Research Article

Chemical genetic discovery of PARP targets reveals a role for PARP-1 in transcription elongation

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Science  01 Jul 2016:
Vol. 353, Issue 6294, pp. 45-50
DOI: 10.1126/science.aaf7865

Mapping ADP-ribosylation by PARPs

During many cell processes, ADP-ribose is transferred from NAD+ onto protein substrates by poly(ADP-ribose) polymerases (PARPs). Gibson et al. developed a method to track ribose transfer events and mapped hundreds of sites of ADP-ribosylation for PARPs 1, 2, and 3 across the proteome and genome. One PARP-1 target is NELF, a protein complex that regulates pausing by RNA polymerase II. If NELF is ribosylated, pausing is released and productive transcription elongation resumes.

Science, this issue p. 45


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.

Adenosine diphosphate (ADP)–ribosylation of proteins is an important modulator of cellular processes, from the regulation of chromatin and transcription to protein translation and stability (1). Most of the 17 poly(ADP-ribose) polymerase (PARP) family members encoded in the human genome are enzymes with either mono- or poly(ADP-ribosyl) transferase activities, which covalently link ADP-ribose derived from the oxidized form of nicotinamide adenine dinucleotide (NAD+) to their target proteins, primarily at glutamate, aspartate, and lysine residues (2). PARPs 1, 2, and 3, collectively referred to as the DNA-dependent PARPs, are a group of nuclear proteins with DNA-dependent mono- (PARP-3) or poly- (PARPs 1 and 2) ADP-ribosyl transferase activities involved in DNA repair, chromosome maintenance, chromatin regulation, and gene expression (2, 3). Previous studies using immune-based enrichment, various affinity resins, or protein microarrays to identify the targets of ADP-ribosylation lacked specificity for individual PARP family members (4). A recent chemical genetics approach targeting a conserved residue in the nicotinamide binding site of the PARP catalytic domain was a technological advance but unfortunately ablated the poly(ADP-ribosyl) transferase activity of PARP enzymes, while preserving mono(ADP-ribosyl) transferase activity (5, 6). A single chemical genetic approach that preserves the natural mono- and poly(ADP-ribosyl) transferase activities of PARP enzymes and is broadly applicable across the PARP family should be of great utility.

An analog-sensitive PARP (asPARP) approach targeting the adenine moiety of NAD+

Our previous studies indicated that the adenine moiety of NAD+ is a useful target for chemical modification to alter the catalytic activity and chemistry of PARP family members (7). In this regard, we developed an adenine-focused, NAD+ analog–sensitive approach for PARPs that preserves their poly(ADP-ribosyl) transferase activity (Fig. 1A and fig. S1A) and is capable of identifying the specific targets of individual PARP family members. Analog sensitivity is achieved by mutation of a large “gatekeeper” amino acid in the active site of a protein to a smaller residue, creating a pocket that fits a bulky R group on an engineered substrate, whereas interaction of the bulky R group with the wild-type (WT) enzyme would have been sterically blocked (8). We initially focused on PARP-1, an abundant and ubiquitously expressed PARP protein in metazoans. To identify a gatekeeper residue in PARP-1, we changed 10 large residues buried within the active site and facing the adenine ring of NAD+ to glycine or alanine on the basis of a molecular model (Fig. 1A and figs. S1B and S2). We selected the eight position of the adenine ring of NAD+ as the site for R group addition because its modification precludes ADP-ribosylation with WT PARP-1 (wtPARP-1) or other PARPs (7), a feature critical to the analog-sensitive approach. We then synthesized a library of 11 NAD+ analogs, each with a different R group at position eight, from 8-methylamino-NAD+ to 8-benzylamino-NAD+ (Fig. 1B). In a screen of the 20 PARP-1 mutants versus the 11 NAD+ analogs, we identified two different gatekeeper residues, L877 and I895 (9), whose mutation to alanine results in analog-sensitive activity in a PARP-1 automodification assay (Fig. 1C and figs. S1C and S3). Critically, both of these residues are buried within the active site of the enzyme; thus, their mutation is unlikely to affect protein-protein interactions that might influence substrate selectivity. Although L877 and I895 are separated by 18 amino acids in the PARP-1 linear sequence, they are adjacent to one another and proximal to the eight position of the adenine ring in three-dimensional (3D) space (Fig. 1D). These results support our molecular model of PARP-1 interactions with NAD+ (figs. S1B and S2), as well as the structural basis for our asPARP approach.

