PAG1- a Novel Hypoxia Regulated Gene

Abstract

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 mutation of the HRE. By applying chromatin conformation capture (3C) technology we could provide evidence that this enhancer element forms a HIF independent chromatin loop with the promoter region in a cell line specific manner.
To further investigate the impact of HIF-1 and HIF-2 on the transcriptional regulation of PAG1, we created HIF-α knockdowns in different cell lines and tested the response of our reporters in these cells. In addition, we overexpressed hydroxylation-insensitive versions of the HIF-α isoforms in the same cell lines. Our analysis revealed that PAG1 is HIF-2α specific target gene in the low invasive breast cancer cell line MCF-7 and the pVHL deficient clear cell renal cell carcinoma cell line 768-O. For the other two investigated cell lines, the cervical cancer cell line HeLa and the high invasive breast cancer line MDA-MB-231 PAG1 seems to be regulated by both HIF-α isoforms. With an overexpression screen of transcription factors, binding to the PAG1 enhancer region, we wanted to identify HIF interaction partners contributing to the transcriptional regulation of PAG1. This further characterisation of the PAG1 enhancer element did not bring any conclusive results.
PAG1 is an ubiquitously expressed transmembrane adaptor protein, participating in the negative regulation of Src family kinases. It has been shown, that PAG1 itself plays a role in cell signalling processes, independent of Src family kinases. The second aim of this thesis hence was the characterization of the connection between PAG1-regulated Src signalling and cellular adaptation to hypoxia. Our studies using PAG1 knockdown cells revealed a PAG1-independent regulation of Src signalling in hypoxia.
In conclusion, this work provided a functional characterisation of the distal enhancer element regulating the response of the novel hypoxia target gene PAG1.