Project description:The metabolic flexibility of tissues dictates the extent and reversibility of organ damage during inflammatory challenges. However, there are currently no countermeasures for myocardial metabolic breakdown in the treatment of septic cardiomyopathy (SCM). Nicotinamide adenine dinucleotide (NAD+) signaling is crucial for maintaining cellular metabolic homeostasis and modulating inflammatory responses. Through the use of single-cell sequencing technology, specifically the 10x Genomics scRNAseq approach, we have investigated the role of NAD+ signaling-related genes in SCM. Our findings reveal that both genetic and pharmacological inhibition of the mono-ADP-ribosyl hydrolase MacroD1, which is highly expressed in cardiomyocytes, can effectively counteract the myocardial metabolic impairment, inflammation, dysfunction, and mortality risk triggered by lipopolysaccharide exposure and cecal ligation and puncture in mice.
Project description:ADP-ribosylation is a common modification that occurs in proteins and nucleic acids, regulating many cellular processes ranging from DNA repair to inflammatory signaling. ADP-ribosylation plays an important role in cancer biology, infectious diseases, and obesity, but its role in the development of type 1 diabetes (T1D) is not well understood. Here, we studied the role of ADP-ribosyltransferase PARP12 in T1D development. PARP12 expression is highly induced in human islets treated with pro-inflammatory cytokines or β cells from diabetic donors. Proteomics analysis of MIN6 insulin-producing cells identified that the RNA machinery is regulated by PARP12 during inflammation. PARP12 also ADP-ribosylates 150 mRNAs, including the insulin mRNA. This mRNA ADP-ribosylation in turn modifies transcript localization and halts translation. Overall, our data identified a role for PARP12 in ADP-ribosylation and translation halting of mRNAs, which may affect insulin production during insulitis.
Project description:Chromatin ADP-ribosylation regulates important cellular processes. However, the exact location and magnitude of chromatin ADP-ribosylation are largely unknown. A robust and versatile method for assessing chromatin ADP-ribosylation is therefore crucial for further understanding its function. Here, we present a chromatin affinity precipitation method based on the high specificity and avidity of two well-characterized ADP-ribose binding domains to map chromatin ADP-ribosylation at the genome-wide scale and at specific loci. Our ADPr-ChAP method revealed that in cells exposed to oxidative stress, ADP-ribosylation of chromatin scaled with histone density, with highest levels at heterochromatic sites and depletion at active promoters. Furthermore, in growth factor-induced adipocyte differentiation, increased chromatin ADP-ribosylation was observed at PPARγ target genes, whose expression is ADP-ribosylation-dependent. In combination with deep-sequencing and conventional ChIP, the established ADPr-ChAP provides a valuable resource for the bioinformatic comparison of ADP-ribosylation with other chromatin modifications and for addressing its role in other biologically important processes.
Project description:The aim of this project was to identify the auto mono-ADP-ribosylation sites on SIRT6 and SIRT7 to study the effect of the point mutations S56A and N189A respectively on their ADP-ribosylation activity. In addition, the mono-ADP-ribosylation pattern of SIRT7 was obtained in cells under different stress conditions (UV, H2O2, ionizing Irradiation and glucose starvation).
Project description:A key determinant of the pro-inflammatory responses in macrophages is the Signal Transducers and Activators of Transcription (STAT) family member, STAT1α. STAT1α activation by interferon-gamma (IFNγ) leads to induction of a transcriptional program that is coordinated in a large part by post-translational modifications such as phosphorylation. Poly(ADP-ribose) Polymerases (PARPs), which include PARP-1, catalyze the addition of ADP-ribose moieties (ADP-ribosylation) to target proteins and modulate their function. We found that PARP-1 mediates IFNγ-stimulated transcription by regulating genome-wide binding of STAT1α and its IFNγ-dependent phosphorylation. We identified STAT1α as a target of PARP-1 and found sites of ADP-ribosylation on its DNA-binding (DBD) and Transcription Activation (TA) domains. Surprisingly, ADP-ribosylation on the DBD and TA domains had distinct functional consequences on STAT1α transcriptional activity. Moreover, loss of ADP-ribosylation on either site led to diminished pro-inflammatory responses. These results suggest that PARP-1-driven ADP-ribosylation of STAT1α is a critical mediator of inflammation in macrophages.
Project description:Although ADP-ribosylation of histones by PARP-1 has been linked to genotoxic stress responses, its role in physiological processes has remained elusive. We found that ADPribosylation of histone H2B-Glu35 inhibits the differentiation of adipocyte precursors, leading to a reduction of newly formed adipocytes in white adipose tissue. ADP-ribosylation of Glu35 by PARP-1 inhibits phosphorylation of adjacent H2B-Ser36, which is required for the proadipogenic gene expression program. It also inhibits adipogenesis in cultured cells and a mouse lineage tracing genetic model. The activity of PARP-1 on H2B requires NMNAT-1, a nuclear NAD+ synthase, which serves as a specificity factor to direct PARP-1 catalytic activity to Glu and Asp residues. Collectively, our results demonstrate a functional interplay between H2B-Glu35 ADP-ribosylation and H2B-Ser36 phosphorylation that controls adipogenesis and cancer cell proliferation.
