The role of poly (ADP-ribose) polymerase 1 in DNA damage-induced downregulation of the ubiquitin ligase CHFR

Abstract

CHFR (Checkpoint with Forkhead-associated and RING finger domains) is involved in a prophase checkpoint upon microtubule stress and is suppressed by DNA methylation in many cancer cell lines. Apart from its checkpoint functions and E₃ ubiquitin ligase activity, CHFR contains PAR-binding zinc-fingers which can interact with poly(ADP-ribose) (PAR) modifications. Poly(ADP-ribose) Polymerase1 (PARP1) is the most important member of PARP family. It is activated by DNA strand breaks, which then synthesizes a long and branched PAR network at DNA damage sites by auto-PARylation. This coordinates the recruitment of various downstream proteins involved in DNA damage response through affinity to PARylation or protein-protein interactions. Thus CHFR is also speculated to play a role in DNA damage responses through its PBZ motif. Previously, our group observed that CHFR was downregulated upon DNA damage induced by ionizing radiation or alkylating agent treatment, at the same time when PARP1 was activated. This downregulation could be inhibited by proteasome inhibitors or PARP inhibitors, indicating the involvement of a PARylation- and ubiquitin-dependent pathway. In my studies, I found that CHFR was downregulated not at the transcriptional level but dependent on auto-ubiquitination. Elevation of PARylation by PARG depletion could enhance the downregulation of CHFR. Collated with the fact that PARP inhibition stabilized CHFR but not its PBZ mutant, I speculated that CHFR downregulation was PARylation dependent. Then I demonstrated PARP1 depletion by siRNA or overexpression of PARP1 increased or decreased CHFR level, respectively, suggesting a role of PARP1 in this regulation. Co-immunoprecipitation results indicated an increased binding affinity between CHFR and PARP1, which could be abolished by PARP inhibitors or PBZ mutation. Finally, I utilized an in vivo ubiquitination assay to assess the ubiquitination status of CHFR and found that CHFR is more poly-ubiquitinated after alkylating agent treatment than after PARP inhibitor treatment. Taken together, I proposed a model that after DNA damage, CHFR is downregulated by auto-ubiquitination-mediated degradation through the binding to PARylation synthesized by PARP1.

Date of Award	2014
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology