enhancer formation. The mechanism by which pioneer TFs gain access to chromatin remains an important unanswered question. Here we show that PARP-1, a nucleosome-binding protein, cooperates with intrinsic properties of the pioneer TF Sox2 to facilitate its binding to intractable genomic loci in embryonic stem cells. These actions of PARP-1 occur independently of its poly(ADP-ribosyl) transferase activity. PARP-1-dependent Sox2-binding sites reside in euchromatic regions of the genome with relatively high nucleosome occupancy and low co-occupancy by other transcription factors. PARP-1 stabilizes Sox2 binding to nucleosomes at suboptimal sites through cooperative interactions on DNA. Our results define intrinsic and extrinsic features that determine Sox2 pioneer activity. The conditional pioneer activity observed with Sox2 at a subset of binding sites may be a key feature of other pioneer TFs operating at intractable genomic loci.

PARP-1 Controls the Adipogenic Transcriptional Program by PARylating C/EBPß and Modulating Its Transcriptional Activity.

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins mediated by PARP family members, such as PARP-1. Although PARylation has been studied extensively, few examples of definitive biological roles for site-specific PARylation have been reported. Here we show that C/EBPß, a key pro-adipogenic transcription factor, is PARylated by PARP-1 on three amino acids in a conserved regulatory domain. PARylation at these sites inhibits C/EBPß's DNA binding and transcriptional activities and attenuates adipogenesis in various genetic and cell-based models. Interestingly, PARP-1 catalytic activity drops precipitously during the first 48 hr of differentiation, corresponding to a release of C/EBPß from PARylation-mediated inhibition. This promotes the binding of C/EBPß at enhancers controlling the expression of adipogenic target genes and continued differentiation. Depletion or chemical inhibition of PARP-1, or mutation of the PARylation sites on C/EBPß, enhances these early adipogenic events. Collectively, our results provide a clear example of how site-specific PARylation drives biological outcomes.

PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes.

The discovery of poly(ADP-ribose) >50 years ago opened a new field, leading the way for the discovery of the poly(ADP-ribose) polymerase (PARP) family of enzymes and the ADP-ribosylation reactions that they catalyze. Although the field was initially focused primarily on the biochemistry and molecular biology of PARP-1 in DNA damage detection and repair, the mechanistic and functional understanding of the role of PARPs in different biological processes has grown considerably of late. This has been accompanied by a shift of focus from enzymology to a search for substrates as well as the first attempts to determine the functional consequences of site-specific ADP-ribosylation on those substrates. Supporting these advances is a host of methodological approaches from chemical biology, proteomics, genomics, cell biology, and genetics that have propelled new discoveries in the field. New findings on the diverse roles of PARPs in chromatin regulation, transcription, RNA biology, and DNA repair have been complemented by recent advances that link ADP-ribosylation to stress responses, metabolism, viral infections, and cancer. These studies have begun to reveal the promising ways in which PARPs may be targeted therapeutically for the treatment of disease. In this review, we discuss these topics and relate them to the future directions of the field.

Dynamic assembly and activation of estrogen receptor α enhancers through coregulator switching.

Although many features of active transcriptional enhancers have been defined by genomic assays, we lack a clear understanding of the order of events leading to enhancer formation and activation as well as the dynamics of coregulator interactions within the enhancer complex. Here, we used selective loss- or gain-of-function mutants of estrogen receptor α (ERα) to define two distinct phases of ligand-dependent enhancer formation. In the first phase (0-20 min), p300 is recruited to ERα by Mediator as well as p300's acetylhistone-binding bromodomain to promote initial enhancer formation, which is not competent for sustained activation. In the second phase (20-45 min), p300 is recruited to ERα by steroid receptor coregulators (SRCs) for enhancer maturation and maintenance. Successful transition between these two phases ("coregulator switching") is required for proper enhancer function. Failure to recruit p300 during either phase leads to abortive enhancer formation and a lack of target gene expression. Our results reveal an ordered and cooperative assembly of ERα enhancers requiring functional interplay among p300, Mediator, and SRCs, which has implications for hormone-dependent gene regulation in breast cancers. More broadly, our results demonstrate the unexpectedly dynamic nature of coregulator interactions within enhancer complexes, which are likely to be a defining feature of all enhancers.

Location, Location, Location: Compartmentalization of NAD+ Synthesis and Functions in Mammalian Cells.

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.

Synthesis, Detection, and Metabolism of Pyridone Ribosides, Products of NAD Overoxidation.

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 1H 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.

New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs.

Poly(ADP-ribose) polymerases (PARPs) are enzymes that transfer ADP-ribose groups to target proteins and thereby affect various nuclear and cytoplasmic processes. The activity of PARP family members, such as PARP1 and PARP2, is tied to cellular signalling pathways, and through poly(ADP-ribosyl)ation (PARylation) they ultimately promote changes in gene expression, RNA and protein abundance, and the location and activity of proteins that mediate signalling responses. PARPs act in a complex response network that is driven by the cellular, molecular and chemical biology of poly(ADP-ribose) (PAR). This PAR-dependent response network is crucial for a broad array of physiological and pathological responses and thus is a good target for chemical therapeutics for several diseases.