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Repression of RAD51 gene expression by E2F4/p130 complexes in hypoxia

R S Bindra and P M Glazer

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Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Figure 1.

Specific repression of the RAD51 gene in hypoxia is mediated by an E2F site in the proximal promoter in a region homologous to the BRCA1 promoter. (a) Transcriptome response of MCF7 cells to hypoxia after a 24 h exposure to 0.5% O2. H/N mRNA expression ratios of DNA repair genes are listed (Array) and quantitative real-time PCR validation for selected genes is also shown for reference (qPCR), with respective pathways listed vertically on left: NER, BER and HR. H/N ratios are averaged from duplicate microarray experiments. Common gene names for the listed gene symbols: Excision Repair Cross-Complementing group 1 and 5 (ERCC1 and ERCC5, respectively), Cockayne Syndrome B (CSB), Apurinic/apyrimidinic Endonuclease/redox factor 1, 2 (APEX1 and APEX2, respectively), DNA Ligase III (LIGIII), RAD51 homolog B (RAD51B), RAD54 homolog B (RAD54B), RAD52 homolog (RAD52), RAD50 homolog (RAD50), RAD51 homolog (RAD51), Breast Cancer gene 1 (BRCA1). (b) Northern blot analyses were performed on total RNA extracted from cells grown after a 48-h exposure to normoxia (N) or hypoxia (H; 0.01% O2) in A549 cells. VEGF expression is shown for comparison to verify that physiologically relevant levels of hypoxia were present in the treated cells, and the expression of 28S is presented to confirm equal sample loading. (c) ClustalW multi-species alignment of the RAD51 proximal promoter region using human (Homo sap.), chimpanzee (Pan trogl.), dog (Canis fam.), mouse (Mus musc.) and rat (Rattus norv.). The E2F site consensus sequence is shown in the upper left panel (Wells et al., 2002). Shaded areas in the alignment indicate maximal cross-species conservation, and the overlapping E2FA and E2FB sites are indicated above the alignment (located on the positive (+) and negative (-) strands, respectively). Sites of mutagenesis in the promoter luciferase studies described in (f) are shown below the alignment. (d) Effect of mutagenesis of sequences within and adjacent to the E2F sites on repression of RAD51 promoter luciferase activity by hypoxia (0.01% O2, 48 h) in RKO cells. The specific site mutations are shown in (c). Results are expressed as the fold change in normalized luciferase activity in hypoxia compared to normoxia (H/N) for each construct. Error bars are based on standard errors calculated from six pairs of H/N replicates. Asterisks indicate statistically significant differences (P<0.05) in H/N-fold changes (NS; not statistically significant). The activity of a luciferase vector driven by a hypoxia-inducible promoter (5X-HRE) is shown as a control to confirm physiologically relevant levels of hypoxia. (e) Sequence comparison of the BRCA1 and RAD51 proximal promoters. Transcription start sites for each gene are indicated (+1), and the identified region of sequence homology is shaded (which contains the previously described E2FB site in BRCA1; Bindra et al., 2005). (f) Effect of E2FB site mutation (similar to the E2FB mutation in the RAD51 promoter as shown for mutant 3 in (c)) on the repression of BRCA1 WT promoter activity by hypoxia in RKO cells. Results are expressed as in (d) for each construct, and error bars are based on standard errors calculated from six pairs of N/H replicates.

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Figure 2.

In vivo analysis of RAD51 promoter occupancy by E2Fs and pocket proteins in normoxic and hypoxic cells. (a) ChIP assays were performed in MCF7 cells following a 48 h exposure to normoxia (N) or hypoxia (H) with antibodies to E2F1, E2F4 or p130 to determine RAD51 proximal promoter occupancy by these factors. A representative agarose gel containing RAD51 promoter amplification products obtained by PCR is shown. (b) Quantification of RAD51 promoter occupancy by these factors in normoxia and hypoxia as assessed by qChIP analysis, expressed as the fold change relative to that observed in normoxia (H/N). Error bars are based on standard errors calculated from a total of six H/N pairs of independent ChIP assay replicates, and P-values were calculated based on the difference in promoter occupancy in normoxia and hypoxia for each transcription factor. (c) Localization of E2Fs to the proximal RAD51 promoter region in vivo. Schematic of approximate primer locations (primer sets 1–7 kb) in the RAD51 gene locus used in ChIP analyses to localize E2F binding within the RAD51 promoter (left panel). The approximate location for the proximal promoter region of RAD51 is shown for reference. Putative E2F sites with >80% similarity to the consensus E2F sequence are shown (E2F) and were identified using the on-line promoter analysis promoter program, JASPAR (Sandelin et al., 2004). The approximate location of the E2F site in the proximal promoter region shown to mediate repression in hypoxia (as shown in Figure 1c and d) is indicated in bold. Nucleotide positions are shown relative to the first (untranslated) exon of the RAD51 gene, and the ATG start codon is also indicated. Quantification of occupancy by E2F1 and E2F4 in normoxic MCF7 cells as assessed by qChIP analysis at regions 1–7 in the RAD51 gene locus is shown in the bar graphs (middle and right panels, respectively). Relative promoter occupancies are shown and error bars are based on standard errors calculated from a total of three independent ChIP assay replicates. (d) qChIP analysis of E2F4 and p130 binding to a selection of known E2F-target genes in normoxic and hypoxic MCF7 cells. Promoter regions containing the putative E2F sites (shaded; negative or positive strand location shown as in [1e]) for each gene are listed in the left panel. Relative promoter occupancies in normoxia and hypoxia for each gene promoter are shown (as described in the Materials and methods section). Error bars and P-values are shown and were calculated as in (b).

