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.

Figure 1. Structure-based engineering of an NAD⁺ analog-sensitive PARP-1 (asPARP-1) mutant

(A) (Left) Schematic illustrating NAD⁺ analog-sensitivity in PARP proteins. (Right) Residues in PARP-1 selected for mutation to glycine or alanine for discovery of gatekeeper residues. (B) Chemical structures of the 11 NAD⁺ analogs used for screening for asPARP-1. (C) Western blot for ADP-ribose from automodification reactions containing PARP-1 or PARP-1 mutants (L877A and I895A) and NAD⁺ or NAD⁺ analogs. (D) Depiction of the spatial relationship between position 8 of the adenine ring in NAD⁺ and the gatekeeper residues.

Figure 2. Activity of asPARPs 1, 2, and 3 with a clickable NAD⁺ analog

(A) Chemical structure of the bi-functional NAD⁺ analog 8-Bu(3-yne)T-NAD⁺ with the clickable analog sensitivity-inducing, alkyne-containing R group highlighted in red. (B) Schematic illustrating asPARP activity-dependent, click chemistry-mediated covalent attachment of fluorophores, biotin, or agarose resin to 8-Bu(3-yne)T-ADP-ribosylated proteins. (C) Automodification reactions with wild-type or analog-sensitive PARP-1, PARP-2, and PARP-3 analyzed by Western blotting for ADP-ribose (top) or click chemistry-based in-gel fluorescence (bottom).

Figure 3. Using analog-sensitive PARP-1 mutants to unambiguously identify the ADP-ribosylation targets of DNA-dependent PARPs

(A) In-gel fluorescence of HeLa cell nuclear extract proteins conjugated to azido-TAMRA following reactions with 8-Bu(3-yne)T-NAD⁺ in the presence of wild-type (wt) or analog-sensitive (as) PARP-1, PARP-2, or PARP-3. (B) Depiction of the strategy for LC-MS/MS detection of PARP-specific ADP-ribosylation sites. (Left) asPARP-dependent labeling of HeLa cell nuclear extract (N.E.) proteins (represented by various shapes) using 8-Bu(3-yne)T-NAD⁺ (red). (Right) Post-labeling sample processing for LC-MS/MS. The 8-Bu(3-yne)T-ADP-ribosylated proteins are covalently linked to azide-agarose by copper-catalyzed cycloaddition (‘click’ chemistry; represented by pentagons), washed, and digested with trypsin to release peptides for protein identification. The remaining covalently linked peptides are eluted using hydroxyl amine (NH₂OH) with a mass shift of 15.0109 Da, which allows for identification of ADP-ribosylation sites. (C) Venn diagram depicting the overlap of the protein targets of PARP-1, PARP-2, and PARP-3. (D) Gene ontology terms enriched for the sets of PARP-1, PARP-2 and PARP-3 targets. (E) Histogram of the two-dimensional relationship between previously identified ADP-ribosylation sites (9) with those identified herein.

Figure 4. P-TEFb-dependent ADP-ribosylation of NELF by PARP-1

(A) Schematic showing the distribution of PARP-1 ADP-ribosylation sites (red), P-TEFb phosphorylation sites, and a PARP target-enriched 7-mer RSRSRDR (green) on proteins in the NELF complex. (B) Western blot analysis of immunoprecipitated FLAG-tagged NELF-E or GFP from 293T cells. (C) Silver stained SDS-PAGE gel (left) and ADP-ribose Western blot (right) of immunopurified NELF complex. Asterisk = automodified PARP-1. (D) Western blot for ADP-ribose of in vitro modification reactions containing GST, GST-tagged wild-type NELF-E, or GST-tagged ADP-ribosylation site point mutant NELF-E, PARP-1, and NAD⁺ as indicated. (E) NELF-E/TAR RNA electrophoretic mobility shift assay with or without PARP-1-mediated ADP-ribosylation. GST or GST-NELF-E was titrated between 0.1 to 1.0 μM and NAD⁺ was added at 25 μM (+) or 100 μM (++) during the ADP-ribosylation reaction. (F) Histogram of the relationship between ADP-ribosylation sites identified herein and the nearest incidence of known phosphorylation modifications on PARP target proteins. (G) Western blot analysis of immunoprecipitated FLAG-tagged NELF-E from 293T cells treated with vehicle, the PARP inhibitor PJ34, or the P-TEFb/CDK9 inhibitor flavopiridol.

Figure 5. Click-ChIP-seq, an asPARP-1-based method for identifying the genome-wide distribution of ADP-ribosylation events catalyzed by a specific PARP protein

(A) Schematic representation of Click-ChIP-seq. Nuclei are isolated from Parp^−/− MEFs expressing asPARP-1, labeled with 8-Bu(3-yne)T-NAD⁺, subjected to crosslinking with formaldehyde, and then processed for chromatin immunoprecipitation. The enriched DNA is subjected to deep sequencing. (B) Genome browser view of a multi-gene locus of the mouse genome showing PARP-1-catalyzed ADP-ribosylation (from Click-ChIP-seq) with other genomic features. (C) Heat map showing pairwise clustered correlations between genomic features and PARP-1-mediated ADP-ribosylation from click-ChIP-seq. (D) Heat map representations showing PARP-1-catalyzed ADP-ribosylation (from Click-ChIP-seq) in comparison to PARP-1 (from ChIP-seq) and transcription (from GRO-seq) at the promoters of all RefSeq genes.

Figure 6. Functional links between PARP-1-catalyzed ADP-ribosylation, NELF binding, and RNA polymerase II pausing genome-wide

(A) Metagenes of GRO-seq read density at the promoters of all expressed RefSeq genes from MCF-7 cells subjected to shRNA-mediated knockdown with either control/luciferase or PARP-1 shRNAs (top) or treatment with the PARP inhibitor (PARPi) PJ34 (bottom). (B) RNA polymerase II pausing indices at the promoters of all transcribed RefSeq genes from MCF-7 cells subjected to shRNA-mediated knockdown with either control/luciferase or PARP-1 shRNAs or treatment with PJ34. (C) Boxplots of promoter proximal Pol II “pausing efficacy” determined by Pol II and NELF ChIP-seq in MCF-7 cells under the different experimental conditions indicated for the top quartile of expressed RefSeq genes. Bars marked with different letters are significantly different (p < 2.16 × 10⁻¹⁶; t-test).