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.

Glucocorticoid receptor represses proinflammatory genes at distinct steps of the transcription cycle.

Widespread anti-inflammatory actions of glucocorticoid hormones are mediated by the glucocorticoid receptor (GR), a ligand-dependent transcription factor of the nuclear receptor superfamily. In conjunction with its corepressor GR-interacting protein-1 (GRIP1), GR tethers to the DNA-bound activator protein-1 and NF-κB and represses transcription of their target proinflammatory cytokine genes. However, these target genes fall into distinct classes depending on the step of the transcription cycle that is rate-limiting for their activation: Some are controlled through RNA polymerase II (PolII) recruitment and initiation, whereas others undergo signal-induced release of paused elongation complexes into productive RNA synthesis. Whether these genes are differentially regulated by GR is unknown. Here we report that, at the initiation-controlled inflammatory genes in primary macrophages, GR inhibited LPS-induced PolII occupancy. In contrast, at the elongation-controlled genes, GR did not affect PolII recruitment or transcription initiation but promoted, in a GRIP1-dependent manner, the accumulation of the pause-inducing negative elongation factor. Consistently, GR-dependent repression of elongation-controlled genes was abolished specifically in negative elongation factor-deficient macrophages. Thus, GR:GRIP1 use distinct mechanisms to repress inflammatory genes at different stages of the transcription cycle.

Role of transcriptional coregulator GRIP1 in the anti-inflammatory actions of glucocorticoids.

Inhibition of cytokine gene expression by the hormone-activated glucocorticoid receptor (GR) is the key component of the anti-inflammatory actions of glucocorticoids, yet the underlying molecular mechanisms remain obscure. Here we report that glucocorticoid repression of cytokine genes in primary macrophages is mediated by GR-interacting protein (GRIP)1, a transcriptional coregulator of the p160 family, which is recruited to the p65-occupied genomic NFκB-binding sites in conjunction with liganded GR. We created a mouse strain enabling a conditional hematopoietic cell-restricted deletion of GRIP1 in adult animals. In this model, GRIP1 depletion in macrophages attenuated in a dose-dependent manner repression of NFκB target genes by GR irrespective of the upstream Toll-like receptor pathway responsible for their activation. Furthermore, genome-wide transcriptome analysis revealed a broad derepression of lipopolysaccharide (LPS)-induced glucocorticoid-sensitive targets in GRIP1-depleted macrophages without affecting their activation by LPS. Consistently, conditional GRIP1-deficient mice were sensitized, relative to the wild type, to a systemic inflammatory challenge developing characteristic signs of LPS-induced shock. Thus, by serving as a GR corepressor, GRIP1 facilitates the anti-inflammatory effects of glucocorticoids in vivo.

Glucocorticoid receptor coordinates transcription factor-dominated regulatory network in macrophages.

BACKGROUND: Inflammation triggered by infection or injury is tightly controlled by glucocorticoid hormones which signal via a dedicated transcription factor, the Glucocorticoid Receptor (GR), to regulate hundreds of genes. However, the hierarchy of transcriptional responses to GR activation and the molecular basis of their oftentimes non-linear dynamics are not understood. RESULTS: We investigated early glucocorticoid-driven transcriptional events in macrophages, a cell type highly responsive to both pro- and anti-inflammatory stimuli. Using whole transcriptome analyses in resting and acutely lipopolysaccharide (LPS)-stimulated macrophages, we show that early GR target genes form dense networks with the majority of control nodes represented by transcription factors. The expression dynamics of several glucocorticoid-responsive genes are consistent with feed forward loops (FFL) and coincide with rapid GR recruitment. Notably, GR binding sites in genes encoding members of the KLF transcription factor family colocalize with KLF binding sites. Moreover, our gene expression, transcription factor binding and computational data are consistent with the existence of the GR-KLF9-KLF2 incoherent FFL. Analysis of LPS-downregulated genes revealed striking enrichment in multimerized Zn-fingers- and KRAB domain-containing proteins known to bind nucleic acids and repress transcription by propagating heterochromatin. This raises an intriguing possibility that an increase in chromatin accessibility in inflammatory macrophages results from broad downregulation of negative chromatin remodelers. CONCLUSIONS: Pro- and anti-inflammatory stimuli alter the expression of a vast array of transcription factors and chromatin remodelers. By regulating multiple transcription factors, which propagate the initial hormonal signal, GR acts as a coordinating hub in anti-inflammatory responses. As several KLFs promote the anti-inflammatory program in macrophages, we propose that GR and KLFs functionally cooperate to curb inflammation.

Combinatorial Treatment with PARP-1 Inhibitors and Cisplatin Attenuates Cervical Cancer Growth through Fos-Driven Changes in Gene Expression.

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.

Nuclear ADP-ribosylation drives IFNγ-dependent STAT1α enhancer formation in macrophages.

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.

PARPs in lipid metabolism and related diseases.

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.

Nuclear receptors in inflammation control: repression by GR and beyond.

Inflammation is a protective response of organisms to pathogens, irritation or injury. Primary inflammatory sensors activate an array of signaling pathways that ultimately converge upon a few transcription factors such as AP1, NFκB and STATs that in turn stimulate expression of inflammatory genes to ultimately eradicate infection and repair the damage. A disturbed balance between activation and inhibition of inflammatory pathways can set the stage for chronic inflammation which is increasingly recognized as a key pathogenic component of autoimmune, metabolic, cardiovascular and neurodegenerative disorders. Nuclear receptors (NRs) are a large family of transcription factors many of which are known for their potent anti-inflammatory actions. Activated by small lipophilic ligands, NRs interact with a wide range of transcription factors, cofactors and chromatin-modifying enzymes, assembling numerous cell- and tissue-specific DNA-protein transcriptional regulatory complexes with diverse activities. Here we discuss established and emerging roles and mechanisms by which NRs and, in particular, the glucocorticoid receptor (GR) repress genes encoding cytokines, chemokines and other pro-inflammatory mediators.