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 copper-catalyzed 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.
authors.affiliation: The Laboratory of Signaling and Gene Expression, Cecil H. and Ida Green Center for Reproductive Biology Sciences and The Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Gibson
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
authors.forename: Yajie
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Howard Hughes Medical Institute and Department of Chemistry, Cornell University, Ithaca, NY 14850, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Sarepta Therapeutics, Cambridge, MA 02142, USA.
authors.forename: Kristine M
authors.initials: KM
authors.lastname: Hussey
authors.affiliation: Howard Hughes Medical Institute and Department of Chemistry, Cornell University, Ithaca, NY 14850, USA.
authors.forename: Jonathan H
authors.initials: JH
authors.lastname: Shrimp
authors.affiliation: Howard Hughes Medical Institute and Department of Chemistry, Cornell University, Ithaca, NY 14850, USA.
authors.forename: Hening
authors.initials: H
authors.lastname: Lin
authors.affiliation: Biolog Life Science Institute, D-28199 Bremen, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Schwede
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
authors.forename: Yonghao
authors.initials: Y
authors.lastname: Yu
authors.affiliation: The Laboratory of Signaling and Gene Expression, Cecil H. and Ida Green Center for Reproductive Biology Sciences and The Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. lee.kraus@utsouthwestern.edu.
authors.forename: W Lee
authors.initials: WL
authors.lastname: Kraus
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry*; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D009243:NAD / Q000737:chemistry*; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D012266:Ribose / Q000737:chemistry*; D061805:Transcription Elongation, Genetic; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D014157:Transcription Factors; C118437:negative elongation factor; D009243:NAD; D000244:Adenosine Diphosphate; D012266:Ribose; C499320:PARP1 protein, human; C483041:PARP2 protein, human; C480979:PARP3 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D042925:Positive Transcriptional Elongation Factor B; D012319:RNA Polymerase II