Fig. 1 Structure-based engineering of an asPARP-1 mutant.

(A) (Left) Schematic illustrating NAD+ analog sensitivity in PARP proteins. R, unnatural chemical moieties added to NAD+. (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 (WB) for ADP-ribose from automodification reactions containing PARP-1 or PARP-1 mutants (L877A and I895A) and NAD+ or NAD+ analogs. M.W., molecular weight. (D) Depiction of the spatial relationship between position eight of the adenine ring in NAD+ and the gatekeeper residues.

To extend the utility of our asPARP approach, we functionalized the R group of 8-butylthio-NAD+ (NAD+ analog 6 in Fig. 1B) with an alkyne moiety to generate 8-Bu(3-yne)T-NAD+ (Fig. 2A). 8-Bu(3-yne)T-NAD+ is an NAD+ analog with a single bifunctional R group at position 8, which facilitates asPARP-selective ADP-ribosylation. The R group also allows incorporation of alkynes into the posttranslationally modified substrates, for subsequent use in azide-alkyne cycloaddition (“click” chemistry) reactions to label or purify the PARP targets (Fig. 2B). asPARP-1 [L877→A877 (L877A)] with 8-Bu(3-yne)T-NAD+ acted similarly to the previously screened analogs, nearing WT enzyme kinetics when compared to PARP-1 and NAD+ (fig. S4). Critically, this “clickable” NAD+ analog also supports activity with asPARP-2 and asPARP-3 mutants (L443A and L394A, respectively, homologous to L877 of PARP-1) (Fig. 2C and fig. S5, A and B). The ability to transfer this analog-sensitive activity to the other PARPs by mutation of the conserved gatekeeper residue (Fig. 2C and fig. S5, C to G) suggests broad utility of this approach across the PARP family, for both mono- and poly(ADP-ribosyl) transferases.

Fig. 2 Activity of asPARPs 1, 2, and 3 with a “clickable” NAD+ analog.

(A) Chemical structure of the bifunctional 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. THPTA, tris(3-hydroxypropyltriazolylmethyl)amine. (C) Automodification reactions with WT 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). TAMRA, tetramethylrhodamine.

Identification of site-specific nuclear substrates of PARPs 1, 2, and 3

We used the asPARP approach to identify site-specific ADP-ribosylation of glutamate and aspartate residues for nuclear substrates of PARPs 1, 2, and 3 (10). We incubated purified recombinant asPARPs 1, 2, or 3 with HeLa cell nuclear extract in the presence of 8-Bu(3-yne)T-NAD+, which resulted in PARP-specific labeling of extract proteins (Fig. 3A). We then “clicked” the 8-Bu(3-yne)T-ADP-ribose–labeled proteins to azide-agarose, which led to their covalent attachment to the agarose resin, allowing extensive washing with denaturants, strong detergents, and organic solvents. We performed trypsin-based peptide identification of the ADP-ribosylated proteins by liquid chromatography–tandem mass spectrometry (LC-MS/MS) (protein ID), washed extensively again, and eluted the ADP-ribosylated peptides using hydroxylamine to identify the sites of ADP-ribosylation by LC-MS/MS (site ID) (10) (Fig. 3B). This approach revealed distinct and overlapping sites of PARP-1–, PARP-2–, and PARP-3–mediated ADP-ribosylation (Fig. 3C and fig. S6A). Ontological analyses of the target proteins revealed enrichment of terms related to transcription and DNA repair—consistent with the known biology of PARPs 1, 2, and 3—as well as additional terms, suggesting previously unknown functions (Fig. 3D and fig. S6B).

Fig. 3 Using asPARP 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 after 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) Postlabeling 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 2D relationship between previously identified ADP-ribosylation sites (10) with those identified herein. D/E, glutamate or aspartate residues.

