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.ADP-ribosylation is a reversible post-translational modification (PTM) of proteins resulting in the covalent attachment of a single ADP-ribose unit [i.e., mono(ADP-ribose) (MAR)] or polymers of ADP-ribose units [i.e., poly(ADP-ribose) (PAR)] on a variety of amino acid residues on target proteins (Gibson and Kraus 2012;Daniels et al. 2015a). This modification is mediated by a diverse group of ADPribosyl transferase (ADPRT) enzymes that use ADP-ribose units derived from β-NAD + to catalyze the ADP-ribosylation reaction. These enzymes include bacterial ADPRTs (e.g., cholera toxin and diphtheria toxin) as well as members of three different protein families in yeast and animals: (1) arginine-specific ecto-enzymes (ARTCs), (2) sirtuins, and (3) PAR polymerases (PARPs) . Surprisingly, a recent study showed that the bacterial toxin DarTG can ADP-ribosylate DNA . How this fits into the broader picture of cellular ADP-ribosylation has yet to be determined.In this review, we focus on the mono(ADP-ribosyl)ation (MARylation) and poly(ADP-ribosyl)ation (PARylation) of glutamate, aspartate, and lysine residues by PARP family members. While many reviews have been written on PARPs in the past decade, we highlight the current trends and ideas in the field, in particular those discoveries that have been published in the past 2-3 years.

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that modulate diverse biological processes through covalent transfer of ADP-ribose from NAD+ onto substrate proteins. Here, we report a robust NAD+ analog-sensitive approach for PARPs, which allows PARP-specific ADP-ribosylation of substrates that is suitable for subsequent copper-catalyzed azide-alkyne cycloaddition reactions. Using this approach, we mapped hundreds of sites of ADP-ribosylation for PARPs 1, 2, and 3 across the proteome, as well as thousands of PARP-1-mediated ADP-ribosylation sites across the genome. We found that PARP-1 ADP-ribosylates and inhibits NELF, a protein complex that regulates promoter-proximal pausing by RNA polymerase II (Pol II). Depletion or inhibition of PARP-1, or mutation of the ADP-ribosylation sites on NELF-E, promotes Pol II pausing, providing a clear functional link between PARP-1, ADP-ribosylation, and NELF. This analog-sensitive approach should be broadly applicable across the PARP family, and has the potential to illuminate the ADP-ribosylated proteome and the molecular mechanisms used by individual PARPs to mediate their responses to cellular signals.

Nuclear receptors activate transcription by recruiting multiple coactivators to the promoters of specific target genes. The functional synergy of the p160 coactivators [steroid receptor coactivator-1, glucocorticoid receptor interacting protein (GRIP1), or the activator for thyroid hormone and retinoid receptors], the histone acetyltransferases cAMP response element binding protein binding protein (CBP) and p300 and the histone methyltransferase coactivator-associated arginine methyltransferase (CARM1) depends on the methyltransferase activity of CARM1. CARM1 methylates histone H3 and other factors including the N-terminal region of p300. Here, we report that CARM1 also methylates Arg-2142 within the C-terminal GRIP1 binding domain (GBD) of p300. In the GBD, both Arg-2088 and Arg-2142 are important for binding GRIP1. Methylation of Arg-2142 inhibits the bimolecular interaction of GRIP1 to p300 in vitro and in vivo. This methylation mark of p300 GBD is removed by peptidyl deiminase 4, thereby enhancing the p300 -GRIP1 interaction. These methylation and demethylimination events also alter the conformation and activity of the coactivator complex and regulate estrogen receptor-mediated transcription, and they thus represent unique mechanisms for regulating coactivator complex assembly, conformation, and function.transcriptional regulation H ormone-activated nuclear receptors (NRs) activate transcription by binding to specific DNA regulatory elements in target gene promoters; there they recruit coactivator proteins to remodel chromatin structure and assemble the transcription complex containing RNA polymerase II (1, 2). The p160 coactivators [e.g., steroid receptor coactivator-1, glucocorticoid receptor interacting protein (GRIP1), and the activator for thyroid hormone and retinoid receptors] bind directly to hormone-activated NR and recruit secondary coactivators such as p300͞cAMP response element binding protein (CREB) binding protein (CBP) and coactivatorassociated arginine methyltransferase (CARM1), which acetylate and methylate (respectively) histones and other proteins in the transcription complex (3-5). These and other covalent modifications of histones and cofactors play important roles in chromatin remodeling and transcriptional regulation (6 -10).

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.

Using a variety of biochemical and cell-based approaches, we show that estrogen receptor alpha (ERalpha) is acetylated by the p300 acetylase in a ligand- and steroid receptor coactivator-dependent manner. Using mutagenesis and mass spectrometry, we identified two conserved lysine residues in ERalpha (Lys266 and Lys268) that are the primary targets of p300-mediated acetylation. These residues are acetylated in cells, as determined by immunoprecipitation-Western blotting experiments using an antibody that specifically recognizes ERalpha acetylated at Lys266 and Lys268. The acetylation of ERalpha by p300 is reversed by native cellular deacetylases, including trichostatin A-sensitive enzymes (i.e. class I and II deacetylases) and nicotinamide adenine dinucleotide-dependent/nicotinamide-sensitive enzymes (i.e. class III deacetylases, such as sirtuin 1). Acetylation at Lys266 and Lys268, or substitution of the same residues with glutamine (i.e. K266/268Q), a residue that mimics acetylated lysine, enhances the DNA binding activity of ERalpha in EMSAs. Likewise, substitution of Lys266 and Lys268 with glutamine enhances the ligand-dependent activity of ERalpha in a cell-based reporter gene assay. Collectively, our results implicate acetylation as a modulator of the ligand-dependent gene regulatory activity of ERalpha. Such regulation is likely to play a role in estrogen-dependent signaling outcomes in a variety of estrogen target tissues in both normal and pathological states.

