Mechanism and function of bHLH/PAS transcription factors in development, stress response and disease

The basic-Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors comprise a set of proteins which function in development and homeostasis, with many having key roles in sensing and responding to environmental stress. Defects in these proteins can lead to a wide range of diseases, with family members critical during ischemic diseases and stroke, development of various cancers, mental disorders and early onset obesity. For example:

1) the Hypoxia Inducible Factor (HIF-1a) is essential for embryonic angiogenesis, metabolism and  adaption to low oxygen stress;

 
2) the Dioxin Receptor is critical for fetal liver angiogenesis and toxin degradation;

3) NPAS4 is induced by neuron activity to generate inhibitory synapses and prevent excitotoxicity during neural signalling;

 
4) SIM1 functions in hypothalamic neuron development and satiety signalling;

5) SIM2 has unknown function, but is essential for postnatal survival and is prevalent in a colon, pancreatic and prostate cancers.

Our projects broadly centre around two main aims:

A) understanding the signalling mechanisms which control activity of the bHLH/PAS transcription factors

B) defining direct target genes of these transcription factors and understanding the mechanisms by which they either activate or repress those target genes

Projects available for Honours in 2010

1. The Dioxin Receptor can be activated by a range of small organic molecules and also by switching cells from adherent to suspension culture. Once activated, the DR functions not only as a transcription factor, but also in a ubiquitin ligase complex to initiate degradation of proteins such as the estrogen and androgen receptors and b-catenin. The project will explore how different signalling mechanisms trigger the DR to function in these separate pathways. We have found that the DR has extensive posttranslational modification, so we will investigate if particular modifications are directing the DR towards specific functions.

2. Sim1 is essential for terminal differentiation of distinct neuroendocrine secreting cells of the hypothalamus. Sim1 deficiency is linked to severe childhood obesity, resultant from defects in satiety signalling in the hypothalamus. The project will study the role of Sim1 in neuron maturation, using in vitro systems for differentiating ES cells to neurons. How Sim1 participates in the satiety signalling pathway will also be explored.

3. Sim2 is one of the most commonly disregulated genes in prostate cancer and high Sim2 levels correlate with poor prognosis. We have found that Sim2 can function as a transcription activator of some genes, but a repressor of other genes. The project will seek to understand these dual functions of Sim2 and discover novel Sim2 regulated genes which are involved in the cancer process.

4. NPAS4 is highly induced during epileptic seizure to dampen the hyperexcitation of neurons, and also induced during stroke to function as a neuroprotective agent. The role of NPAS4 in maintaining homeostasis between excitatory and inhibitory synapses has lead to the hypothesis that defective in NPAS4 may be a component of autism spectrum disorders. The project will explore the signalling pathways that induce NPAS4 and the downstream mechanisms by which NPAS4 functions in synapse development and neuroprotection.

5. Biotechnology Project. As described above, many bHLH/PAS transcription factors are critical for progression of a variety of diseases. The PAS domain is a three dimensional protein fold which contains an open pocket at its core, thus making these proteins opportune drug targets. This project will develop systems for discovering small organic ligands which are able to alter function of the above listed members of the bHLH/PAS family. The aim will be to subject these proteins to high throughput screening assays using natural product and synthetic chemical libraries, in attempt to discover molecules that can be developed as pharmaceuticals.

The projects in our lab use many techniques of modern cell and molecular biology, eg DNA cloning and manipulation, reporter gene construction, generation of stable cells lines which exhibit inducible expression of cDNAs for ectopic expression of proteins, inducible expression of shRNA for gene knockdown, RNA analysis by real time PCR and microarray, isolation of protein complexes by Ab bound resins and mass spec identification of proteins residing in the complexes, ChIP assay to determine DNA sites bound by bHLH/PAS proteins (including ChIP-sequencing for global analysis of DNA binding sites), bacterial expression and purification of proteins, dissection and culture of primary neurons from rodent embryos, FACS sorting of cell populations, analysis of point mutant proteins in cell based and in vitro biochemical assays.

Please contact Murray Whitelaw for any further details