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NAD+ Analog-sensitive PARPs Reveal a Role for PARP-1 in Transcription Elongation

ABSTRACT: 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. Overall design: Using GRO-seq (knockdown of Luciferase [control] with or without PARP inhibitor [PJ34] treatment and knockdown of PARP-1) and RNA-seq (knockdown of Luciferase [control] with or without CDK inhibitor [DRB] treatment and knockdown of PARP-1) in MCF-7 human breast cancer cells.

INSTRUMENT(S): Illumina Genome Analyzer (Homo sapiens)

SUBMITTER: W. Lee Kraus  

PROVIDER: GSE74142 | GEO | 2016-06-09



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Chemical genetic discovery of PARP targets reveals a role for PARP-1 in transcription elongation.

Gibson Bryan A BA   Zhang Yajie Y   Jiang Hong H   Hussey Kristine M KM   Shrimp Jonathan H JH   Lin Hening H   Schwede Frank F   Yu Yonghao Y   Kraus W Lee WL  

Science (New York, N.Y.) 20160602 6294

Poly[adenosine diphosphate (ADP)-ribose] polymerases (PARPs) are a family of enzymes that modulate diverse biological processes through covalent transfer of ADP-ribose from the oxidized form of nicotinamide adenine dinucleotide (NAD(+)) onto substrate proteins. Here we report a robust NAD(+) analog-sensitive approach for PARPs, which allows PARP-specific ADP-ribosylation of substrates that is suitable for subsequent copper-catalyzed azide-alkyne cycloaddition reactions. Using this approach, we m  ...[more]

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