The bHLH/PAS (basic Helix-Loop-Helix/Per-Arnt-Sim homology) protein family comprise a broad set of transcription factors with diverse roles in development and homeostasis. The bHLH domain is a well known dimerisation and DNA binding domain prevalent in developmental transcription factors. PAS domains are found in proteins which sense and transmit signals relating to environmental or metabolic status (e.g. PAS domains have been found to detect oxygen status, light, environmental pollutants and redox or nutrient status). Our laboratory studies mechanisms of signal transduction and gene regulation by several members of this family.
- Mechanism and Function of Nxf
The Nxf protein is a brain specific member of the bHLH/PAS family that was found by the Koblar group to undergo a rapid increase in expression following chemically induced epileptic seizure in rats. We have found that the Nxf protein can function as a strong transcription activator, although this varies markedly in different cell lines and is totally dependent on the co-expression of either the Arnt1 or Arnt2 partner protein. In collaboration with the Koblar group, we are currently investigating the roles of Nxf in brain development and stress response.
- Gene Regulation by the Single Minded Transcription Factors
Single Minded 1 and 2 (Sim1 & 2) are encoded by separate genes and expressed predominantly in the brain, muscle and kidney. Sim1 null 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. Sim2 null mice die at birth due to a poorly characterised breathing defect. We are currently deciphering target genes for each of the Sim proteins and investigating signalling mechanisms which may regulate their function.
- Deciphering the Posttranslational code of the Dioxin Receptor
The Dioxin Receptor (DR) is activated by environmental pollutants and notorious for mediating the severe toxicity associated with dioxin and PCB poisoning. DR null mice survive, but have impaired liver function due to incomplete vascularisation and exhibit poor fertility. We are mapping a complete profile of posttranslational modifications in the DR during its multi-step activation process and attempting to link these to functions of early development and toxin metabolism.
- Novel functions of the Hypoxia Inducible Factor HIF-1a
HIF-1a is well established as a key transcription factor for inducing genes involved in angiogenesis, erythropoiesis and enhanced glycolysis during hypoxic stress. Somewhat surprisingly, HIF-1a is also found to induce some pro-apoptotic genes and in certain circumstances can function as a transcription repressor. We are investigating the mechanisms of these lesser known functions of HIF-1a and relating these to new biological roles.
Mechanisms of Specificity of the bHLH.PAS Proteins
The bHLH.PAS proteins are often coexpressed and dimerisation behaviour is highly regulated. The protein dimers bind to closely related DNA sequences, but are functionally distinct, activating specific and discrete sets of target genes. The molecular properties that drive this specific dimer formation and DNA recognition in bHLH.PAS proteins, which are critical to maintain the precision of gene expression networks, are poorly understood. We want to decipher the features of the bHLH and PAS domains that direct the interactions involved in macromolecular recognition, for both protein-protein and protein-DNA complexes. Ultimately, we hope to understand at the molecular level how these features confer specificity and function for closely related proteins.
Our experiments include:
- biochemical analysis of dimerisation and DNA binding of native, mutant and constructed chimeric forms of bHLH.PAS proteins.
- isolating and characterising novel proteins that interact with the bHLH.PAS region to enhance DNA binding by the HIFalpha/s and Sim proteins.
- structural characterisation of the protein/DNA complex by x-ray crystallography.
The techniques used include protein expression and purification, electrophoretic mobility shift assays, two-hybrid assays systems and reporter gene assays in mammalian cells in culture.
NHMRC, ARC Special Research Centre for the Genetics of Molecular Development, Cancer Council of SA.