M Lee - Structural biology in gene regulation and DNA damage repair pathway
How can we explain the complexity of the human species when we have a comparably similar number of genes as the simple roundworm, C. elegans? It is apparent that the attribute distinguishing humans from the less complex organisms is not the number of genes itself but the regulation of gene expression. More complex gene regulation, therefore, is required for higher organisms and aberrant regulation of gene expression leads to various disorders such as developmental abnormalities, cancer and metabolic disorders.
Our laboratory's research interests lie in the area of gene regulation and DNA damage repair pathway. We are particularly interested in characterising the macromolecular complexes (protein-protein and protein-nucleic acid complexes) in the nucleus to understand their roles in gene regulation and DNA damage repair pathway.
Research areas
SFPQ, multifunctional nuclear protein
Splicing factor proline-glutamine rich (SFPQ) is an abundant and essential nuclear protein involved in various aspects of gene expression by interacting with DNA, RNA, and other protein interaction partners. The resulting biological implications are wide ranging, including neural cell development, regulation of circadian rhythm, regulation of viral expression and tumour repression and progression. Capitalising on the recent success in determination of the structure of the core domain of human SFPQ, the current research in our laboratory aims to understand the multifunction of SFPQ by characterising the complexes of SFPQ with its interaction partners.
The main technique to be used in our laboratory is X-ray crystallography, complemented by molecular biology techniques (cloning and mutagenesis), protein overexpression and protein purification. Other biophysical techniques employed in the laboratory include small-angle X-ray scattering in collaboration with Dr Andrew Whitten at Australian Nuclear Science and Technology Organisation (ANSTO), microscale thermophoresis (MST), and electrophoretic mobility shift assay (EMSA). In vivo macromolecular interaction is currently being explored using yeast two-hybrid system in collaboration with Dr Christine Hawkins in the department.
Understanding dual nucleic acid specificities of SFPQ in reversible gene regulation
SFPQ has been reported to repress the transcription of several genes via direct binding to the promoter regions. Despite the presence of two RNA recognition motifs, the cognate RNA sequences of SFPQ are yet to be identified. However, several long noncoding RNAs (lncRNAs) that interact with SFPQ have been identified in the recent reports on the reversible regulatory roles of SFPQ. In the proposed mechanism of reversible gene regulation, the transcription repression exerted by SFPQ via direct binding to the promoter region can be reversed by its interactions with lncRNAs. This project aims to solve the structure of SFPQ in complex with nucleic acids to provide the structural basis of the reversible gene regulation.
SFPQ, a universal nuclear hormone receptor modulator?
Recent studies have revealed that SFPQ interacts with a range of nuclear hormone receptors including peroxisome proliferator-activated receptor, retinoid X receptor, glucocorticoid receptor, thyroid hormone receptors, progesterone receptor, and androgen receptor. Some of these interactions have been implicated in cancer cell proliferation and apoptosis. These observations lead to the working hypothesis that SFPQ is a universal nuclear hormone receptor modulator.
This project aims to characterise the interactions between SFPQ and nuclear hormone receptors biophysically and structurally. The outcome of this project may offer a new platform to facilitate rational drug design for many metabolic diseases including diabetes by modulating the interactions between SFPQ and nuclear hormone receptors.
Understanding the roles of SFPQ in DNA damage repair pathway
SFPQ has been reported to play a role in DNA repair by direct interaction with the homologous recombinase, Rad51 and modulates its homologous-pairing and strand-exchange activity. The recruitment of SFPQ to DNA damage sites has been shown in vivo and the attenuation of SFPQ expression sensitises the cells to ionising radiation.
Interestingly, the region of Rad51 required for SFPQ binding overlaps with that required for interaction with BRCA2, the breast cancer susceptibility gene 2. This observation leads to the working hypothesis that SFPQ may compete with BRCA2 for interaction with Rad51 and that SFPQ may be an important regulator to modulate the interaction between Rad51 and BRCA2. This project focuses on the production of SFPQ-Rad51 and SFPQ-Rad51-BRCA2 complexes, and the biophysical and structural characterisation of these complexes.
