Project description:Reward-related memory is an important factor in cocaine seeking. One necessary signaling mechanism for long-term memory formation is the activation of poly(ADP-ribose) polymerase-1 (PARP-1), via poly(ADP-ribosyl)ation. We demonstrate herein that auto-poly(ADP-ribosyl)ation of activated PARP-1 was significantly pronounced during retrieval of cocaine-associated contextual memory, in the central amygdala (CeA) of rats expressing cocaine-conditioned place preference (CPP). Intra-CeA pharmacological and shRNA depletion of PARP-1 activity during cocaine-associated memory retrieval abolished CPP. In contrast, PARP-1 inhibition after memory retrieval did not affect CPP reconsolidation process and subsequent retrievals. Chromatin Immuoprecipitation (ChIP) sequencing revealed that PARP-1 binding in the CeA is highly enriched in genes involved in neuronal signaling. We identified amongst PARP targets in CeA a single gene, yet uncharacterized and encoding a putative transposase inhibitor, at which PARP-1 enrichment dramatically increases during cocaine-associated memory retrieval and positively correlates with CPP. Our findings have important implications for understanding drug-related behaviors, and suggest possible future therapeutic targets for drug abuse. 4 samples, each is pooled central amygdalae tissues collected from 2 rats. Rats were trained for cocaine-conditioned place-preference (CPP), tissues were harvested immediately following cocaine-CPP retrieval. Three groups of rats were used: high cocaine CPP, low cocaine CPP and control saline only trained rats.
Project description:Drosophila Insulator proteins mediate long-range chromosomal interactions. ChIP-seq revealed that binding of insulator proteins to some specific DNA sites was regulated by poly(ADP-ribosyl)ation in S2 cells. Three insulator sites regulated by poly(ADP-ribosyl)ation were used as baits to map their distant interacting sites using 4C assay in control S2 cells. Mapping the chromosomal interactions of three specific insulator binding sites with 4C assay in control S2 cells.
Project description:Reward-related memory is an important factor in cocaine seeking. One necessary signaling mechanism for long-term memory formation is the activation of poly(ADP-ribose) polymerase-1 (PARP-1), via poly(ADP-ribosyl)ation. We demonstrate herein that auto-poly(ADP-ribosyl)ation of activated PARP-1 was significantly pronounced during retrieval of cocaine-associated contextual memory, in the central amygdala (CeA) of rats expressing cocaine-conditioned place preference (CPP). Intra-CeA pharmacological and shRNA depletion of PARP-1 activity during cocaine-associated memory retrieval abolished CPP. In contrast, PARP-1 inhibition after memory retrieval did not affect CPP reconsolidation process and subsequent retrievals. Chromatin Immuoprecipitation (ChIP) sequencing revealed that PARP-1 binding in the CeA is highly enriched in genes involved in neuronal signaling. We identified amongst PARP targets in CeA a single gene, yet uncharacterized and encoding a putative transposase inhibitor, at which PARP-1 enrichment dramatically increases during cocaine-associated memory retrieval and positively correlates with CPP. Our findings have important implications for understanding drug-related behaviors, and suggest possible future therapeutic targets for drug abuse.
Project description:Drosophila Insulator proteins mediate long-range chromosomal interactions. ChIP-seq revealed that binding of insulator proteins to some specific DNA sites was regulated by poly(ADP-ribosyl)ation in S2 cells. Three insulator sites regulated by poly(ADP-ribosyl)ation were used as baits to map their distant interacting sites using 4C assay in control S2 cells.
Project description:The autosomal recessive immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is a genetically heterogeneous disorder. Despite recent successes in the identification of the underlying gene defects, it is currently unclear how mutations in any of the four known ICF genes cause a primary immunodeficiency. Here we demonstrate that loss of ZBTB24 in B cells from ICF2 patients impairs non-homologous end-joining (NHEJ) during immunoglobulin class-switch recombination and consequently impairs immunoglobulin production and subtype balance. Mechanistically, we found that ZBTB24 associates with poly(ADP-ribose) polymerase 1 (PARP1) and stimulates auto-poly(ADP-ribosyl)ation of this enzyme. The zinc finger in ZBTB24 binds PARP1-associated poly(ADP-ribose) chains and mediates the PARP1-dependent recruitment of ZBTB24 to DNA breaks. Moreover, by binding to poly(ADP-ribose) chains ZBTB24 protects these moieties from degradation by poly(ADP-ribose) glycohydrolase (PARG). This enhances the poly(ADP-ribose)-dependent interaction between PARP1 and the LIG4/XRCC4 NHEJ complex and promotes NHEJ by facilitating the assembly of this repair complex at DNA breaks. Thus, we uncover ZBTB24 as a regulator of PARP1-dependent NHEJ and class-switch recombination, providing a molecular basis for the immunodeficiency in ICF syndrome.
