Jinshan He

Ubiquitin-specific Protease 7 Regulates Nucleotide Excision Repair through Deubiquitinating XPC Protein and Preventing XPC Protein from Undergoing Ultraviolet Light-induced and VCP/p97 Protein-regulated Proteolysis

Background: XPC protein is ubiquitinated, but the ubiquitination does not lead to significant proteolysis of XPC. Results: Ubiquitin-specific protease 7 is a deubiquitinating enzyme (DUB) for XPC. Conclusion: USP7 deubiquitination prevents XPC from undergoing UV-induced and VCP/p97-regulated XPC proteolysis. Significance: USP7 and VCP/p97 involvement in XPC regulation is crucial for understanding how nucleotide excision repair is executed and regulated in eukaryotic cells. Ubiquitin specific protease 7 (USP7) is a known deubiquitinating enzyme for tumor suppressor p53 and its downstream regulator, E3 ubiquitin ligase Mdm2. Here we report that USP7 regulates nucleotide excision repair (NER) via deubiquitinating xeroderma pigmentosum complementation group C (XPC) protein, a critical damage recognition factor that binds to helix-distorting DNA lesions and initiates NER. XPC is ubiquitinated during the early stage of NER of UV light-induced DNA lesions. We demonstrate that transiently compromising cellular USP7 by siRNA and chemical inhibition leads to accumulation of ubiquitinated forms of XPC, whereas complete USP7 deficiency leads to rapid ubiquitin-mediated XPC degradation upon UV irradiation. We show that USP7 physically interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces the ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further show that valosin-containing protein (VCP)/p97 is involved in UV light-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and colocalizes with XPC. Chemical inhibition of the activity of VCP/p97 ATPase causes an increase in ubiquitinated XPC on DNA-damaged chromatin. Moreover, USP7 deficiency severely impairs the repair of cyclobutane pyrimidine dimers and, to a lesser extent, affects the repair of 6-4 photoproducts. Taken together, our findings uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis.

USP7 modulates UV-induced PCNA monoubiquitination by regulating DNA polymerase eta stability

DNA polymerase eta (Polη) has unique and pivotal functions in several DNA damage-tolerance pathways. Steady-state level of this short-lived protein is tightly controlled by multiple mechanisms including proteolysis. Here, we have identified the deubiquitinating enzyme (DUB), ubiquitin-specific protease 7 (USP7), as a novel regulator of Polη stability. USP7 regulates Polη stability through both indirect and direct mechanisms. Knockout of USP7 increased the steady-state level of Polη and slowed down the turnover of both Polη and p53 proteins through destabilizing their E3 ligase murine double minute 2 (Mdm2). Also, USP7 physically binds Polη in vitro and in vivo. Overexpression of wild-type USP7 but not its catalytically-defective mutants deubiquitinates Polη and increases its cellular steady-state level. Thus, USP7 directly serves as a specific DUB for Polη. Furthermore, ectopic expression of USP7 promoted the UV-induced proliferating cell nuclear antigen (PCNA) monoubiquitination in Polη-proficient but not in Polη-deficient XPV (Xeroderma pigmentosum variant) cells, suggesting that USP7 facilitates UV-induced PCNA monoubiquitination by stabilizing Polη. Taken together, our findings reveal a modulatory role of USP7 in PCNA ubiquitination-mediated stress-tolerance pathways by fine-tuning Polη turnover.

USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing RNF168*

During DNA damage response (DDR), histone ubiquitination by RNF168 is a critical event, which orchestrates the recruitment of downstream DDR factors, e.g. BRCA1 and 53BP1. Here, we report USP7 deubiquitinase regulates the stability of RNF168. We showed that USP7 disruption impairs H2A and ultraviolet radiation (UVR)-induced γH2AX monoubiquitination, and decreases the levels of pBmi1, Bmi1, RNF168 and BRCA1. The effect of USP7 disruption was recapitulated by siRNA-mediated USP7 depletion. The USP7 disruption also compromises the formation of UVR-induced foci (UVRIF) and ionizing radiation-induced foci (IRIF) of monoubiquitinated H2A (uH2A) and polyubiquitinated H2AX/A, and subsequently affects UVRIF and IRIF of BRCA1 as well as the IRIF of 53BP1. USP7 was shown to physically bind RNF168 in vitro and in vivo. Overexpression of wild-type USP7, but not its interaction-defective mutant, prevents UVR-induced RNF168 degradation. The USP7 mutant is unable to cleave Ub-conjugates of RNF168 in vivo. Importantly, ectopic expression of RNF168, or both RNF8 and RNF168 together in USP7-disrupted cells, significantly rescue the formation of UVRIF and IRIF of polyubiquitinated H2A and BRCA1. Taken together, these findings reveal an important role of USP7 in regulating ubiquitin-dependent signaling via stabilization of RNF168.

Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair.

Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3′ side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4Cdt2. Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4Cdt2 for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER.

Valosin-containing Protein (VCP)/p97 Segregase Mediates Proteolytic Processing of Cockayne Syndrome Group B (CSB) in Damaged Chromatin.

Cockayne syndrome group A and B (CSB) proteins act in transcription-coupled repair, a subpathway of nucleotide excision repair. Here we demonstrate that valosin-containing protein (VCP)/p97 segregase functions in ultraviolet radiation (UVR)-induced ubiquitin-mediated CSB degradation. We show that VCP/p97 inhibition and siRNA-mediated ablation of VCP/p97 and its cofactors UFD1 and UBXD7 impair CSB degradation. VCP/p97 inhibition also results in the accumulation of CSB in chromatin. Moreover, VCP/p97 interacts with both native and ubiquitin-conjugated forms of CSB. The localized cellular UVR exposures lead to VCP/p97 accumulation at DNA damage spots, forming distinct UVR-induced foci. However, manifestation of VCP/p97 foci is independent of CSB and UBXD7. Furthermore, VCP/p97 and UBXD7 associate with the Cockayne syndrome group A-DDB1-Cul4A complex, an E3 ligase responsible for CSB ubiquitination. Compromising proteasome and VCP/p97 function allows accumulation of both native and ubiquitinated CSB and results in an increase of UBXD7, proteasomal RPN2, and Sug1 in the chromatin compartment. Surprisingly, both biochemical inhibition and genetic defect of VCP/p97 enhance the recovery of RNA synthesis following UVR, whereas both VCP/p97 and proteasome inhibitions decrease cell viability. Our findings reveal a new role of VCP/p97 segregase in the timely processing of ubiquitinated CSB from damaged chromatin.

DDB2 modulates TGF-β signal transduction in human ovarian cancer cells by downregulating NEDD4L

The expression of DNA damage-binding protein 2 (DDB2) has been linked to the prognosis of ovarian cancer and its underlying transcription regulatory function was proposed to contribute to the favorable treatment outcome. By applying gene microarray analysis, we discovered neural precursor cell expressed, developmentally downregulated 4-Like (NEDD4L) as a previously unidentified downstream gene regulated by DDB2. Mechanistic investigation demonstrated that DDB2 can bind to the promoter region of NEDD4L and recruit enhancer of zeste homolog 2 histone methyltransferase to repress NEDD4L transcription by enhancing histone H3 lysine 27 trimethylation (H3K27me3) at the NEDD4L promoter. Given that NEDD4L plays an important role in constraining transforming growth factor β signaling by targeting activated Smad2/Smad3 for degradation, we investigated the role of DDB2 in the regulation of TGF-β signaling in ovarian cancer cells. Our data indicate that DDB2 enhances TGF-β signal transduction and increases the responsiveness of ovarian cancer cells to TGF-β-induced growth inhibition. The study has uncovered an unappreciated regulatory mode that hinges on the interaction between DDB2 and NEDD4L in human ovarian cancer cells. The novel mechanism proposes the DDB2-mediated fine-tuning of TGF-β signaling and its downstream effects that impinge upon tumor growth in ovarian cancers.

Damaged DNA-binding protein down-regulates epigenetic mark H3K56Ac through histone deacetylase 1 and 2.

Acetylated histone H3 lysine 56 (H3K56Ac) is one of the reversible histone post-translational modifications (PTMs) responsive to DNA damage. We previously described a biphasic decrease and increase of epigenetic mark H3K56Ac in response to ultraviolet radiation (UVR)-induced DNA damage. Here, we report a new function of UV damaged DNA-binding protein (DDB) in deacetylation of H3K56Ac through specific histone deacetylases (HDACs). We show that simultaneous depletion of HDAC1/2 compromises the deacetylation of H3K56Ac, while depletion of HDAC1 or HDAC2 alone has no effect on H3K56Ac. The H3K56Ac deacetylation does not require functional nucleotide excision repair (NER) factors XPA and XPC, but depends on the function of upstream factors DDB1 and DDB2. UVR enhances the association of DDB2 with HDAC1 and, enforced DDB2 expression leads to translocation of HDAC1 to UVR-damaged chromatin. HDAC1 and HDAC2 are recruited to UVR-induced DNA damage spots, which are visualized by anti-XPC immunofluorescence. Dual HDAC1/2 depletion decreases XPC ubiquitination, but does not affect the recruitment of DDB2 to DNA damage. By contrast, the local accumulation of γH2AX at UVR-induced DNA damage spots was compromised upon HDAC1 as well as dual HDAC1/2 depletions. Additionally, UVR-induced ATM activation decreased in H12899 cells expressing H3K56Ac-mimicing H3K56Q. These results revealed a novel role of DDB in H3K56Ac deacetylation during early step of NER and the existence of active functional cross-talk between DDB-mediated damage recognition and H3K56Ac deacetylation.

