Abstract:
This thesis concerns some mechanistic properties of the basic helix-loop-helix/PAS
(bHLH-PAS) factors aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1
alpha (HIF-1α). The bHLH-PAS family of proteins is a family of factors that controls a
variety of developmental and physiological events. A common feature for this family of
proteins is that they act mainly as intracellular transcription factors. They bind to DNA
as a heterodimeric complex, usually together with a bHLH-PAS protein belonging to
the aryl hydrocarbon receptor nuclear translocator (ARNT) subfamily. The AhR bind
ligands that are environmental pollutants, as well as possibly physiological compounds
occurring in the diet. Known functions of the ligand-activated AhR include activation of
genes involved in xenobiotic metabolism and an ubiquitin ligase activity targeting
nuclear receptors (such as the estrogen receptor) and beta-catenin. HIF-1α mediates
signal transduction and gene regulation in cells exposed to deprived oxygen conditions
(hypoxia).
In paper I, we have shown that the Ah-receptor can be activated by stimulus other than
xenobiotics, e.g dioxin. AhR is recruited to target genes in both ligand treated and in
suspension culture, suggesting a common mechanism of activation between these two
routes of AhR activation. The gene expression profiles critically differ between
xenobiotic and suspension activated AhR signaling. The classical xenobiotic
metabolizing AhR targets such as Cyp 1a1, Cyp 1b1 and Nqo were regulated by both
ligand and suspension conditions. Sequence analysis coupled with ChIP assays and
reporter gene analysis identified a functional xenobiotic response element (XRE) within
the mouse TIPARP gene that features a concatamer of 4 XRE cores residing in the first
intron.
In paper II we have shown that ectopic expression of ARNT, in mammalian cells and
yeast cells, was sufficient to promote nuclear accumulation of the Ah-receptor in a
ligand-independent manner. We further observed that overexpression of ARNT
promotes derepression of Ah-receptor function in the absence of ligand, thereby
possibly representing an alternative mechanism of activation that is distinct from
activation by xenobiotic ligands and thus may be of physiological relevance. We also
describe that an excess of ARNT in relation to the Ah-receptor and HIF-1α promotes
derepression of the receptor and stabilization of HIF-1α in vivo and in vitro,
representing a possible alternative mechanism of activation of bHLH-PAS proteins.
