Identification of novel pathways of regulation of AHR and HIF-­1 function

Abstract

Mammalian bHLH-Pas (basic HLH (helix-loop-helix)-PER-ARNT-SIM) proteins belong to the bHLH superfamily of transcription factors. Members of the family have a broad spectrum of functions that among others sense and regulate the cellular response to physiological signals such as low oxygen levels (hypoxia), or environmental signals such as toxins. The bHLH-Pas proteins consist of a signal-induced subunit, for example, aryl hydrocarbon receptor (AhR) or hypoxia inducible factor (HIF). To respond to environmental (AhR) or physiological (HIF) stimuli, the signal-induced subunits dimerize with their ubiquitously expressed, unregulated dimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT), bind to DNA, and induce the activation of a cascade of target genes. At normal oxygen levels (normoxia), proline hydroxylases hydroxylate HIF, and the subsequent interaction of HIF with the von Hippel-Lindau tumor suppressor gene (pVHL) leads to ubiquitination and degradation of HIF. Asparagine hydroxylation by the factor inhibiting HIF (FIH) leads under normoxia to suppressed HIF transactivation. According to the model, proline and asparagine hydroxylation reactions are inhibited under hypoxia.

In paper I, we established a mechanism that allows us to explain the molecular switch of the aryl hydrocarbon receptor (AhR) from its transcription factor to its E3 ubiquitin ligase function. We used the breast cancer cell line MCF7 to demonstrate that the availability of ARNT modulates the dual functions of AhR. Upon ARNT availability, the AhR functions as a ligand-induced transcription factor. If, however, other binding partners, such as the repressor of AhR (AhRR), occupy ARNT, the AhR functions as an E3 ubiquitin ligase. In paper II, we revealed significantly elevated FIH expression in skeletal muscle compared to other tissues. We also demonstrated that FIH loss leads to an induced oxidative metabolism, and an increased glycolytic capacity, resulting in elevated oxygen consumption. Loss of FIH further correlates with a decreased metabolic efficiency, an increased oxidative rate and an accelerated HIF-mediated response to hypoxia. In paper III (manuscript), we investigated the role FIH plays in epigenetic regulation, among others to explain the underlying mechanism behind the results presented in paper II. Muscle cells show the highest FIH levels on tissue level and we observed elevated histone 3 lysine 9 dimethyl (H3K9me2) levels in FIH null mice. Further work showed significantly increased methylation levels at the H3K9 dimethyl repression mark at promoter regions of various metabolic HIF target genes in FIH null mouse embryonic fibroblasts, and subsequently correlations of loss of FIH and Jmjd1a dysregulated protein levels. In the following, despite promising preliminary results to date no FIH-Jmjd1a protein-protein interaction could be demonstrated.