ARNT is a potential direct HIF-1 target gene in human Hep3B hepatocellular carcinoma cells

Markus Mandl, Reinhard Depping

The transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT) participates in the hypoxia-inducible factor (HIF) pathway which senses a decline in cellular oxygen tension. In hypoxia, HIF-1α and ARNT form the transcriptional active complex HIF-1 followed by the expression of target genes. ARNT is considered as constitutively expressed and unaffected by hypoxia. However, certain tumour cell lines derived from different entities are capable to elevate ARNT expression under hypoxic conditions which implies a survival benefit. It was demonstrated that high ARNT protein levels mediate radioresistance in tumour cells. Furthermore, a HIF-1α-driven feed-forward loop leading to augmented HIF signalling was discovered in Hep3B cells. Herein HIF-1α elevates the mRNA and protein expression of its binding partner ARNT in hypoxia. However, the detailed mechanism remained unclear. The objective of this study was to test whether HIF-1α might directly regulate ARNT expression by recruitment to the ARNT promoter. Chromatin immunoprecipitation (ChIP), CRISPR/Cas9 genome editing, Western blotting, quantitative RT-PCR and reporter gene assays were applied. The unpaired t test was used for statistical analysis. ChIP assays revealed the binding of both HIF-1α and ARNT to the ARNT promoter in hypoxia. The relevance of this particular region for hypoxic ARNT induction was confirmed by CRISPR/Cas9 genome editing. ARNT normoxic basal expression and hypoxic inducibility was reduced in genome-edited Hep3B cells. This phenotype was accompanied with impaired HIF signalling and was rescued by ARNT overexpression. The results indicate ARNT to be a putative HIF-1 target gene and a limiting factor in this model.