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PARP1 proximity proteomics reveals interaction partners at stressed replication forks

ABSTRACT: PARP1 mediates poly-ADP-ribosylation of proteins on chromatin in response to different types of DNA lesions. PARP inhibitors are used for the treatment of BRCA1/2-deficient breast, ovarian, and prostate cancer. Loss of DNA replication fork protection is proposed as one mechanism that contributes to the vulnerability of BRCA1/2-deficient cells to PARP inhibitors. However, the mechanisms that regulate PARP1 activity at stressed replication forks remain poorly understood. Here, we performed proximity proteomics of PARP1 and isolation of proteins on stressed replication forks to map putative PARP1 regulators. We identified TPX2 as a direct PARP1-binding protein that regulates the auto-ADP-ribosylation activity of PARP1. TPX2 interacts with DNA damage response proteins and promotes homology-directed repair of DNA double-strand breaks. Moreover, TPX2 mRNA levels are increased in BRCA1/2-mutated breast and prostate cancers, and high TPX2 expression levels correlate with the sensitivity of cancer cells to PARP-trapping inhibitors. We propose that TPX2 confers a mitosis-independent function in the cellular response to replication stress by interacting with PARP1.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Petra Beli

LAB HEAD: Petra Beli

PROVIDER: PXD037154 | Pride | 2022-10-11

REPOSITORIES: Pride

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			Action	DRS
	20141230_QEP1_PBE_JY_U2OS_SILAC_EdU_30min_HU_2h_ingel__01.raw	Raw
	20141230_QEP1_PBE_JY_U2OS_SILAC_EdU_30min_HU_2h_ingel__02.raw	Raw
	20141230_QEP1_PBE_JY_U2OS_SILAC_EdU_30min_HU_2h_ingel__03.raw	Raw
	20141230_QEP1_PBE_JY_U2OS_SILAC_EdU_30min_HU_2h_ingel__04.raw	Raw
	20141230_QEP1_PBE_JY_U2OS_SILAC_EdU_30min_HU_2h_ingel__05.raw	Raw

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Project description:Epstein-Barr virus (EBV) establishes life-long latency in human B-cells by maintaining its chromatinized episomes within the nucleus. These circularized mini-chromosomes do not integrate into the host genome. Therefore, it is essential for EBV to organize its chromatin in a manner suitable for genomic stability, DNA replication, and efficient gene expression. Poly [ADP-ribose] polymerase 1 (PARP1) activity is significantly higher in B-cells infected with EBV than those without, and considerably higher in the transcriptionally active type III latency compared to the immunoevasive type I. In addition to its role in DNA damage response, PARP1 has been implicated in transcriptional regulation and structural maintenance of both the human and EBV genome at specific regions. To better understand PARP1's role in the regulation of the EBV episome, we have functionally characterized the effect of PARP enzymatic inhibition on total episomal structure through in situ Hi-C mapping, generating the first complete 3D structure of the EBV genome. We have also mapped intragenomic contact changes after PARP inhibition to global binding of the chromatin looping factors CTCF and cohesin across the EBV genome. Additionally, PARP inhibition was shown to alter gene expression at the regions where chromatin looping was most effected. Finally, we have identified a novel function of PARP, which regulates cohesin complex chromatin binding. In conclusion, PARP1 inhibition does not alter the location of cohesin binding but does increase its frequency of binding at these regions. Despite this, there are fewer overall unique intragenomic interactions after PARP inhibition, while some areas have new chromatin loops not seen in the untreated EBV episome, leading us to conclude that PARP does have an essential role in the regulation of global EBV chromatin structure. The altered expression profile after the structural rearrangement induced by PARP inhibition also supports the idea that PARP1 helps maintain EBV latency programs.

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PARP1 proximity proteomics reveals interaction partners at stressed replication forks

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