Gene expression before or 3 hours after t-RA treatment in HeLa cells expressing an shRNA control or shRNA against PARG
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE40883 | GEO | 2012/09/15
SECONDARY ACCESSION(S): PRJNA175254
REPOSITORIES: GEO
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Project description:The goal of this experiment was to compare gene expression after t-RA treatment in cells with or without the presence of the PolyADP ribose Glycohydrolase protein (PARG) Microarray analysis of gene expression before or 3 hours after t-RA treatment in Hela cells expressing an shRNA control or shRNA against PARG
2012-09-15 | E-GEOD-40883 | biostudies-arrayexpress
Project description:Poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes that modify target proteins in the nucleus by the addition and removal, respectively, of ADP-ribose polymers. Although a role for PARP-1 in gene regulation has been well established, the role of PARG is less clear. To investigate how PARP-1 and PARG coordinately regulate global patterns of gene expression, we used short hairpin RNAs (shRNAs) to stably knockdown PARP-1 or PARG in MCF-7 cells, followed by expression microarray analyses.
2009-10-23 | GSE12952 | GEO
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.
2024-01-26 | PXD041119 | Pride
Project description:A subset of cancer cells are intrinsically sensitive to inhibitors targeting PARG, the poly(ADP-ribose) glycohydrolase that degrades PAR chains. Sensitivity is accompanied by persistent DNA replication stress, and can be induced by inhibition of TIMELESS, a replisome accelerator. However, the nature of the oncogenic vulnerability responsible for intrinsic sensitivity remains undetermined. To understand PARG activity dependency, we analyzed Timeless model systems and intrinsically sensitive ovarian cancer cells. We show that nucleoside supplementation rescues all phenotypes associated with PARG inhibitor sensitivity, including replisome speed and fork-restart ability, S-phase completion and mitotic entry, proliferation dynamics and clonogenic potential, in both a TIMELESShaploinsufficient model and intrinsically sensitive cells. Importantly nucleoside supplementation restores PARG inhibitor resistance without reverting PAR chain accumulation, indicating that sensitivity correlates with PAR chain intolerance. We show that inhibition of thymidylate synthase, an enzyme required for dNTP homeostasis, induces PARG-dependency, and, using label-free, quantitative proteomics, we show that PKMYT1, a negative regulator of CDK1, is overexpressed in PARG inhibitor-sensitive cells. Together, these observations suggest that PARG inhibitor sensitivity reflects an inability to control replisome speed and/or maintain helicase-polymerase coupling in response to nucleotide imbalances, in turn applying selective pressure on mitotic entry controls to maintain genome integrity.
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Project description:Poly (ADP-ribose) glycohydrolase (PARG) is a dePARylating enzyme which promotes DNA repair by removal of poly (ADP-ribose) (PAR) from PARP1-parylated proteins. Loss or inhibition of PARG results in replication stress and sensitizes cancer cells to DNA damaging agents, suggesting that PARG inhibitors may provide a therapeutic option for cancer therapy. Indeed, PARG inhibitors (PARGi) are now undergoing clinical development for patients having tumors with homologous recombination deficiency (HRD), such as ovarian and breast cancer patients with germline or somatic BRCA1/2-mutations. Biomarkers of PARGi response will be required for the optimal development of these important new drugs. PARP inhibitors kill BRCA-deficient cancer cells by increasing ssDNA gaps during replication. Here, we report that, similar to PARP inhibitors, PARGi treatment can also cause an accumulation of ssGAPs in sensitive cells. PARGi exposure increased accumulation of S-Phase specific PAR, a marker for Okazaki fragment processing (OFP) defects on lagging strands, in sensitive cells but not in resistant cells. PARGi also caused accumulation of PAR at the replication fork and increased pRPA, a marker for replication stress, at the replication fork in sensitive cells. Additionally, PARGi exhibited monotherapy activity in some HR-deficient, as well as HR-proficient, patient-derived organoids of ovarian cancer, and drug sensitivity directly correlated with the accumulation of ssDNA gaps. Taken together, PARGi treatment results in toxic accumulation of PAR at replication forks resulting in ssDNA gaps due to OFP defects during replication. Regardless of the BRCA-status, the induction of ssDNA gaps in preclinical models of ovarian cancer cells correlates with PARGi sensitivity. Patient-derived organoids will be a useful model system for testing PARGi sensitivity and functional biomarkers
