PARP1 regulates DNA damage-induced nucleolar-nucleoplasmic shuttling of WRN and XRCC1 in a toxicant and protein-specific manner

Abstract

The prime function of nucleoli is ribogenesis, however, several other, non-canonical functions have recently been identified, including a role in genotoxic stress response. Upon DNA damage, numerous proteins shuttle dynamically between the nucleolus and the nucleoplasm, yet the underlying molecular mechanisms are incompletely understood. Here, we demonstrate that PARP1 and PARylation contribute to genotoxic stress-induced nucleolar-nucleoplasmic shuttling of key genome maintenance factors in HeLa cells. Our work revealed that the RECQ helicase, WRN, translocates from nucleoli to the nucleoplasm upon treatment with the oxidizing agent H 2 O 2 , the alkylating agent 2-chloroethyl ethyl sulfide (CEES), and the topoisomerase inhibitor camptothecin (CPT). We show that after treatment with H 2 O 2 and CEES, but not CPT, WRN translocation was dependent on PARP1 protein, yet independent of its enzymatic activity. In contrast, nucleolar-nucleoplasmic translocation of the base excision repair protein, XRCC1, was dependent on both PARP1 protein and its enzymatic activity. Furthermore, gossypol, which inhibits PARP1 activity by disruption of PARP1-protein interactions, abolishes nucleolar-nucleoplasmic shuttling of WRN, XRCC1 and PARP1, indicating the involvement of further upstream factors. In conclusion, this study highlights a prominent role of PARP1 in the DNA damage-induced nucleolar-nucleoplasmic shuttling of genome maintenance factors in HeLa cells in a toxicant and protein-specific manner.

The Nucleolus and PARP1 in Cancer Biology

“…In addition, phosphorylation was proposed to modulate WRN's subnuclear localization [39,68]. Consistent with the idea that such DNA damage-induced protein dynamics are regulated in a stress-specific manner, we recently found that after treatment with H 2 O 2 and the alkylating agent 2-chloroethyl ethyl sulfide (CEES), but not the topoisomerase inhibitor camptothecin (CPT), nucleolar-nucleoplasmic translocation of WRN was dependent on the PARP1 protein, yet independent of its enzymatic activity [69].…”

“…We hypothesized that in case of WRN, PARP1 and an additional unknown factor mediate the release of WRN from nucleoli, while XRCC1 requires nucleoplasmic DNA damage-bound and PARylated PARP1 as a loading platform, which leads to XRCC1 retention in the nucleoplasm until its tasks in BER are completed. This notion is supported by findings revealing that in cells without PARP1 activity, XRCC1 relocates quickly to nucleoli upon DNA damage induction [69].…”

“…Since TARG1 was reported to bind to RNA, ribosomal proteins, as well as proteins associated with rRNA proteins and ribosomal assembly factors, it is conceivable that in nucleoli, TARG1 plays a role in ribosome assembly or quality control, which is stalled when TARG1 is recruited to sites of DNA damage in a PAR-dependent manner [153]. With regards to a role of PARP1 and PARylation in nucleolar DNA damage response, we recently showed that upon H 2 O 2 treatment of HeLa cells, WRN and XRCC1 translocate from nucleoli to the nucleoplasm, however interestingly enough, probably by different mechanisms [69]. Thus, while the release of WRN from nucleoli was purely dependent on the presence of PARP1 protein without any obvious involvement of its catalytic activity, we found that relocalization of XRCC1 upon H 2 O 2 -induced DNA damage was dependent on both PARP1 protein and its enzymatic activity (Figure 1).…”

PARP1 catalytic variants reveal branching and chain length-specific functions of poly(ADP-ribose) in cellular physiology and stress response

“…In a previous study, we revealed that XRCC1 translocates from nucleoli to the nucleoplasm upon H 2 O 2 treatment. In the presence of PARP1, XRCC1 is retained in the nucleoplasm, while in PARP1 KO cells, XRCC1 swiftly relocates back into nucleoli ( 65 ). In accordance with this, Figure 8 shows that in PARP1\WT-reconstituted cells, XRCC1 was released from nucleoli in response to H 2 O 2 -treatment.…”

“…Microscopic images were acquired using either a Zeiss Axiovert 200M widefield microscope (for PAR detection) or a Zeiss LSM700 confocal microscope (for XRCC1 and fibrillarin detection). Image data was analyzed using automated KNIME workflows as described previously ( 34 , 65 ). Only cells with a GFP fluorescence signal >1.5-fold of the mean background fluorescence were considered as GFP-positive and thus analyzed for PAR, XRCC1 and fibrillarin.…”