AbstractsBiology & Animal Science

An imbalance between oxygen supply and consumption leads to low oxygen conditions, also called hypoxia. This event is an essential feature in certain diseases like cancer, arteriosclerosis, stroke and inflammation. The cellular adaptation to hypoxia is mastered by the hypoxia-inducible transcription factors (HIFs). HIFs consist of a heterodimer between the constitutively expressed form HIF-β, also called aryl hydrocarbon receptor nuclear translocator (ARNT), and the tightly oxygen-controlled subunit HIF-α. In humans, three HIF-α isoforms have been described: HIF-1α, HIF-2α and HIF-3α. Under well oxygenated conditions, the prolyl-4-hydroxylases (PHD) 1-3 hydroxylate the HIF-α subunit on two prolyl residues within their oxygen- dependent degradation domain. This leads to recognition of HIF-α by the von Hippel-Lindau protein (pVHL) and subsequent proteasomal degradation. In addition, the factor inhibiting HIF (FIH) hydroxylates the HIF-α subunits at an asparagine residue, preventing the interaction with transcriptional cofactors. Under low oxygen conditions HIF-α is stabilised and translocates into the nucleus where it heterodimerizes with ARNT to form the HIF complex. This complex activates more than 200 direct target genes via binding to cis-regulatory regions comprising the consensus 5’-RCGTG-3’ sequence, also referred to as hypoxia response element (HRE), which leads to cellular adaptation to hypoxia. The phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG1), also called the Csk-binding protein (Cbp), is an ubiquitously expressed transmembrane adaptor protein. In a microarray study comparing normoxic and hypoxic HeLa cells, we found this gene to be up-regulated. The aim of this thesis was to describe the molecular mechanism behind the hypoxic regulation. We confirmed the microarray data in a panel of additional cancer cell lines from different origin. Furthermore, Pag1 was induced in tissues derived from mice exposed to hypoxia. Genome-wide HIF ChIP-sequencing analysis revealed an HRE 82 kb upstream of the transcriptional start site of the PAG1 gene. Analysis of the region surrounding this site revealed chromatin modifications like H3K3me1, H3K27ac, overlapping with a DNase I hypersensitivity cluster and binding of a variety of transcription factors. All the aforementioned modifications indicate a potential regulatory region in surrounding the HRE. We confirmed the HIF binding to the HRE by ChIP-qPCR analysis and we detected the binding of the transcriptional coactivator p300. Using reporter gene assays, we independently confirmed that this region consists of a 2 kb enhancer element, responding to HIF binding in hypoxia and specifically regulating PAG1 but not the neighbouring FABP5 gene. Mutation of the HRE sequence by site-directed mutagenesis led to the loss of hypoxic induction. We mutated the HRE in cellula using the genome editing tool transcription activator-like effector endonucleases (TALEN) in HeLa and MCF-7 cells. No induction of the PAG1 gene could be observed any longer after…