Abstract
BMI1 facilitates DNA damage response (DDR) induced by double strand DNA breaks; however, it remains unknown whether BMI1 functions in single strand DNA (ssDNA) lesions-initiated DDR. We report here that BMI1 reduces hydroxyurea-elicited ATR activation, thereby reducing the S-phase checkpoints. Hydroxyurea induces ssDNA lesions, which activate ATR through binding TOPBP1 as evidenced by phosphorylation of ATR at threonine 1989 (ATRpT1989). ATR subsequently phosphorylates H2AX at serine 139 (γH2AX) and CHK1 at serine 345 (CHK1pS345), leading to phosphorylation of CDK1 at tyrosine 15 (CDK1pY15) and S-phase arrest. BMI1 overexpression reduced γH2AX, CHK1pS345, CDK1pY15, S-phase arrest, and ATR activation in HU-treated MCF7 and DU145 cells, whereas BMI1 knockdown enhanced these events. BMI1 contains a ring finger, helix-turn, proline/serine domain and two nuclear localization signals (NLS). Individual deletion of these domains did not abolish BMI1-derived reductions of CHK1pS345 in MCF7 cells following HU exposure, suggesting that these structural features are not essential for BMI1 to attenuate ATR-mediated CHK1pS345. BMI1 interacts with both TOPBP1 and ATR. Furthermore, all of our BMI1 mutants associate with endogenous TOPBP1. It has previously been established that association of TOPBP1 and ATR is required for ATR activation. Thus, our results suggest that BMI1 decreases ATR activation through a mechanism that involves binding to TOPBP1 and/or ATR.
Highlights
BMI1 is a polycomb group (PcG) protein of the polycomb repressive complex 1 (PRC1) [1], and is required for formation of E3 ubiquitin ligase activity of PRC1 via binding to the catalytic subunit RING2 [2,3,4,5]
To investigate whether BMI1 is involved in single strand DNA (ssDNA)-stimulated DNA damage response (DDR), we have constructed MCF7 breast cancer and DU145 prostate cancer cell lines in which BMI1 was either stably overexpressed or knocked-down (Supplementary Figure 1)
A major consequence of checkpoint activation, is commonly examined at 24 hour following treatment [50, 51], we focused this study on the late phase of CHK1 activation and DDR induced by HU at doses ≤ 1 mM
Summary
BMI1 is a polycomb group (PcG) protein of the polycomb repressive complex 1 (PRC1) [1], and is required for formation of E3 ubiquitin ligase activity of PRC1 via binding to the catalytic subunit RING2 [2,3,4,5]. E4F1 inhibits cell proliferation, in part, through promoting p53 and CHK1 functions [12,13,14]. Suppression of these loci contributes to BMI1-derived maintenance of the self-renewal of hematopoietic and neural stem cells [8, 15, 16]. Inhibition of INK4A and ARF-mediated tumor suppression is critical for tumorigenesis [10, 11] and upregulation of BMI1 occurs in numerous cancer types including non-small cell lung cancer [17], colon cancer [18], breast cancer [19], and nasopharyngeal carcinoma [20]. Expression of BMI1 can synergize with c-Myc in transgenic mouse models for leukemogenesis [26, 27]
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