An Analog-Sensitive Approach Reveals Specific Functions of PARP1

The PARP family of enzymes regulate diverse cellular processes including chromatin maintenance, DNA repair, and gene expression by mono- or poly-ADP–ribosylating target proteins by transferring an ADP ribose derived from NAD⁺. Previous approaches used to identify PARP family targets have not been able to sufficiently distinguish specific targets of individual PARP family members, prompting Gibson and colleagues to develop an analog-sensitive PARP (asPARP) approach. This was initially accomplished with PARP1 by identifying mutations in the PARP1 active site that could accommodate various NAD⁺ analogs. The specific NAD⁺ analogs prevented wild-type PARP enzymes from recognizing the NAD⁺ analog and ADP-ribosylating substrates while allowing asPARP substrates to be labeled or purified in subsequent chemical reactions. Using this approach, PARP1, PARP2, and PARP3–mediated ADP-ribosylation sites were mapped, and both unique and overlapping substrates were identified. PARP1 was found to ADP ribosylate components of the negative elongation factor (NELF) complex at specific glutamate residues. The NELF complex normally promotes RNA polymerase (Pol) II pausing, limiting transcriptional elongation, and it was found that ADP ribosylation prevented its NELF RNA binding ability and prevented Pol II pausing. Consistent with these findings, PARP1 ADP ribosylation was enriched at the promoters of actively transcribed genes, suggesting that ADP ribosylation may promote Pol II pause release and productive transcription elongation. Global run-on (GRO) sequencing was used to identify transcriptionally engaged RNA polymerases, and revealed a global increase in paused Pol II peaks when PARP1 was suppressed, with increased pausing at active promoters and decreased pausing in gene bodies, indicating that PARP1 is required for efficient release of Pol II into productive elongation. Altogether, these findings reveal a role for PARP1 in promoting Pol II pause release and transcription elongation, and indicate that the asPARP approach can be used to elucidate the specific roles of PARP family proteins.

Gibson BA, Zhang Y, Jiang H, Hussey KM, Shrimp JH, Lin H, et al. Chemical genetic discovery of PARP targets reveals a role for PARP-1 in transcription elongation. Science 2016 June 2 [Epub ahead of print].