DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the errorprone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.DNA damage response | UbcH7 | 53BP1 | protein degradation | DSB repair P rompt response to double-strand breaks (DSBs) caused by, for example, ionization radiation (IR), requires sequential and coordinated assembly of DNA damage response (DDR) proteins at damage sites (1). Recent research findings reveal key roles of the tumor suppressor p53-binding protein 1 (53BP1) and BRCA1 in the decision making of DSB repair. 53BP1, together with Rif1, suppress BRCA1-dependent homologous recombination (HR), thereby promoting nonhomologous end-joining (NHEJ) in G1 phase (2-6). Conversely, BRCA1 antagonizes 53BP1/Rif1, favoring HR in S and G2 phases (7,8). In the absence of BRCA1 or with enhanced retention of 53BP1 at DSB sites, cells primarily use the error-prone NHEJ to repair DSBs throughout the cell cycle, which leads to gene rearrangement, cell death, and increased sensitivity to anticancer therapies (9-11). Consistently, BRCA1-null mice are early embryonic lethal (12, 13) and codepletion of TP53BP1 rescued the lethality phenotype of BRCA1-null mice (12)(13)(14).Low expression level of 53BP1 was found to be associated with poor clinical outcome in triple negative breast cancer patients with BRCA1 mutation (12, 15), as well as resistance to genotoxins and poly(ADP-ribose) polymerase inhibitors (12,16,17). This finding is probably because loss of 53BP1 restored HR and promoted cell survival (12-14). Reduced expression of 53BP1 was also observed in tumors from the brain (18), lymph node (19), and pancreas (20). These data indicate that loss of 53BP1 might be a common mechanism for advanced tumors to evade from radiotherapy or chemotherapy. However, molecular mechanisms controlling the protein level of 53BP1 remain less well understood.Here we show that UbcH7, an E2 enzyme involved in the ubiquitin (Ub) pathway, controls the protein stability of 53BP1, thereby determining the DSB repair choice. Loss of UbcH7 sta...
Cited2 is a transcriptional modulator involved in various biologic processes including fetal liver hematopoiesis. In the present study, the function of Cited2 in adult hematopoiesis was investigated in conditional knockout mice. Deletion of Cited2 using Mx1-Cre resulted in increased hematopoietic stem cell (HSC) apoptosis, loss of quiescence, and increased cycling, leading to a severely impaired reconstitution capacity as assessed by 5-fluorouracil treatment and long-term transplantation. Transcriptional profiling revealed that multiple HSC quiescence-and hypoxia-related genes such as Egr1, p57, and Hes1 were affected in Cited2-deficient HSCs. Because Cited2 is a negative regulator of HIF-1, which is essential for maintaining HSC quiescence, and because we demonstrated previously that decreased HIF-1␣ gene dosage partially rescues both cardiac and lens defects caused by Cited2 deficiency, we generated Cited2 and HIF-1␣ doubleknockout mice. Additional deletion of HIF-1␣ in Cited2-knockout BM partially rescued impaired HSC quiescence and reconstitution capacity. At the transcriptional level, deletion of HIF-1␣ restored expression of p57 and Hes1 but not Egr1 to normal levels. Our results suggest that Cited2 regulates HSC quiescence through both HIF-1-dependent and HIF-1-independent pathways. (Blood. 2012;119(12):2789-2798) IntroductionHematopoietic stem cells (HSCs) are thought to be localized in the hypoxic microenvironment of the BM and remain quiescent or differentiate into multiple blood cell lineages. Several factors have been found to regulate HSC quiescence in either a cell-intrinsic (eg, p21, p57, Bmi1, Egr1, GATA2, Gfi1, Pbx1, and others) or a cell-extrinsic (eg, Tie2/Angpt1, c-Mpl/Tpo, CXCR4/CXCL12, and others) manner.CBP/p300-interacting transactivator with glutamic acid (E) and aspartic acid (D)-rich tail 2 (Cited2), a member of the Cited family of transcriptional modulators, is a cytokine-inducible gene 1 that plays various roles during mouse development. [2][3][4][5][6] In particular, Cited2 plays an important role in fetal liver hematopoiesis, which is supported by severely impaired fetal liver HSC function and decreased expression of genes known to be essential for hematopoiesis in Cited2-deficient fetal liver HSC/progenitors. 7 Cited2 has also been implicated in tumor cell invasion and progression. 8,9 Cited2 is a negative regulator of hypoxia-inducible factor 1 (HIF-1). Bhattacharya et al first demonstrated in vitro that Cited2 competes with HIF-1␣ for binding to p300-CH1 and inhibits HIF-1-mediated transactivation. 10 Bakker et al also showed that FOXO3a inhibits HIF-1-induced apoptosis by stimulating the transcription of Cited2, which reduces the expression of the pro-apoptotic HIF-1 target genes NIX (also called "Bnip3l") and RTP801 (also called "Ddit4"). 11 In our previous studies, we showed that deletion of HIF-1␣ partially rescues defects in heart and lens caused by Cited2 deficiency. 4,12 In addition, at the structural level, Freedman et al revealed that Cited2 and HIF-1␣ share a conserved...
Background: Chk1 regulates both nuclear and cytoplasmic checkpoints. Results: The Chk1 polypeptide contains mechanisms mediating its cellular localization. Conclusion: The nuclear (but not cytoplasmic) pool of Chk1 is essential for cell viability. Significance: Dissecting mechanisms regulating the mobilization of Chk1 within different cellular compartments is critical for understanding checkpoint activation and its roles in cell biology.
Chk1, a serine/threonine protein kinase, is centrally involved in cell cycle checkpoints and cellular response to DNA damage. Phosphorylation of Chk1 at two Ser/Gln (SQ) sites, Ser-317 and Ser-345, by the upstream kinase ATR is critical for checkpoint activation. However, the precise molecular mechanisms controlling Chk1 phosphorylation and subsequent checkpoint activation are not well understood. Here, we report unique auto-regulatory mechanisms that control protein phosphorylation of human Chk1, as well as checkpoint activation and cell viability. Phosphorylation of Ser-317 is a prerequisite, but not sufficient, for maximal phosphorylation at Ser-345. The amino (N)- terminal kinase domain of Chk1 prevents Chk1 phosphorylation at the carboxyl (C) terminus by ATR in the absence of DNA damage. Loss of the inhibitory effect imposed by the N-terminus causes constitutive phosphorylation of Chk1 by ATR under normal growth conditions, which in turn triggers artificial checkpoints that suppress the S phase progression. Using mutagenesis, we identified two point mutations that render Chk1 constitutively active. Unexpectedly, we found that expression of the constitutively active mutant form of Chk1 inhibited cancer cell proliferation, representing a novel strategy in suppressing tumor growth. Together, these studies revealed unique regulatory mechanisms of Chk1 phosphorylation and its implications in cancer therapy.
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