Chemical Genetic Discovery of PARP Targets Reveals a Role for PARP-1 in Transcription Elongation

Abstract

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that modulate diverse biological processes through covalent transfer of ADP-ribose from 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 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.