UV-induced proteolysis of RNA polymerase II is mediated by VCP/p97 segregase and timely orchestration by Cockayne syndrome B protein

RNA polymerase II (RNAPII) acts as a damage sensor for transcription-coupled nucleotide excision repair (TC-NER) and undergoes proteolytic clearance from damaged chromatin by the ubiquitin-proteasome system (UPS). Here, we report that Valosin-containing protein (VCP)/p97, a druggable oncotarget, is essential for RNAPIIs proteolytic clearance in mammalian cells. We show that inhibition of VCP/p97, or siRNA-mediated ablation of VCP/p97 and its cofactors UFD1 and UBXD7 severely impairs ultraviolet radiation (UVR)-induced RNAPII degradation. VCP/p97 interacts with RNAPII, and the interaction is enhanced by Cockayne syndrome B protein (CSB). However, the VCP/p97-mediated RNAPII proteolysis occurs independent of CSB. Surprisingly, CSB enhances UVR-induced RNAPII ubiquitination but delays its turnover. Additionally, VCP/p97-mediated RNAPII turnover occurs with and without Von Hippel-Lindau tumor suppressor protein (pVHL), a known substrate receptor of Elongin E3 ubiquitin ligase for RNAPII. Moreover, pVHL re-expression improves cell viability following UVR. Whereas, VCP/p97 inhibition decreases cell viability and enhances a low-dose UVR killing in presence of pVHL. These findings reveal a function of VCP/p97 segregase in UVR-induced RNAPII degradation in mammalian cells, and suggest a role of CSB in coordinating VCP/p97-mediated extraction of ubiquitinated RNAPII and CSB itself from chromatin.

Human CRL4 DDB2 ubiquitin ligase preferentially regulates post-repair chromatin restoration of H3K56Ac through recruitment of histone chaperon CAF-1

Acetylated histone H3 lysine 56 (H3K56Ac) diminishes in response to DNA damage but is restored following DNA repair. Here, we report that CRL4DDB2 ubiquitin ligase preferentially regulates post-repair chromatin restoration of H3K56Ac through recruitment of histone chaperon CAF-1. We show that H3K56Ac accumulates at DNA damage sites. The restoration of H3K56Ac but not H3K27Ac, H3K18Ac and H3K14Ac depends on CAF-1 function, whereas all these acetylations are mediated by CBP/p300. The CRL4DDB2 components, DDB1, DDB2 and CUL4A, are also required for maintaining the H3K56Ac and H3K9Ac level in chromatin, and for restoring H3K56Ac following induction of DNA photolesions and strand breaks. Depletion of CUL4A decreases the recruitment of CAF-1 p60 and p150 to ultraviolet radiation- and phleomycin-induced DNA damage. Neddylation inhibition renders CRL4DDB2 inactive, decreases H3K56Ac level, diminishes CAF-1 recruitment and prevents H3K56Ac restoration. Mutation in the PIP box of DDB2 compromises its capability to elevate the H3K56Ac level but does not affect XPC ubiquitination. These results demonstrated a function of CRL4DDB2 in differential regulation of histone acetylation in response to DNA damage, suggesting a novel role of CRL4DDB2 in repair-driven chromatin assembly.

UV radiation-induced SUMOylation of DDB2 regulates nucleotide excision repair.

Subunit 2 of DNA damage-binding protein complex (DDB2) is an early sensor of nucleotide excision repair (NER) pathway for eliminating DNA damage induced by UV radiation (UVR) and cisplatin treatments of mammalian cells. DDB2 is modified by ubiquitin and poly(ADP-ribose) (PAR) in response to UVR, and these modifications play a crucial role in regulating NER. Here, using immuno-analysis of irradiated cell extracts, we have identified multiple post-irradiation modifications of DDB2 protein. Interestingly, although the DNA lesions induced by both UVR and cisplatin are corrected by NER, only the UV irradiation, but not the cisplatin treatment, induces any discernable DDB2 modifications. We, for the first time, show that the appearance of UVR-induced DDB2 modifications depend on the binding of DDB2 to the damaged chromatin and the participation of functionally active 26S proteasome. The in vitro and in vivo analysis revealed that SUMO-1 conjugations comprise a significant portion of these UVR-induced DDB2 modifications. Mapping of SUMO-modified sites demonstrated that UVR-induced SUMOylation occurs on Lys-309 residue of DDB2 protein. Mutation of Lys-309 to Arg-309 diminished the DDB2 SUMOylation observable both in vitro and in vivo. Moreover, K309R mutated DDB2 lost its function of recruiting XPC to the DNA damage sites, as well as the ability to repair cyclobutane pyrimidine dimers following cellular UV irradiation. Taken together, our results indicate that DDB2 is modified by SUMOylation upon UV irradiation, and this post-translational modification plays an important role in the initial recognition and processing of UVR-induced DNA damage occurring within the context of chromatin.