Project description:Mycobacterium tuberculosis maintains long-term infections characterised by the need to regulate growth and adapt to contrasting in vivo environments. Here we show that M. tuberculosis complex bacteria utilise reversible ADP-ribosylation of single-stranded DNA as an epigenetic mechanism to coordinate stationary phase growth with transcriptional adaptation. The DNA-modification is controlled by DarT, an ADP-ribosyl transferase, which adds ADP-ribose to thymidine, and DarG, which enzymatically removes this base modification. Using darG-knockdown M. bovis BCG, we map the first DNA-ADP-ribosylome from any organism. We show that inhibition of replication by DarT is reversible and accompanied by extensive ADP-ribosylation at the origin of replication (OriC). In addition, we observe ADP-ribosylation across the genome and demonstrate that ADP-ribose-thymidine alters the transcriptional activity of M. tuberculosis RNA polymerase. Furthermore, we demonstrate that during stationary phase, DarT-dependent ADP-ribosylation of M. tuberculosis DNA is required to optimally induce expression of the Zur regulon including the ESX-3 secretion system and multiple alternative ribosome proteins. Thus, ADP-ribosylation of DNA can provide an epigenetic link through every aspect of DNA biology from replication to transcription to translation.
Project description:Mycobacterium tuberculosis maintains long-term infections characterised by the need to regulate growth and adapt to contrasting in vivo environments. Here we show that M. tuberculosis complex bacteria utilise reversible ADP-ribosylation of single-stranded DNA as an epigenetic mechanism to coordinate stationary phase growth with transcriptional adaptation. The DNA-modification is controlled by DarT, an ADP-ribosyl transferase, which adds ADP-ribose to thymidine, and DarG, which enzymatically removes this base modification. Using darG-knockdown M. bovis BCG, we map the first DNA-ADP-ribosylome from any organism. We show that inhibition of replication by DarT is reversible and accompanied by extensive ADP-ribosylation at the origin of replication (OriC). In addition, we observe ADP-ribosylation across the genome and demonstrate that ADP-ribose-thymidine alters the transcriptional activity of M. tuberculosis RNA polymerase. Furthermore, we demonstrate that during stationary phase, DarT-dependent ADP-ribosylation of M. tuberculosis DNA is required to optimally induce expression of the Zur regulon including the ESX-3 secretion system and multiple alternative ribosome proteins. Thus, ADP-ribosylation of DNA can provide an epigenetic link through every aspect of DNA biology from replication to transcription to translation.
Project description:Mycobacterium tuberculosis maintains long-term infections characterised by the need to regulate growth and adapt to contrasting in vivo environments. Here we show that M. tuberculosis complex bacteria utilise reversible ADP-ribosylation of single-stranded DNA as an epigenetic mechanism to coordinate stationary phase growth with transcriptional adaptation. The DNA-modification is controlled by DarT, an ADP-ribosyl transferase, which adds ADP-ribose to thymidine, and DarG, which enzymatically removes this base modification. Using darG-knockdown M. bovis BCG, we map the first DNA-ADP-ribosylome from any organism. We show that inhibition of replication by DarT is reversible and accompanied by extensive ADP-ribosylation at the origin of replication (OriC). In addition, we observe ADP-ribosylation across the genome and demonstrate that ADP-ribose-thymidine alters the transcriptional activity of M. tuberculosis RNA polymerase. Furthermore, we demonstrate that during stationary phase, DarT-dependent ADP-ribosylation of M. tuberculosis DNA is required to optimally induce expression of the Zur regulon including the ESX-3 secretion system and multiple alternative ribosome proteins. Thus, ADP-ribosylation of DNA can provide an epigenetic link through every aspect of DNA biology from replication to transcription to translation.
Project description:A key determinant of the pro-inflammatory responses in macrophages is the Signal Transducers and Activators of Transcription (STAT) family member, STAT1α. STAT1α activation by interferon-gamma (IFNγ) leads to induction of a transcriptional program that is coordinated in a large part by post-translational modifications such as phosphorylation. Poly(ADP-ribose) Polymerases (PARPs), which include PARP-1, catalyze the addition of ADP-ribose moieties (ADP-ribosylation) to target proteins and modulate their function. We found that PARP-1 mediates IFNγ-stimulated transcription by regulating genome-wide binding of STAT1α and its IFNγ-dependent phosphorylation. We identified STAT1α as a target of PARP-1 and found sites of ADP-ribosylation on its DNA-binding (DBD) and Transcription Activation (TA) domains. Surprisingly, ADP-ribosylation on the DBD and TA domains had distinct functional consequences on STAT1α transcriptional activity. Moreover, loss of ADP-ribosylation on either site led to diminished pro-inflammatory responses. These results suggest that PARP-1-driven ADP-ribosylation of STAT1α is a critical mediator of inflammation in macrophages.