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Figure 3.

Analysis of pocket protein expression and association with E2F4 in hypoxic and normoxic cells. (a) Western blot analyses were performed to determine the expression levels and phosphorylation status of the pocket proteins, p130 and p107, after exposure to normoxia (N) or hypoxia (H; 0.01% O2) from total cell (TCL), cytoplasmic (Cyto) and nuclear (Nuc) extracts of A549 cells. The expression of MSH6 in A549 cells is not regulated by hypoxia (Mihaylova et al., 2003; Bindra et al., 2004) and is presented to confirm equal sample loading, and tubulin expression is also shown to confirm successful isolation of nuclear fractions. (b) Western blot analyses were performed to assess the expression levels and phosphorylation status of the pocket proteins, p130 and p107, as well as the expression of E2F4, after exposure to normoxia or hypoxia as in (a) from total cell extracts of RKO cells. (c) Analysis of associations between the pocket proteins, p130, p107, and Rb, with E2F4 by co-immunoprecipitation using an antibody specific to E2F4 in total cell extracts from normoxic and hypoxia RKO cells, followed by immunoblot with antibodies to either p130, p107, Rb or E2F4, as indicated.

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Figure 4.

Downregulation of RAD51 and BRCA1 expression in normal diploid fibroblast cells and p130 dephosphorylation in response to hypoxia. (a) qPCR analysis of endogenous RAD51 and BRCA1 expression in normoxic and hypoxic BJ fibroblasts (normal human diploid cells), normalized to 18S rRNA expression and expressed as fold changes relative to normoxia (H/N) for each transcript. The expression of DEC1, a hypoxia-inducible gene, is shown as a control to confirm physiologic levels of hypoxia. Error bars are based on standard deviations calculated from duplicate assays. (b) Western blot analyses were performed to assess the expression levels and phosphorylation status of p130 as well as the expression of E2F4, after exposure to normoxia or hypoxia from total cell extracts of BJ cells. HIF-1alpha and Glut1 expression are shown as controls to confirm physiologic levels of hypoxia.

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Figure 5.

Elucidation of the role of E2F4/p130 binding in the represssion of RAD51 and BRCA1 gene expression by hypoxia. (a) Effect of HPV E7 expression on RAD51 repression by hypoxia. qPCR analysis of RAD51 and DEC1 expression in RKO-E7 (clones 6 and 14) and RKO-Neo cells following exposure to normoxia or hypoxia (48 h, 0.01% O2), normalized to 18S rRNA expression. Error bars are based on standard errors calculated from triplicate experiments. (b) ChIP assays were performed in RKO-Neo and RKO-E7 in parallel to the experiments shown in (a) to assess changes in p130 and E2F4 occupancy at the RAD51 proximal promoter in normoxia and hypoxia. Representative agarose gels containing RAD51 promoter amplification products obtained by PCR are shown for p130 and E2F4 (left and right panels, respectively). (c) qChIP analysis of RAD51 promoter occupancy by p130 and E2F4 in normoxia and hypoxia in RKO-Neo and RKO-E7 cells. Relative promoter occupancies and corresponding error bars are shown and were calculated as in (3c), based on duplicate ChIP assays. (d) The analyses in (c) were extended to the BRCA1 promoter in RKO-Neo and RKO-E7 cells.

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Figure 6.

Mechanism of coordinated HR gene repression by E2F4/p130 in hypoxia. Model of the molecular mechanism by which hypoxia induces the co-repression of RAD51 and BRCA1 expression via hypophosphorylation and nuclear accumulation of p130, the induction of E2F4/p130 complex formation and recruitment of these repressive complexes to specific elements (GCGGGAAT) in the RAD51 and BRCA1 gene promoters. This leads to transcriptional repression and decreased expression of these factors and diminished cellular capacity for recombinational repair, resulting in genetic instability (Bindra et al., 2004, 2005) and possibly altered therapy response (Farmer et al., 2005).

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