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Project description:Poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes that modify target proteins in the nucleus by the addition and removal, respectively, of ADP-ribose polymers. Although a role for PARP-1 in gene regulation has been well established, the role of PARG is less clear. To investigate how PARP-1 and PARG coordinately regulate global patterns of gene expression, we used short hairpin RNAs (shRNAs) to stably knockdown PARP-1 or PARG in MCF-7 cells, followed by expression microarray analyses. Experiment Overall Design: Two shRNAs targeting Luciferase (Luc, control), PARP-1, or PARG were cloned into the pSUPER.retro vector (puro or neo resistant) and sequentially introduced into parental MCF-7 cells using a standard retroviral infection technique. Luc, PARP-1, and PARG control-matched cell lines were generated three independent times. Experiment Overall Design: Total RNA was isolated from stable Luc, PARP-1, and PARG knockdown cells was analyzed for global patterns of gene expression. Briefly, 7 μg of total RNA was labeled using Affymetrix's standard one-cycle amplification and labeling protocol. The labeled cRNA was then hybridized to Affymetrix Human U133A 2.0 GeneChips, which were scanned using a GeneChip Scanner 3000. Each biological replicate (1, 2 or 3) represents a single RNA isolation from each set of cell lines. Experiment Overall Design: These 9 samples were samples were part of a 15-sample microarray analysis (GSE12971) examining expression regulation by SIRT1, PARP-1, PARG and macroH2A. All 15 samples were included for the data normalization steps. Experiment Overall Design: The raw array data was processed by Affymetrix GeneChip Operating Software (GCOS) to obtain detection calls (ABS_CALL) and signal values (VALUE_2). The signals were then normalized by scaling to a target value of 500 using GCOS. To adjust for batch effects due to day-to-day differences in RNA isolations, the empirical Bayes method was applied to the data set. After adjusting any values less than 0.01 to 0.01, the data was log2 transformed, median centered for each array, and median centered for each individual probe set (VALUE). Filters were then applied to obtain final gene lists. Experiment Overall Design: Specifically, the criteria for a gene to be considered regulated by PARP-1 or PARG was: (1) detection call flagged as present or marginal in 2 out of 3 array replicates for both control and factor knockdown cell lines and (2) significance of values between control and knockdown cell lines for any given gene had a two-tailed Studentâ??s t-test with a p-value < 0.05. To determine the genes most robustly regulated by PARP-1 or PARG, a fold change criteria was applied, namely log2 fold change > 0.5 or < -0.5 when compared to the Luc control knockdown cells.
2009-10-30 | E-GEOD-12952 | biostudies-arrayexpress
Project description:Ascites or solid tumour samples from patients with ovarian cancer were collected and grown in culture as ex vivo models of purified tumour cells. RNA-seq was performed on these models to establish gene expression profiles, which allow identification of genes that are differentially expressed between patients with differing tumour intrinsic properties. These samples have been interrogated for the presence of a gene expression signature indicative of sensitivity to an inhibitor of poly(ADP-ribose) glycohydrolase (PARG). These samples are processed in the same manner as previous studies: “E-MTAB-7223 - RNA-seq of human ex vivo ovarian cancer models with matched stromal cells” and “E-MTAB-10801 - RNA-seq of human ex vivo ovarian cancer models with matched stromal cells - part II” with no stromal counterparts included in this current sequencing batch.
2021-10-10 | E-MTAB-11000 | biostudies-arrayexpress
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2020-08-19 | PXD014741 | Pride
Project description:Affymetrix expression arrays were used to compare expression patterns upon knockdown of PARP-1, PARG, SIRT1, or macroH2A in comparison to Luciferase control.
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