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Immunoprecipitated PARP1 interactome

ABSTRACT: Poly-(ADP-ribose) polymerase inhibitors (PARPi) elicit anti-tumour activity in homologous recombination defective cancers by promoting cytotoxic, chromatin-bound, “trapped” PARP1 DNA lesions. How cells process trapped PARP1 remains unclear. By exploiting wild-type or trapping-resistant PARP1 transgenes combined with rapid immunoprecipitation mass-spectrometry of endogenous proteins (RIME) and Apex2-proximity labelling, we generated proteomic profiles of trapped and non-trapped PARP1 complexes. This combined approach identified an interaction between PARP1 and the ubiquitin system including its central component - the ubiquitin-regulated p97 ATPase (aka VCP).

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: James Wright

LAB HEAD: Jyoti Choudhary

PROVIDER: PXD024337 | Pride | 2021-10-05

REPOSITORIES: Pride

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Project description:BACKGROUND:Chromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored. RESULTS:We compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50 ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1 ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1 ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents. CONCLUSIONS:This study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.

			Action	DRS
	Fusion_DK06-1.msf	Msf
	Fusion_DK06-1.raw	Raw
	Fusion_DK06-2.msf	Msf
	Fusion_DK06-2.raw	Raw
	Fusion_DK06-3.msf	Msf

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Immunoprecipitated PARP1 interactome

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