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Series GSE74141 Query DataSets for GSE74141
Status Public on Jun 09, 2016
Title NAD+ Analog-sensitive PARPs Reveal a Role for PARP-1 in Transcription Elongation
Organisms Homo sapiens; Mus musculus
Experiment type Genome binding/occupancy profiling by high throughput sequencing
Summary 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.
Overall design Using ChIP-seq (knockdown of Luciferase [control] with or without CDK inhibitor [DRB] treatment, knockdown of PARP-1, knockdown of NELF-E, and re-expression of GFP [control], wild-type NELF-E, mutant NELF-E, or viral NELF inhibitor HDAg-S) in MCF-7 human breast cancer cells and using ChIP-seq (PARP-1 and click-ChIP-seq) in mouse embryonic fibroblasts (MEFs)
Contributor(s) Gibson B, Kraus WL
Citation(s) 27256882
Submission date Oct 19, 2015
Last update date May 15, 2019
Contact name W. Lee Kraus
Organization name UT Southwestern Medical Center
Street address 5323 Harry Hines Blvd.
City Dallas
State/province TX
ZIP/Postal code 75390-8511
Country USA
Platforms (2)
GPL13112 Illumina HiSeq 2000 (Mus musculus)
GPL18573 Illumina NextSeq 500 (Homo sapiens)
Samples (66)
GSM1911176 ChIP-seq_Input_MEF_1
GSM1911177 ChIP-seq_Input_MEF_2
GSM1911178 PARP-1_ChIP-seq_MEF_1
BioProject PRJNA299154
SRA SRP064969

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Supplementary file Size Download File type/resource
GSE74141_RAW.tar 18.6 Gb (http)(custom) TAR (of BED)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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