Project description:O-GlcNAc glycosylation is a prevalent protein post-translational modification (PTM) that occurs intracellularly. Previous investigations have demonstrated that O-GlcNAcylation crosstalks with phosphorylation and ubiquitination, but it is unclear whether it interplays with other PTMs. Here we studied its relationship with ADP-ribosylation, which involves decorating target proteins with the ADP-ribose moiety. We discovered that one ADP-ribosylation “eraser”- ADP-ribose glycohydrolase (PARG)- is O-GlcNAcylated at Ser26. O-GlcNAcylation of PARG is essential to maintain its nuclear localization and chromatin association. In hepatocellular carcinoma (HCC) cells, PARG O-GlcNAcylation enhances DNA damage-binding protein 1 (DDB1) poly(ADP-ribosyl)ation (PARylation) and attenuates its ubiquitination. DDB1 is thus stabilized and degrades its downstream targets, such as c-Myc. We further utilized mouse xenograft models and demonstrated that PARG-S26A promoted HCC. Our study thus revealed that PARG O-GlcNAcylation inhibits HCC, and we propose that O-GlcNAc glycosylation may crosstalk with many other PTMs.
Project description:Arabidopsis Nudix hydrolases, AtNUDX6 and 7, exhibit pyrophosphohydrolase activities toward NADH and contribute to the modulation of various defense responses, such as the poly(ADP-ribosyl)ation (PAR) reaction and salicylic acid (SA)-induced Nonexpresser of Pathogenesis-Related genes 1 (NPR1)-dependent defense pathway, against biotic and abiotic stresses. To identify genes (NRGs) whose expression levels positively and negatively correlated with NADH levels, a transcriptome analysis using KO-nudx6, KO-nudx7, and double KO-nudx6/7 plants, in which intracellular NADH levels increased in a step-wise manner. Arabidopsis wild-type, KO-nudx6, KO-nudx7, and double KO-nudx6/7 plants were grown on MS medium in a growth chamber kept at 23°C during 16 h of light (100 µmol/m2/s) and at 22°C during 8 h of darkness. Two-week-old cotyledons were collected as samples for microarray analysis.
Project description:Arabidopsis Nudix hydrolases, AtNUDX6 and 7, exhibit pyrophosphohydrolase activities toward NADH and contribute to the modulation of various defense responses, such as the poly(ADP-ribosyl)ation (PAR) reaction and salicylic acid (SA)-induced Nonexpresser of Pathogenesis-Related genes 1 (NPR1)-dependent defense pathway, against biotic and abiotic stresses. To identify genes (NRGs) whose expression levels positively and negatively correlated with NADH levels, a transcriptome analysis using KO-nudx6, KO-nudx7, and double KO-nudx6/7 plants, in which intracellular NADH levels increased in a step-wise manner.
Project description:PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. Here we show that PARP14 is dual function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a dramatic increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response.
Project description:Protein poly(ADP-ribosyl)ation (PARylation) primarily catalyzed by poly(ADP-ribose) polymerases (PARPs) plays a crucial role in controlling various cellular responses. However, PARylation targets and their functions remain largely elusive. In this study, we deployed an Arabidopsis protein microarray coupled with in vitro PARylation to globally identify PARylation targets in plants. In line with nuclear localization of Arabidopsis PARPs, 56% of PARylation targets are predicted to localize in nucleus. Consistent with the essential role of protein PARylation in plant innate immunity, forkhead-associated (FHA) domain protein DAWDLE (DDL), one of the PARylation targets, positively regulates plant defense to both adapted and non-adapted pathogens. Arabidopsis PARP2 interacts and PARylates DDL, which was enhanced upon treatment of microbe-associated molecular pattern. Mass spectrometry and mutagenesis analysis identified multiple PARylation sites of DDL by PARP2. Genetic complementation assays indicate that DDL PARylation is required for its function in plant immunity. In contrast, DDL PARylation appears to be dispensable for its previously reported function in plant development likely mediated by the regulation of microRNA biogenesis. Our study uncovers many previously unknown PARylation targets and points to the distinct functions of DDL in plant immunity and development mediated by protein PARylation and small RNA biogenesis respectively.