Project description:The PARP family of proteins comprises 17 members, about two thirds of which are active mono- or poly(ADP-ribosyl)transferase enzymes that transfer the ADP-ribose moiety of NAD+ onto target proteins. In many cases, ADP-ribosylation, which plays critical roles in human diseases (e.g., cancer, heart disease, and neuropathies) is associated with abrogation of the molecular functions of the target. Discerning ADP-ribosylation events mediated by a specific PARP is challenging, since all PARPs use the same substrate (i.e., NAD+) and the available inhibitors lack the specificity needed to make such conclusions. In order to identify the direct and specific targets of individual PARP family members, we have developed a chemical and genetic approach known as analog sensitivity, in which alteration of a single conserved amino acid in the active site of the PARP protein creates a pocket that allows use of an unnatural NAD+ analog containing a steric moiety. We have functionalized the steric moiety with alkyne for use in click chemistry reactions. This approach, which is transferable to other PARP family members, creates substrate specificity where none previously existed, allowing PARP-specific post-translational modification followed by target visualization, or isolation of ADP-ribosylated targets using click chemistry techniques. We have used this technology in conjunction with mass spectrometry to identify hundreds of targets both unique to, as well as shared among, the nuclear PARP proteins, PARP-1, PARP-2, and PARP-3. We have also determined the genome-wide distribution of PARP-1-specific ADP-ribosylation by coupling this analog-sensitive PARP approach with chromatin cross-linking in method that we call “Click-ChIP-seq”. We observed that PARP-1-specific ADP-ribosylation is enriched at transcriptionally active promoters in proximity to sites of PARP-1 enrichment. In addition, we observed that NELF, an important regulator of RNA Polymerase II (Pol II) pausing, is not only a target of ADP-ribosylation by PARP-1 but also spatially correlated with chromatin-associated ADP-ribose and PARP-1 in Click-ChIP-seq and ChIP-seq assays, respectively. Given these observations, we hypothesized that ADP-ribosylation might modulate NELF function and result altered Pol II pausing. We have explored this possibility using global run-on coupled with deep sequencing (GRO-seq) in MCF-7 cells in which PARP-1 was depleted by RNAi-mediated knockdown. PARP-1 depletion caused an increase in Pol II pausing genome-wide. Taken together, these results suggest the intriguing possibility that ADP-ribosylation of NELF by PARP-1 may be an important and heretofore unknown step in the release of Pol II into productive elongation.

Project description:Many of the long-term effects of cocaine on the brain's reward circuitry have been shown to be mediated by alterations in gene expression. Several chromatin modifications, including histone acetylation and methylation, have been implicated in this regulation, but the effect of other histone modifications remains poorly understood. Poly(ADP-ribose) polymerase-1 (PARP-1), a ubiquitous and abundant nuclear protein, catalyzes the synthesis of a negatively charged polymer called poly(ADP-ribose) or PAR on histones and other substrate proteins and forms transcriptional regulatory complexes with several other chromatin proteins. Here, we identify an essential role for PARP-1 in cocaine-induced molecular, neural, and behavioral plasticity. Repeated cocaine administration, including self-administration, increased global levels of PARP-1 and its mark PAR in mouse nucleus accumbens (NAc), a key brain reward region. Using PARP-1 inhibitors and viral-mediated gene transfer, we established that PARP-1induction in NAc mediates enhanced behavioral responses to cocaine, including increased self-administration of the drug. Using chromatin immunoprecipitation sequencing, we demonstrated a global, genome-wide enrichment of PARP-1 in NAc of cocaine-exposed mice and identified several PARP-1 target genes that could contribute to the lasting effects of cocaine. Specifically, we identified sidekick-1-important for synaptic connections during development-as a critical PARP-1 target gene involved in cocaine's behavioral effects as well as in its ability to induce dendritic spines on NAc neurons. These findings establish the involvement of PARP-1 and PARylation in the long-term actions of cocaine. c57bl/6 mice were given IP injections of chronic cocaine 20mg/kg once per day for 7 days and sacrificed 30 minutes after the final dose of cocaine. Control animals were given saline for 7 days and sacrificed 30 minutes after the final dose of saline. Nucleus accumbens (NAc) tissue was collected and then PARP-1 ChIP-seq was performed. Three sequencing replicates were performed on each group.

Dataset Information

Examination of functional interplay between NMNAT-1 (a nuclear NAD+ synthase) and PARP-1 (a nuclear poly(ADP-ribose) polymerase)

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