Motif analyses at the sites of PARP-1–, PARP-2–, and PARP-3–mediated ADP-ribosylation indicate some similarities in sequence preference among the three PARPs (e.g., glutamate residue proximal to the site of modification) but point out differences as well (fig. S6C). The sites of PARP-1–, PARP-2–, and PARP-3–mediated ADP-ribosylation that we identified herein partially overlapped and were more numerous than sites of ADP-ribosylation identified using other approaches (fig. S7), with excellent agreement for the specific sites of ADP-ribosylation in common targets when compared to a previous cell-based bulk ADP-ribosylation assay (10) (Fig. 3E). We also observed considerable overlap with an asPARP-1 data set that we generated from intact mouse embryonic fibroblast (MEF) nuclei (fig. S8). Collectively, these results show that our asPARP approach robustly and faithfully identifies sites of ADP-ribosylation mediated by a specific PARP family member.

Negative elongation factor (NELF) is ADP-ribosylated in a positive transcription elongation factor (P-TEFb)–dependent manner

Previous reports implicating the Drosophila melanogaster homolog of PARP-1 as a key modulator of RNA polymerase II (Pol II) pause release at heat shock loci (11, 12), together with the identification of NELF-A and -E as ADP-ribosylated proteins (Fig. 4A and fig. S6B), led us to explore whether PARP-1 activity and ADP-ribosylation of the NELF complex might play a role in the control of transcription elongation. The negative elongation factor complex (NELF-A, -B, -C or -D, and -E) functions to restrict transcriptional elongation and stimulate promoter-proximal pausing by Pol II (13). Immunoaffinity purification of NELF from mammalian cells expressing FLAG epitope–tagged NELF-E demonstrated that PARP-1 interacts with the NELF complex (Fig. 4B) and that NELF-E and NELF-A are ADP-ribosylated in mammalian cells (Fig. 4C). Mutation of the four NELF-E glutamate residues identified in our proteomic screen (E122, E151, E172, and E374) (Fig. 4A) to glutamines, a structurally similar residue refractory to ADP-ribosylation, resulted in a substantial reduction in NELF-E modification by PARP-1 (Fig. 4D). Finally, using an electrophoretic mobility shift assay with a model NELF-E–interacting RNA [i.e., HIV trans-activation response element (TAR)], we found that ADP-ribosylation of NELF-E ablates its ability to bind RNA, a function necessary for the establishment paused Pol II (14) (Fig. 4E and fig. S9).

Fig. 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-nucleotide oligomer 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 the immunopurified NELF complex. The asterisk indicates automodified PARP-1. (D) Western blot for ADP-ribose of in vitro modification reactions containing glutathione S-transferase (GST), GST-tagged WT 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 the ADP-ribosylation sites identified herein and the nearest incidence of known phosphorylation modifications on PARP target proteins. aa, amino acids. (G) Western blot analysis of immunoprecipitated FLAG-tagged NELF-E from 293T cells treated with vehicle, the PARPi PJ34, or the P-TEFb/CDK9 inhibitor flavopiridol.

We found that phosphorylation sites (Fig. 4F) and, to a lesser extent, sites of other posttranslational modifications (fig. S10A) are frequently located at or near ADP-ribosylation sites across the proteome. This suggests a broad role for ADP-ribosylation as a modulator at hubs of regulatory activity, as well as a more specific regulatory role for ADP-ribosylation (and PARPs) in cooperation with phosphorylation (and kinases) across the proteome. In fact, using a PARP inhibitor (PARPi) (i.e., PJ34) and a cyclin-dependent kinase 9 (CDK9) inhibitor (i.e., flavopiridol), we observed that ADP-ribosylation of NELF-E in mammalian cells is dependent on phosphorylation by CDK9/P-TEFb (Fig. 4G), a kinase that phosphorylates Pol II, DSIF, and NELF-E (15). Furthermore, both inhibitors reduced the extent of serine-2 phosphorylation (Ser2P) in the C-terminal domain heptapeptide repeat of the Pol II RPB1 subunit in cells (fig. S10B). Because elevated Ser2P is associated with actively elongating Pol II (15), these results indicate a reduction in elongating Pol II upon inhibition of PARP-1 and CDK9. Interestingly, a 7-nucleotide oligomer amino acid sequence (RSRSRDR) enriched in targets of PARP-1 (fig. S10C) is located within the previously identified phosphorylation target site for P-TEFb in NELF-E, near a cluster of PARP-1–mediated ADP-ribosylation sites (Fig. 4A). Together, these results highlight the functional links between PARP-1–mediated ADP-ribosylation and transcription-related phosphorylation.

