USP1 deubiquitinase maintains phosphorylated CHK1 by limiting its DDB1-dependent degradation

Genome Instability and Carcinogenesis UPR3081 CNRS, IGC, IMM, 31 chemin Joseph Aiguier, 13402 Marseille, France.
Human Molecular Genetics (Impact Factor: 6.39). 03/2011; 20(11):2171-81. DOI: 10.1093/hmg/ddr103
Source: PubMed


The maintenance of genetic stability depends on the fine-tuned initiation and termination of pathways involved in cell cycle
checkpoints and DNA repair. Here, we describe a new pathway that regulates checkpoint kinase 1 (CHK1) activity, a key element
controlling both checkpoints and DNA repair. We show that the ubiquitin-specific peptidase 1 (USP1) deubiquitinase participates
in the maintenance of both total and phosphorylated levels of CHK1 in response to genotoxic stress. We establish that USP1
depletion stimulates the damage-specific DNA-binding protein 1-dependent degradation of phosphorylated CHK1 in both a monoubiquitinylated
Fanconi anaemia, complementation group D2 (FANCD2)-dependent and -independent manner. Our data support the existence of a
circuit in which CHK1 activates checkpoints, DNA repair and proliferating cell nuclear antigen and FANCD2 monoubiquitinylation.
The latter two events, in turn, switch off activated CHK1 by negative feedback inhibition, which contributes to the downregulation
of the DNA damage response. This pathway, which is compromised in the cancer-prone disease Fanconi anaemia (FA), likely contributes
to the hypersensitivity of cells from FA patients to DNA damage and to the clinical phenotype of the syndrome; it may also
represent a pharmacological target to improve patient care and develop new cancer therapies.

