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Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping.

ABSTRACT: A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1?/2?/? DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) to localize and functionally characterize a 82 kb upstream HRE that solely drives oxygen-regulated expression of the newly identified HIF target gene PAG1. PAG1, a transmembrane adaptor protein involved in Src signalling, was hypoxically induced in various cell lines and mouse tissues. ChIP and reporter gene assays demonstrated that the -82 kb HRE regulates PAG1, but not an equally distant gene further upstream, by direct interaction with HIF. Ablation of the consensus HRE motif abolished the hypoxic induction of PAG1 but not general oxygen signalling. 3C assays revealed that the -82 kb HRE physically associates with the PAG1 promoter region, independent of HIF-DNA interaction. These results demonstrate a constitutive interaction between the -82 kb HRE and the PAG1 promoter, suggesting a physiologically important rapid response to hypoxia.

SUBMITTER: Schorg A

PROVIDER: S-EPMC4499134 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping.

Schörg Alexandra A Santambrogio Sara S Platt James L JL Schödel Johannes J Lindenmeyer Maja T MT Cohen Clemens D CD Schrödter Katrin K Mole David R DR Wenger Roland H RH Hoogewijs David D

Nucleic acids research 20150524 12

A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1α/2α/β DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) t ...[more]

PMID: 26007655

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Project description:In multiple myeloma (MM), hypoxia-inducible transcription factor-1 (HIF-1) is overexpressed in the MM cells of the hypoxic bone marrow (BM) microenvironment. Herein, we explored in MM cells the in vitro and in vivo effects of persistent HIF-1 inhibition by expression of a lentivirus shRNA pool on proliferation, survival and transcriptional and pro-angiogenic profiles. Among the significantly modulated genes (326 and 361 genes in hypoxic and normoxic condition, respectively), we found that HIF-1 inhibition in the human myeloma cell line JJN3 downregulates the pro-angiogenic molecules VEGF, IL8, IL10, CCL2, CCL5, and MMP9. Interestingly, several pro-osteoclastogenic cytokines were also inhibited, such as IL-7 and CCL3/MIP-1. The effect of HIF-1 inhibition was assessed in vivo in NOD/SCID mice both in subcutaneous and intratibial models, indicating in either case a dramatic reduction of weight and volume of the tumor burden as a consequence of HIF-1 knockdown. Moreover, a significant reduction of the number of vessels per field and VEGF immunostaining were observed. Finally, in the intra-tibial experiments, HIF-1 inhibition significantly blocks JJN3-induced bone destruction. Overall, our data indicate that HIF-1 suppression in MM cells significantly blocks MM-induced angiogenesis and reduces both tumor burden and bone destruction in vivo, strongly indicating HIF-1 as an emerging therapeutic target in MM. The transcriptional profiles on JJN3 transduced with shRNA anti-HIF-1 (JJN3-anti-HIF-1), as compared to those infected with the control vector pLKO.1 (JJN3-pLKO.1), have been analyzed either in hypoxic or normoxic conditions. To perform gene expression profiles, total RNA was purified using the RNeasy Total RNA Isolation Kit (Qiagen, Valencia, CA). Preparation of biotin-labeled cRNA, hybridization to GeneChip Human Genome U133 Plus 2.0 Arrays and scanning (GeneChip¨ Scanner 3000 7G, Affymetrix Inc.) were performed according to manufacturer's protocols.

Dataset Information

Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping.

Publications

Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping.

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