NAD+ Analog-sensitive PARPs Reveal a Role for PARP-1 in Transcription Elongation
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ABSTRACT:
PROVIDER: PRJNA299154 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR2724020.fastq.gz | Fastqsanger.gz | |||
| SRR2724021.fastq.gz | Fastqsanger.gz | |||
| SRR2724022.fastq.gz | Fastqsanger.gz | |||
| SRR2724023.fastq.gz | Fastqsanger.gz | |||
| SRR2724024.fastq.gz | Fastqsanger.gz |
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Project description:The PARP family of proteins comprises 17 members, about two thirds of which are active mono- or poly(ADP-ribosyl)transferase enzymes that transfer the ADP-ribose moiety of NAD+ onto target proteins. In many cases, ADP-ribosylation, which plays critical roles in human diseases (e.g., cancer, heart disease, and neuropathies) is associated with abrogation of the molecular functions of the target. Discerning ADP-ribosylation events mediated by a specific PARP is challenging, since all PARPs use the same substrate (i.e., NAD+) and the available inhibitors lack the specificity needed to make such conclusions. In order to identify the direct and specific targets of individual PARP family members, we have developed a chemical and genetic approach known as analog sensitivity, in which alteration of a single conserved amino acid in the active site of the PARP protein creates a pocket that allows use of an unnatural NAD+ analog containing a steric moiety. We have functionalized the steric moiety with alkyne for use in click chemistry reactions. This approach, which is transferable to other PARP family members, creates substrate specificity where none previously existed, allowing PARP-specific post-translational modification followed by target visualization, or isolation of ADP-ribosylated targets using click chemistry techniques. We have used this technology in conjunction with mass spectrometry to identify hundreds of targets both unique to, as well as shared among, the nuclear PARP proteins, PARP-1, PARP-2, and PARP-3. We have also determined the genome-wide distribution of PARP-1-specific ADP-ribosylation by coupling this analog-sensitive PARP approach with chromatin cross-linking in method that we call “Click-ChIP-seq”. We observed that PARP-1-specific ADP-ribosylation is enriched at transcriptionally active promoters in proximity to sites of PARP-1 enrichment. In addition, we observed that NELF, an important regulator of RNA Polymerase II (Pol II) pausing, is not only a target of ADP-ribosylation by PARP-1 but also spatially correlated with chromatin-associated ADP-ribose and PARP-1 in Click-ChIP-seq and ChIP-seq assays, respectively. Given these observations, we hypothesized that ADP-ribosylation might modulate NELF function and result altered Pol II pausing. We have explored this possibility using global run-on coupled with deep sequencing (GRO-seq) in MCF-7 cells in which PARP-1 was depleted by RNAi-mediated knockdown. PARP-1 depletion caused an increase in Pol II pausing genome-wide. Taken together, these results suggest the intriguing possibility that ADP-ribosylation of NELF by PARP-1 may be an important and heretofore unknown step in the release of Pol II into productive elongation.
2016-06-09 | GSE74142 | GEO
Project description:The PARP family of proteins comprises 17 members, about two thirds of which are active mono- or poly(ADP-ribosyl)transferase enzymes that transfer the ADP-ribose moiety of NAD+ onto target proteins. In many cases, ADP-ribosylation, which plays critical roles in human diseases (e.g., cancer, heart disease, and neuropathies) is associated with abrogation of the molecular functions of the target. Discerning ADP-ribosylation events mediated by a specific PARP is challenging, since all PARPs use the same substrate (i.e., NAD+) and the available inhibitors lack the specificity needed to make such conclusions. In order to identify the direct and specific targets of individual PARP family members, we have developed a chemical and genetic approach known as analog sensitivity, in which alteration of a single conserved amino acid in the active site of the PARP protein creates a pocket that allows use of an unnatural NAD+ analog containing a steric moiety. We have functionalized the steric moiety with alkyne for use in click chemistry reactions. This approach, which is transferable to other PARP family members, creates substrate specificity where none previously existed, allowing PARP-specific post-translational modification followed by target visualization, or isolation of ADP-ribosylated targets using click chemistry techniques. We have used this technology in conjunction with mass spectrometry to identify hundreds of targets both unique to, as well as shared among, the nuclear PARP proteins, PARP-1, PARP-2, and PARP-3. We have also determined the genome-wide distribution of PARP-1-specific ADP-ribosylation by coupling this analog-sensitive PARP approach with chromatin cross-linking in method that we call “Click-ChIP-seq”. We observed that PARP-1-specific ADP-ribosylation is enriched at transcriptionally active promoters in proximity to sites of PARP-1 enrichment. In addition, we observed that NELF, an important regulator of RNA Polymerase II (Pol II) pausing, is not only a target of ADP-ribosylation by PARP-1 but also spatially correlated with chromatin-associated ADP-ribose and PARP-1 in Click-ChIP-seq and ChIP-seq assays, respectively. Given these observations, we hypothesized that ADP-ribosylation might modulate NELF function and result altered Pol II pausing. We have explored this possibility using global run-on coupled with deep sequencing (GRO-seq) in MCF-7 cells in which PARP-1 was depleted by RNAi-mediated knockdown. PARP-1 depletion caused an increase in Pol II pausing genome-wide. Taken together, these results suggest the intriguing possibility that ADP-ribosylation of NELF by PARP-1 may be an important and heretofore unknown step in the release of Pol II into productive elongation.