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Available from: Filippo Rosselli
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    • "ed in chromatin remodeling and DNA repair and replication ( Jones et al . , 2002 ; Xirodimas , 2008 ) , in which the FANC pathway is also involved . Inhibition of neddylation increases both CHK1 stability and the activation of the G2 / M checkpoint ( Yang et al . , 2012 ) , which are two important cellular features associated with Fanconi anemia ( Guervilly et al . , 2011 ) . Additionally , neddylation negatively regulates the transcriptional activity of NF - kB and p53 ( Abida et al . , 2007 ; Gao et al . , 2006 ; Xirodimas et al . , 2004 ) , and the activity of these transcription factors is increased in FANC - pathway - deficient cells . These findings further support the hypothesis that there is a po"
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    ABSTRACT: The aim of this study was to identify novel substrates of the FANCcore complex, which inactivation leads to the genetic disorder Fanconi anemia (FA), which associates bone marrow failure, developmental abnormalities and predisposition to cancer. Eight FANC proteins participate in the nuclear FANCcore complex, an E3 ubiquitin-ligase that monoubiquitinates FANCD2 and FANCI in response to replicative stress. Here, we used mass spectrometry to compare proteins from FANCcore complex deficient FA-A and FA-C cells to their ectopically corrected counterparts challenged with hydroxyurea, an inducer of FANCD2 monoubiquitination. FANCD2 and FANCI appear as the only targets of the FANCcore complex. We identified other proteins post-translationally modified in a FANCA- or FANCC-dependent manner. The majority of these potential targets localizes to the cell membrane. Finally, we demonstrated that (a) the chemokine receptor CXCR5 is neddylated; (b) FANCA, but not FANCC, appears to modulate CXCR5 neddylation through an unknown mechanism; (c) CXCR5 neddylation is involved in targeting the receptor to the cell membrane; and (d) CXCR5 neddylation stimulates cell migration/motility. Our work has uncovered a pathway involving FANCA in neddylation and cell motility.
    Full-text · Article · Jul 2014 · Journal of Cell Science
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    • "FANC pathway downregulation or deficiency was correlated with decreased levels of p-RPA32, with decreased frequency of p-RPA32 foci and with enhanced γH2AX accumulation (Figures 3B, 3C and S3A). As previously reported [20], [21], CHK1 was hyperphosphorylated in FANC-deficient cells (Figure 3C). Thus, the deficit in RPA phosphorylation that occurs in these cells cannot merely be ascribed to a defect in ATR activity; it is more likely to represent the consequence of the presence of fewer stretches of ssDNA. "
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    ABSTRACT: To cope with ultraviolet C (UVC)-stalled replication forks and restart DNA synthesis, cells either undergo DNA translesion synthesis (TLS) by specialised DNA polymerases or tolerate the lesions using homologous recombination (HR)-based mechanisms. To gain insight into how cells manage UVC-induced stalled replication forks, we analysed the molecular crosstalk between the TLS DNA polymerases Polη and Rev1, the double-strand break repair (DSB)-associated protein MDC1 and the FANC pathway. We describe three novel functional interactions that occur in response to UVC-induced DNA lesions. First, Polη and Rev1, whose optimal expression and/or relocalisation depend on the FANC core complex, act upstream of FANCD2 and are required for the proper relocalisation of monoubiquitinylated FANCD2 (Ub-FANCD2) to subnuclear foci. Second, during S-phase, Ub-FANCD2 and MDC1 relocalise to UVC-damaged nuclear areas or foci simultaneously but independently of each other. Third, Ub-FANCD2 and MDC1 are independently required for optimal BRCA1 relocalisation. While RPA32 phosphorylation (p-RPA32) and RPA foci formation were reduced in parallel with increasing levels of H2AX phosphorylation and MDC1 foci in UVC-irradiated FANC pathway-depleted cells, MDC1 depletion was associated with increased UVC-induced Ub-FANCD2 and FANCD2 foci as well as p-RPA32 levels and p-RPA32 foci. On the basis of the previous observations, we propose that the FANC pathway participates in the rescue of UVC-stalled replication forks in association with TLS by maintaining the integrity of ssDNA regions and by preserving genome stability and preventing the formation of DSBs, the resolution of which would require the intervention of MDC1.
    Full-text · Article · Jan 2013 · PLoS ONE
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    • "In the lung, Tnc appears at the epithelial-mesenchymal interface during branching morphogenesis where it may promote airway branching [30], [31]. Usp1 depletion impacts the stability and phosphorylation of Chk1 which is a cell cycle regulated and DNA damage checkpoint protein [32]. Given their known biological function, the downregulated expression of these genes is consistent with our observation of impaired BASC proliferation induced by c-Myc depletion. "
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    ABSTRACT: Bronchoalveolar stem cells (BASCs) located in the bronchoalveolar duct junction are thought to regenerate both bronchiolar and alveolar epithelium during homeostatic turnover and in response to injury. The mechanisms directing self-renewal in BASCs are poorly understood. BASCs (Sca-1(+), CD34(+), CD31(-) and, CD45(-)) were isolated from adult mouse lung using FACS, and their capacity for self-renewal and differentiation were demonstrated by immunostaining. A transcription factor network of 53 genes required for pluripotency in embryonic stem cells was assessed in BASCs, Kras-initiated lung tumor tissue, and lung organogenesis by real-time PCR. c-Myc was knocked down in BASCs by infection with c-Myc shRNA lentivirus. Comprehensive miRNA and mRNA profiling for BASCs was performed, and significant miRNAs and mRNAs potentially regulated by c-Myc were identified. We explored a c-Myc regulatory network in BASCs using a number of statistical and computational approaches through two different strategies; 1) c-Myc/Max binding sites within individual gene promoters, and 2) miRNA-regulated target genes. c-Myc expression was upregulated in BASCs and downregulated over the time course of lung organogenesis in vivo. The depletion of c-Myc in BASCs resulted in decreased proliferation and cell death. Multiple mRNAs and miRNAs were dynamically regulated in c-Myc depleted BASCs. Among a total of 250 dynamically regulated genes in c-Myc depleted BASCs, 57 genes were identified as potential targets of miRNAs through miRBase and TargetScan-based computational mapping. A further 88 genes were identified as potential downstream targets through their c-Myc binding motif. c-Myc plays a critical role in maintaining the self-renewal capacity of lung bronchoalveolar stem cells through a combination of miRNA and transcription factor regulatory networks.
    Full-text · Article · Aug 2011 · PLoS ONE
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