RESUMO
In this study, we explored the existence of a transcriptional network co-regulated by E2F7 and HIF1α, as we show that expression of E2F7, like HIF1α, is induced in hypoxia, and because of the previously reported ability of E2F7 to interact with HIF1α. Our genome-wide analysis uncovers a transcriptional network that is directly controlled by HIF1α and E2F7, and demonstrates both stimulatory and repressive functions of the HIF1α -E2F7 complex. Among this network we reveal Neuropilin 1 (NRP1) as a HIF1α-E2F7 repressed gene. By performing in vitro and in vivo reporter assays we demonstrate that the HIF1α-E2F7 mediated NRP1 repression depends on a 41 base pairs 'E2F-binding site hub', providing a molecular mechanism for a previously unanticipated role for HIF1α in transcriptional repression. To explore the biological significance of this regulation we performed in situ hybridizations and observed enhanced nrp1a expression in spinal motorneurons (MN) of zebrafish embryos, upon morpholino-inhibition of e2f7/8 or hif1α Consistent with the chemo-repellent role of nrp1a, morpholino-inhibition of e2f7/8 or hif1α caused MN truncations, which was rescued in TALEN-induced nrp1a(hu10012) mutants, and phenocopied in e2f7/8 mutant zebrafish. Therefore, we conclude that repression of NRP1 by the HIF1α-E2F7 complex regulates MN axon guidance in vivo.
Assuntos
Orientação de Axônios/genética , Fator de Transcrição E2F7/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neurônios Motores/metabolismo , Neuropilina-1/genética , Peixe-Zebra/genética , Animais , Sítios de Ligação , Hipóxia Celular/genética , Linhagem Celular Tumoral , Fator de Transcrição E2F7/metabolismo , Estudo de Associação Genômica Ampla , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hibridização In Situ , Morfolinos/genética , Neuropilina-1/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Transcrição Gênica/genética , Peixe-Zebra/embriologiaRESUMO
The E2F family of transcription factors plays an important role in controlling cell-cycle progression. While this is their best-known function, we report here novel functions for the newest members of the E2F family, E2F7 and E2F8 (E2F7/8). We show that simultaneous deletion of E2F7/8 in zebrafish and mice leads to severe vascular defects during embryonic development. Using a panel of transgenic zebrafish with fluorescent-labelled blood vessels, we demonstrate that E2F7/8 are essential for proper formation of blood vessels. Despite their classification as transcriptional repressors, we provide evidence for a molecular mechanism through which E2F7/8 activate the transcription of the vascular endothelial growth factor A (VEGFA), a key factor in guiding angiogenesis. We show that E2F7/8 directly bind and stimulate the VEGFA promoter independent of canonical E2F binding elements. Instead, E2F7/8 form a transcriptional complex with the hypoxia inducible factor 1 (HIF1) to stimulate VEGFA promoter activity. These results uncover an unexpected link between E2F7/8 and the HIF1-VEGFA pathway providing a molecular mechanism by which E2F7/8 control angiogenesis.
Assuntos
Fatores de Transcrição E2F/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Fisiológica/genética , Ativação Transcricional , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Fatores de Transcrição E2F/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Deleção de Genes , Humanos , Camundongos , Regiões Promotoras Genéticas , Peixe-ZebraRESUMO
We investigated the involvement of Rac1 in Ha-ras-overexpression-induced apoptosis using a murine NIH/3T3-derived cell line (designated 7-4), which contains an inducible Ha-ras oncogene under the regulation of Escherichia coli lac operator-repressor system. Ha-ras overexpression was induced by isopropyl beta-D-thiogalactoside (IPTG). To reveal the role of endogenous Rac1, the dominant negative Rac1(Asn17) gene was transfected into the 7-4 cells. Using two cell lines 7-4 Racd2 and 7-4 Racd3 (7-4 derivates) stably expressing Rac1(Asn17), we demonstrate that suppression of Rac1 activity blocked Ha-ras-overexpression-induced apoptosis under a serum-depleted condition, indicating that Rac1 activity is required for a Ras-mediated apoptosis pathway. Cell-cycle analysis revealed that dominant-negative Rac1 partially shifted cell population from S-phase to G0/G1 phase in the cells overexpressing Ha-ras. In contrast to other reports, we showed activation of the transcription factor NFkappaB in the two cell lines expressing dominant-negative Rac1. All together, our results demonstrate that Ha-ras-overexpression- induced apoptosis can be blocked by dominant-negative Rac1, possibly through decreased S-phase accumulation and increased NFkappaB activity.