SUMMARY The interplay between mitogenic and proinflammatory signaling pathways play key roles in determining the phenotypes and clinical outcomes of breast cancers. Using GRO-seq in MCF-7 cells, we defined the immediate transcriptional effects of crosstalk between estradiol (E2) and TNFα, identifying a large set of target genes whose expression is rapidly altered with combined E2+TNFα treatment, but not with either agent alone. The pleiotropic effects on gene transcription in response to E2+TNFα are orchestrated by extensive remodeling of the ERα enhancer landscape in an NF-κB- and FoxA1-dependent manner. In addition, expression of the de novo and synergistically regulated genes is strongly associated with clinical outcomes in breast cancers. Together, our genomic and molecular analyses indicate that TNFα signaling, acting in pathways culminating in the redistribution of NF-κB and FoxA1 binding sites across the genome, creates latent ERα binding sites that underlie altered patterns of gene expression and clinically relevant cellular responses.

Summary Over 50 years ago, the discovery of poly(ADP-ribose) (PAR) set a new field of science in motion - the field of poly(ADP-ribosyl) transferases (PARPs) and ADP-ribosylation. The field is still flourishing today. The diversity of biological processes now known to require PARPs and ADP-ribosylation was practically unimaginable even two decades ago. From an initial focus on DNA damage detection and repair in response to genotoxic stresses, the field has expanded to include the regulation of chromatin structure, gene expression, and RNA processing in a wide range of biological systems, including reproduction, development, aging, stem cells, inflammation, metabolism, and cancer. This special focus issue of Molecular Cell includes a collection of three Reviews, three Perspectives, and a SnapShot, which together summarize the current state of the field and suggest where it may be headed.

Cyclic nucleotide-gated ionic channels in pho- (9). The mamnima rod and olfactory cyclic nucleotide-gated channels contain a threonine residue at this particular position (6-8; Fig. 1). It has been proposed that this alanine/threonine difference might have been important in the evolutionary divergence of cyclic nucleotidebinding sites and that it provides the structural basis for discriminating between cAMP and cGMP (9). We tested the validity of this hypothesis for cyclic nucleotide-gated channels by mutagenesis and expression of wild-type and mutant channels from rod photoreceptors and olfactory epithelium. MATERIALS AND METHODSConstruction of Recombinant pCHOLF102. PCR (13) was done with pCHOLF100 (8) as template and the following primers: a 5' adapter primer [containing an EcoRV restriction site, a consensus sequence for eukaryotic ribosomal-binding sites (14), and the first nine nucleotides from the coding region of CHOLF1001 and a gene-specific 3' primer. The EcoRV/DraIII-digested PCR product replaced the corresponding fragment of pCHOLF100 to yield pCHOLF101. The insert of pCHOLF101 was subcloned into a pT7T3 vector to yield pCHOLF102.Site-Directed Mutagenesis. The point mutations at positions 560 and 537 of the rod and olfactory channel polypeptides, respectively, were introduced by PCR procedure (13) with synthetic oligonucleotides containing the desired nucleotide substitutions.Rod-channel mutant T560A was constructed by the method of Hemsley et al. (15). A circular plasmid with the wild-type rod-channel sequence from pRCG1 (6) was amplified by a pair of primers located "back-to-back" on opposite DNA strands. The resulting PCR product was recircularized and digested with Nsi I and Sty I. The corresponding Nsi I-Sty I fragment in pRCG1 was replaced by the mutated fragment to create pT560A. For the construction ofrod-channel mutant T560S, we took advantage of a newly introduced Cla I restriction site near codon 560 in pT560A. A PCR fragment was produced by using a mutagenic and a complementary primer and linearized pT560A as template. The Nsi I-Cla Ifagment containing the mutation was exchanged for the corresponding Nsi I-Cla I fragment ofpT560A to generate pT560S.Both olfactory-channel mutants T537A and T537S were constructed by combining two overlapping PCR fragments with the aid of newly introduced restriction sites at the locus of mutation (BssHII for pT537A and Rsr II for pT537S) and other suitable restriction sites in the plasmid. pCHOLF102 was used as template. All mutations were verified by sequencing of the entire insert with the dideoxynucleotide chain-termination method.Functional Expression. mRNA specific for the rodphotoreceptor channel, the olfactory channel, and the mutant channels was synthesized in vitro (16) by using the respective linearized plasmid cDNA as template. Transcription was primed with the cap dinucleotide 7-methylguanosine(5')-triphospho(5')guanosine (0.6 mM) (17). Macroscopic current measurements on excised inside-out patches (18)(19)(20) were made after injection...