PDF file - 1436KB, S1. FANCD2 is ubiquitinated and transcriptionally down-regulated upon treatment with DFX in MCF7 cells. S2. FANCD2 is ubiquitinated and transcriptionally down-regulated upon treatment with DFX in A549 cells. S3. FANCD2 and FANCI ubiquitination does not occur at less severe levels of hypoxia. S4. siRNA depletion of ATR, but not ATM, inhibits FANCD2 and FANCI ubiquitination induced by hypoxia and DFX. S5. FANCD2 and FANCI ubiquitination upon DFX treatment is independent of HIF-1. S6. FANCD2 protein down-regulation by DFX is not due to altered protein stability and transcriptional down-regulation may be related to p130 dephosphorylation and HIF-2. S7. FANCD2 protein level decreases in hypoxia and is restored by HPV16-E7 overexpression. S8. FANCI transcriptional down-regulation upon prolonged exposure to DFX or hypoxia is mitigated by HPV16-E7 overexpression. S9. Increased chromosomal aberrations induced by hypoxia in the absence of FANCD2 are not detected by cytogenetic analysis.
ARTICLE ABSTRACT
Hypoxia induces genomic instability through replication stress and dysregulation of vital DNA repair pathways. The Fanconi anemia (FA) proteins, FANCD2 and FANCI, are key members of a DNA repair pathway that responds to replicative stress, suggesting that they undergo regulation by hypoxic conditions. Here acute hypoxic stress activates the FA pathway via ubiquitination of FANCD2 and FANCI in an ATR-dependent manner. In addition, the presence of an intact FA pathway is required for preventing hypoxia-induced DNA damage measurable by the comet assay, limiting the accumulation of γH2AX (a marker of DNA damage or stalled replication), and protecting cells from hypoxia-induced apoptosis. Furthermore, prolonged hypoxia induces transcriptional repression of FANCD2 in a manner analogous to the hypoxic downregulation of BRCA1 and RAD51. Thus, hypoxia-induced FA pathway activation plays a key role in maintaining genome integrity and cell survival, while FA protein downregulation with prolonged hypoxia contributes to genomic instability.Implications: This work highlights the critical role of the FA pathway in response to hypoxic stress and identifies the pathway as a therapeutic target under hypoxic conditions. Mol Cancer Res; 12(7); 1016–28. ©2014 AACR.
