Dr Anne Chapman-Smith

Research Interests

Understanding specificity of the bHLH.PAS proteins: Molecular mechanisms

The bHLH.PAS structure-function group is directed by Dr Anne Chapman-Smith.

The bHLH.PAS proteins are often coexpressed and dimerisation behaviour is tightly regulated. The various protein dimers in the family 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, which are critical to maintain the precision of gene expression networks, are poorly understood. We want to decipher the elements 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 specificity of function is directed amongst these highly similar proteins.

Our work has identified a novel mode of DNA binding for bHLH proteins by the Dioxin Receptor, where the PAS domain is required for high affinity interaction between protein and DNA. More recently we have shown that the PAS domains of the Dioxin Receptor and the Hypoxia Inducible Factor probably interact with DNA to alter DNA conformation. Both of these functions are new modes of macromolecular interaction for PAS domains, which are commonly found to detect oxygen status, light, environmental pollutants and redox or nutrient status in proteins which sense and transmit signals relating to environmental or metabolic status.

Current experiments are aimed at:
1. biochemical analysis of dimerisation and DNA binding of native, mutant and constructed chimeric forms of bHLH.PAS proteins. 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.

2. isolating and characterising novel proteins that interact with the bHLH.PAS region to enhance DNA binding and regulate specificity for the Hypoxia Inducible Factors and the Single-minded proteins.

3. structural characterisation of bHLH.PAS protein/DNA complexes. This aspect of the work is being carried out in collaboration with Assoc. Prof. Matthew Wilce at Monash University, who is an expert in crystallography of protein/nucleic acid complexes, and Dr. Lisa Martin at Monash University, and includes the use of Small Angle Xray Scattering and Atomic Force Microscopy.

Relevant publications
1. Kewley, R.J., Whitelaw, M.L. and Chapman-Smith, A. (2004) The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. Int. J. Biochem. Cell Biol. 36, 189-204.

2. Chapman-Smith, A., Lutwyche, J.K. and Whitelaw, M.L. (2004) Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators. J. Biol. Chem. 279, 5353-62.

3. Chapman-Smith, A and Whitelaw, M.L. (2006) Novel DNA binding by a basic helix-loop-helix protein: the role of the Dioxin Receptor PAS domain. J. Biol. Chem. 281, 12535-45.

4. Chapman-Smith, A. Whelan, F. Mechler, A. Ng, S. Shearwin, K. Martin L.L. and Whitelaw, M.L.  PAS domains mediate protein-DNA interactions and alter DNA conformation. (manuscript in preparation)

Entry last updated: Wednesday, 19 Sep 2012