Identifying sites of PARP-1–mediated ADP-ribosylation across the genome

Although recent genomic approaches have facilitated the detection of sites of ADP-ribosylation genome-wide in the context of DNA damage (16), they have not allowed unambiguous assignment of genomic ADP-ribosylation events to a specific PARP family member in unstimulated cells. To unambiguously define sites of PARP-1–mediated ADP-ribosylation across the genome, we developed an assay, which we call “Click-ChIP-seq” (click chemistry–based chromatin isolation and precipitation with deep sequencing), using the asPARP-1 approach in nuclei. We expressed green fluorescent protein (GFP) (as a control), wtPARP-1, or asPARP-1 in Parp1−/− MEFs (fig. S11A). ADP-ribosylation after addition of 8-Bu(3-yne)T-NAD+ was clearly evident in the nuclei of Parp1−/− MEFs expressing asPARP-1, but not wtPARP-1 (fig. S11B). We then collected 8-Bu(3-yne)T-NAD+–treated nuclei, cross-linked them with formaldehyde, “clicked” the 8-Bu(3-yne)T-ADP-ribose to biotin, and sheared the chromatin by sonication, finally subjecting the material to an assay analogous to chromatin immunoprecipitation (Fig. 5A and fig. S11C). A quantitative polymerase chain reaction–based assay of the enriched genomic DNA revealed asPARP-1–specific ADP-ribosylation at gene promoters in nuclei isolated from MEFs (fig. S11D).

Fig. 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 cross-linking with formaldehyde, and then processed for chromatin immunoprecipitation. The enriched DNA is subjected to deep sequencing. (B) Genome browser view of a multigene 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. TSS, transcription start site.

Click-ChIP-seq revealed robust enrichment of PARP-1–mediated ADP-ribosylation at the promoters of transcriptionally active genes, which were defined by an enrichment of histone H3 lysine-4 trimethylation (H3K4me3, a mark of active promoters, from ChIP-seq) and actively transcribing Pol II [from global run-on sequencing (GRO-seq)] (Fig. 5B). This assay also revealed genomic loci with significant peaks of PARP-1 lacking enrichment of PARP-1–mediated ADP-ribosylation located in regions of repressed chromatin (fig. S12), which are difficult to discern in the absence of the specificity provided by the asPARP approach. Genome-wide correlation analyses between PARP-1–mediated ADP-ribosylation and a variety of other histone modifications and chromatin or transcription-related factors revealed strong clustering, as well as positive correlations with PARP-1 (correlation coefficient r = 0.606) and NELF-B (r = 0.754) (Fig. 5C). Heat-map representations of the genomic data highlight the relationships at gene promoters among PARP-1–mediated ADP-ribosylation; Pol II accumulation; and H3K4me3, NELF-B, and PARP-1 enrichment (Fig. 5D and fig. S13A). PARP-1–mediated ADP-ribosylation and CDK9 occupancy at promoters strongly correlated with low levels of Pol II pausing (fig. S13B). These results suggest that PARP-1–mediated ADP-ribosylation may act in a manner similar to CDK9/P-TEFb–mediated phosphorylation to promote the release of paused Pol II into productive elongation.