2016-06-09 | GSE74141 | GEO
Project description:To identify the ADPRylated substrates that underlie PARP-7-mediated ovarian cancer cell phenotypes, we developed an NAD+ analog-sensitive approach for PARP-7 comprising a PARP-7 NAD+ binding pocket mutant (S563G) paired with the NAD+ analog 8-Bu(3-yne)T-NAD+. When coupled with mass spectrometry, this approach allowed us to identify the PARP-7 ADP-ribosylated proteome in ovarian cancer cells, including components of the cell-cell adhesion and cytoskeleton organization machinery. Specifically, we found that PARP-7 monoADPRylates alpha-tubulin to promote microtubule instability, which may play a key role in the regulating ovarian cancer cell growth and motility. Collectively, our results point to an extensive PARP-7 ADP-ribosylated proteome with important roles in cancer-related cellular phenotypes.
2020-12-17 | MSV000086611 | MassIVE
Project description:NMNAT-1 and PARP-1, two key enzymes in the NAD+ metabolic pathway, localize to the nucleus where integration of their enzymatic activities has the potential to control a variety of nuclear processes. Using a variety of biochemical, molecular, cell-based, and genomic assays, we show that NMNAT-1 and PARP-1 physically and functionally interact at target gene promoters in MCF-7 cells. Specifically, we show that PARP-1 recruits NMNAT-1 to promoters, where it produces NAD+ to support PARP-1 catalytic activity, but also enhances the enzymatic activity of PARP-1 independent of NAD+ production. Furthermore, using two-photon excitation microscopy, we show that NMNAT-1 catalyzes the production of NAD+ in a nuclear pool that may be distinct from other cellular compartments. In expression microarray experiments, depletion of NMNAT-1 or PARP-1 alters the expression of about 200 protein-coding genes each, with about 10% overlap between the two gene sets. NMNAT-1 enzymatic activity is required for PARP-1-dependent PARylation at the promoters of commonly regulated target genes, as well as the expression of those target genes. Collectively, our studies link the enzymatic activities of NMNAT-1 and PARP-1 to the regulation of a set of common target genes through functional interactions at target gene promoters. We examined the co-localization of NMNAT-1 and PARP-1 at RefSeq promoters in MCF-7 cells using ChIP-chip Five samples:(1) two FLAG-NMNAT-1 IP'd with FLAG antibody from MCF-7 cells ectopically expressig FLAG-NMNAT-1 and (2) three native PARP-1 IP'd from parental MCF-7 cells with PARP-1 antibody
2012-02-15 | E-GEOD-35358 | biostudies-arrayexpress
Project description:NAD+ Analog-sensitive PARPs Reveal a Role for PARP-1 in Transcription Elongation
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Project description:ADP-ribosylation (ADPRylation) is a regulatory posttranslational modification of proteins that results in the covalent attachment of ADP-ribose (ADPR) moieties on target proteins. PARP-7 (TiPARP) is a monoADPR transferase whose proteins substrates and biological activities are poorly understood. Here, we describe a chemical genetics approach that has allowed us to identify substrates of PARP-7 and explore their biological functions. We observed reduced levels of PARP7 mRNA in ovarian cancer patient samples versus normal tissue, but found nonetheless that PARP-7 is required for a number of cancer-related biological endpoints in ovarian cancer cells, including growth, migration, and invasion. Global gene expression and gene ontology analyses in ovarian cancer cells subjected to siRNA-mediated knockdown of PARP7 revealed an enrichment of genes encoding proteins with roles in cell-cell adhesion, cell cycle arrest, apoptosis, and gene regulation. To identify the ADPRylated substrates that underlie PARP-7-mediated ovarian cancer cell phenotypes, we developed an NAD+ analog-sensitive approach for PARP-7 comprising a PARP-7 NAD+ binding pocket mutant (S563G) paired with the NAD+ analog 8-Bu(3-yne)T-NAD+. When coupled with mass spectrometry, this approach allowed us to identify the PARP-7 ADP-ribosylated proteome in ovarian cancer cells, including components of the cell-cell adhesion and cytoskeleton organization machinery. Specifically, we found that PARP-7 monoADPRylates α-tubulin to promote microtubule instability, which may play a key role in the regulating ovarian cancer cell growth and motility. Collectively, our results point to an extensive PARP-7 ADP-ribosylated proteome with important roles in cancer-related cellular phenotypes.
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Project description:NMNAT-1 and PARP-1, two key enzymes in the NAD+ metabolic pathway, localize to the nucleus where integration of their enzymatic activities has the potential to control a variety of nuclear processes. Using a variety of biochemical, molecular, cell-based, and genomic assays, we show that NMNAT-1 and PARP-1 physically and functionally interact at target gene promoters in MCF-7 cells. Specifically, we show that PARP-1 recruits NMNAT-1 to promoters, where it produces NAD+ to support PARP-1 catalytic activity, but also enhances the enzymatic activity of PARP-1 independent of NAD+ production. Furthermore, using two-photon excitation microscopy, we show that NMNAT-1 catalyzes the production of NAD+ in a nuclear pool that may be distinct from other cellular compartments. In expression microarray experiments, depletion of NMNAT-1 or PARP-1 alters the expression of about 200 protein-coding genes each, with about 10% overlap between the two gene sets. NMNAT-1 enzymatic activity is required for PARP-1-dependent PARylation at the promoters of commonly regulated target genes, as well as the expression of those target genes. Collectively, our studies link the enzymatic activities of NMNAT-1 and PARP-1 to the regulation of a set of common target genes through functional interactions at target gene promoters. We examined the co-localization of NMNAT-1 and PARP-1 at RefSeq promoters in MCF-7 cells using ChIP-chip
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