Precise spatial and temporal control of gene expression is essential for the development and survival of all living organisms.
Transcription factors control the “on-off” switches inherent in the regulatory regions of all genes, and are therefore the central overseers of gene expression networks. Many transcription factors are themselves regulated by specific intracellular or extracellular signals and thereby relay information concerning the environmental, metabolic or developmental status of the cell through to the genome. The research focus in our laboratory is the study of how environmental signalling pathways are transduced into genetic reprogramming events. The mechanisms and biological significance of one class of signal regulated transcription factors, the basic-Helix-Loop-Helix/PAS family, are being investigated.
The basic-Helix-Loop-Helix (bHLH) family of transcription factors have the uniform molecular design of a DNA binding basic region being adjunct to a Helix-Loop-Helix dimerisation domain, and exhibit a common mechanistic theme of heterodimerisation to produce active, DNA bound transcription factor complexes. A subclass of this family has recently become evident with the discovery of bHLH/PAS factors, which contain a PAS (Per/Arnt/Sim) homology region contiguous with the bHLH. PAS domains have been conserved through evolution and are found in proteins which sense or transmit signals relating to the extracellular environment or metabolic status of a cell (eg sensing oxygen, light, environmental pollutants, redox or nutrient status). The PAS domain comprises approximately 300 amino acids and harbours two degenerate repeats, designated PAS A and PAS B (Figure 1). While the exact mechanisms of PAS domains are not fully understood, it is clear that they can form dimerisation interfaces, limiting partner selection to other PAS containing proteins.
While the bHLH/PAS proteins are potent transcription factors, there is only rudimentary understanding of how they are controlled and the complement of genes which they regulate has only been marginally investigated. The majority of biological functions and molecular mechanisms of these proteins therefore remain to be elucidated. The current biological relevance of some mammalian bHLH/PAS proteins studied in this laboratory can be summarised as follows:
- Dioxin Receptor
The dioxin receptor is characterised as a ligand dependent inducer of a gene battery encoding enzymes involved in xenobiotic metabolism (eg cytochrome P-4501A1 and glutathione-S-transferase). It is notorious for mediating the severe toxicity associated with dioxin and PCB poisoning. Despite the dioxin receptor being the most studied bHLH/PAS protein, the signal transduction pathways linking dioxin receptor activation to the myriad of toxicological responses observed upon dioxin exposure (including of birth defects, impaired reproductive capacity and tumorigenesis) remain a mystery.
- Hypoxia Inducible Factors
HIF-1alpha and HIF-2alpha proteins are rapidly activated when cells are subjected to oxygen deprivation or hypoglycemia. The key to hypoxic activation seems to be a decreased ability of certain oxygen dependent enzymes to make posttranslational modifications on the proteins when oxygen becomes limiting. Gene knockout studies show both HIFs to be essential for survival. HIF-1alpha -/- mice die due to defective vascularisation, while HIF-2alpha-/- mice die from an inability to regulate heartbeat. The fact that HIF-1alpha and HIF-2alpha cannot compensate for each other establishes that these factors perform separate essential functions during development, and presumably have a distinct set of target genes. HIF-1alpha is known to induce the genes encoding erythropoietin, vascular endothelial growth factor, glucose transport proteins and several glycolytic enzymes. No target genes have been unequivocally assigned for HIF-2alpha.
- Sim1 and Sim2
Homozygous Sim1 knock out mice do not survive birth due to hypocellularity of neuroendocrine lineages in the hypothalamic-pituitary axis, indicating a critical role for Sim1 in terminal differentiation of certain peptide hormone secreting neurons. Sim1 deficiency has also been linked to early onset obesity. Extra dosage of the Sim2 protein is postulated to play a role in etiology of the Down’s Syndrome phenotype. Sim2-/- mice die at birth due to a poorly characterised breathing defect. A splice variant of Sim2 has been found to be prevalent in pancreatic, prostate and colon cancers and linked to the successful growth of these cancers. We are currently exploring the role of this splice variant in cancer development. Interestingly, the two Sim proteins discovered in mice may function as both transcription activators or repressors, depending on context. We are currently exploring the nature of this repression and the target genes under control of the Sim proteins.
Nxf was initially discovered by Simon Koblar’s lab as one of the most highly induced genes following epileptic seizure in rats. Nxf was also reported in the literature as a brain specific member of the bHLH/PAS family of transcription factors. Our studies in mice and zebra fish have now confirmed the brain specific expression pattern of Nxf and verified that it is dramatically induced following chemically initiated epileptic seizure in a second model organism, zebrafish. We are using in vitro and in vivo systems to explore the hypotheses that
- Nxf performs a critical function in development of the embryonic brain and
- Nxf is involved in damage/repair mechanisms in the adult brain.
Arnt is a general partner protein which heterodimerises with all of the above factors. It has also recently been shown that Arnt can homodimerise and function alone as a transcription factor on model reporter genes, although it is not known whether Arnt has any biological role independently of forming heterodimeric transcription factor complexes with other bHLH/PAS proteins. While Sim, the dioxin receptor and HIF-1alpha seemingly operate towards quite disparate biological purposes, they share a common obligate requirement for Arnt as a partner protein.