Role of PARP-1–mediated ADP-ribosylation of NELF in the release of paused Pol II

To determine the role of PARP-1 in the release of paused Pol II, we performed GRO-seq in MCF-7 breast cancer cells to monitor the effects of short hairpin RNA (shRNA)–mediated PARP-1 knockdown or treatment with the PARPi PJ34 on Pol II pausing. GRO-seq is a genomic assay that reveals the location of transcriptionally engaged RNA polymerases globally (17). We observed an accumulation of reads in the peaks of paused Pol II upon PARP-1 knockdown or treatment with PARPi [compared with an untreated luciferase knockdown control (Luc)] at gene promoters (fig. S14). This effect was evident genome-wide (Fig. 6A), with a clear increase in global Pol II pausing indices upon PARP-1 depletion or inhibition (Fig. 6B). At active promoters with a significant accumulation of GRO-seq reads in the paused Pol II peak upon PARP-1 knockdown, we observed decreased GRO-seq reads in the gene bodies (fig. S15, see panel B for the gene body effects), which suggests that PARP-1 activity is necessary to achieve an efficient release of Pol II into productive elongation.

Fig. 6 Genome-wide functional links between PARP-1–catalyzed ADP-ribosylation, NELF binding, and Pol II pausing.

(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 luciferase (control) or PARP-1 shRNAs (top) or treatment with the PARPi PJ34 (bottom). (B) Pol II pausing indices at the promoters of all transcribed RefSeq genes from MCF-7 cells subjected to shRNA-mediated knockdown with either luciferase (control) or PARP-1 shRNAs or treatment with PJ34. (C) Box plots of promoter proximal Pol II “pausing efficacy,” as 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).

We showed that a large fraction of PARP-1–regulated genes (determined by PARP-1 knockdown) are also regulated by P-TEFb [determined by treatment with 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB)] and NELF (determined by NELF knockdown), with respect to expression (by RNA-seq; fig. S16, A and B) and Pol II pausing (by Pol II ChIP-seq; fig. S16C). In addition, we found that the viral NELF inhibitor HDAg-S (18) reverses the inhibitory effects of both PARP-1 knockdown and DRB treatment on Pol II pausing, as determined from a ChIP-seq–based “Pol II pausing efficacy” assay (i.e., promoter proximal Pol II enrichment divided by NELF-E enrichment) (Fig. 6C and figs. S17 and S18). Finally, we showed that a NELF-E ADP-ribosylation site mutant (Mut) produces a NELF complex that is resistant to the inhibitory effects of PARP-1 and is a more potent inducer of Pol II pausing than the NELF complex containing WT NELF-E, in spite of lower cellular expression levels (Fig. 6C and figs. S18 and S19). Collectively, our data point to a functional link between CDK9-mediated phosphorylation, PARP-1–mediated ADP-ribosylation, and NELF-mediated Pol II pausing (fig. S20). Our results indicate that PARP-1–dependent ADP-ribosylation of NELF-E reinforces P-TEFb–mediated Pol II pause release and productive elongation for a subset of NELF-regulated genes, especially those with elevated NELF and Pol II loading, as well as H3K4me3 enrichment (fig. S21). Finally, these mechanisms are functional even at promoters where PARP-1 serves noncatalytic functions, such as the expulsion of the linker histone H1 from nucleosomes (19) (fig. S22).

Conclusions and perspectives

PARP proteins have gained considerable attention as therapeutic targets for the treatment of cancers and other diseases (20), although the broader biology of the PARP family remains largely unexplored. Understanding the biology of PARP proteins requires comprehension of the protein substrates that they modify. In this regard, we have developed an asPARP approach that preserves the natural mono- and poly(ADP-ribosyl) transferase activities of PARP enzymes, which can be coupled with protein mass spectrometry to identify the targets of specific PARP family members. We have also repurposed this asPARP technology for use in genomic assays to identify the genome-wide distribution of ADP-ribosylation events catalyzed by a specific PARP protein. Our studies focusing on NELF illustrate how an integrated approach based on asPARP technology can further the exploration of the biological functions for ADP-ribosylation. Our asPARP approach, which uses a conserved residue in the PARP catalytic domain, should be broadly applicable across the PARP family. This technique will facilitate the rapid, robust, and systematic identification of the molecular targets and mechanisms of action of the entire PARP family, with the potential to transform our understanding of PARP protein functions in physiology and disease.

Supplementary Materials

Materials and Methods

Figs. S1 to S22

Tables S1 and S2

References (2149)

References and Notes

  1. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
  2. Acknowledgments: 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.
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