@article{1dc6b98123034e0186795d31fe50dff2,
title = "Chemical genetic discovery of PARP targets reveals a role for PARP-1 in transcription elongation",
abstract = "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 coppercatalyzed azide-alkyne cycloaddition reactions. Using this approach, we mapped hundreds of sites of ADP-ribosylation for PARPs 1, 2, and 3 across the proteome, as well as thousands of PARP-1-mediated ADP-ribosylation sites across the genome. We found that PARP-1 ADP-ribosylates and inhibits negative elongation factor (NELF), a protein complex that regulates promoter-proximal pausing by RNA polymerase II (Pol II). Depletion or inhibition of PARP-1 or mutation of the ADP-ribosylation sites on NELF-E promotes Pol II pausing, providing a clear functional link between PARP-1, ADP-ribosylation, and NELF. This analog-sensitive approach should be broadly applicable across the PARP family and has the potential to illuminate the ADP-ribosylated proteome and the molecular mechanisms used by individual PARPs to mediate their responses to cellular signals.",
author = "Gibson, {Bryan A.} and Yajie Zhang and Hong Jiang and Hussey, {Kristine M.} and Shrimp, {Jonathan H.} and Hening Lin and Frank Schwede and Yonghao Yu and Kraus, {W. Lee}",
note = "Funding Information: We thank M. Chae, Q. Liang, J. DeBrabander, U. Havemann, and D. Imren for technical assistance and members of the Kraus laboratory for helpful discussions about this project. The asPARP expression constructs and the NAD+ analogs can be obtained from University of Texas (UT) Southwestern Medical Center and Biolog Life Science Institute, respectively, under a material transfer agreement. W.L.K., B.A.G., F.S., and H.L. are inventors on U.S. patent application no. 62/144,711, filed by UT Southwestern Medical Center, related to the asPARP technology. W.L.K. and B.A.G. are inventors on U.S. patent application nos. 62/009,955 and PCT/US2015/034852, filed by UT Southwestern Medical Center, related to the ADP-ribose detection reagents. Y.Y. is an inventor on U.S. patent no. 8828672 B2, filed by UT Southwestern Medical Center, related to technology for the determination of D/E-ADP-ribosylation sites. This work was supported by a predoctoral fellowship from the American Heart Association to B.A.G.; grants from the Cancer Prevention and Research Institute of Texas (CPRIT R1103), the Welch Foundation (I-1800), and the UT Southwestern Endowed Scholars Program to Y.Y., who is the Virginia Murchison Linthicum Scholar in Medical Research and a CPRIT Scholar in Cancer Research; a grant from the NIH National Institute of General Medical Sciences (GM086703) to H.L.; and a grant from the NIH National Institute of Diabetes and Digestive and Kidney Diseases (DK069710) and support from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowments to W.L.K. W.L.K. is a founder and consultant for Ribon Therapeutics. The genomic data sets from this study are available from the National Center for Biotechnology Information Gene Expression Omnibus database under accession numbers GSE74141 (ChIP-seq) and GSE74142 (GRO-seq and RNA-seq). The proteomic data sets generated for these studies are available in table S1. Author contributions: On the basis of (7), B.A.G. conceived the asPARP concept, with input from W.L.K. W.L.K. conceived the Click-ChIP-seq method, which was further developed by B.A.G. B.A.G. performed all experiments and computational analyses, except as follows: H.J., J.H.S., H.L., and F.S. synthesized all precursors and NAD+ analogs used in this study. B.A.G. and H.J. performed the enzyme kinetics assays. B.A.G., Y.Z., and Y.Y. prepared the samples and ran the LC-MS/MS analysis. K.M.H. made the PARP-1 knockdown MCF-7 cells and prepared the GRO-seq samples. W.L.K. secured funding to support this project and provided intellectual support for all aspects of the work. B.A.G. and W.L.K. prepared the figures and wrote the paper",
year = "2016",
month = jul,
day = "1",
doi = "10.1126/science.aaf7865",
language = "English (US)",
volume = "353",
pages = "45--50",
journal = "Science",
issn = "0036-8075",
publisher = "American Association for the Advancement of Science",
number = "6294",
}