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The theme of our research is to understand how high-fidelity chromosome segregation is achieved in normal mitosis and how cell division can go awry to affect human health. We are interested in learning about both execution machinery and regulatory mechanisms during mitosis.The rationale of our cancer research is that aneuploid cancer cells require genetic or epigenetic alterations to survive and proliferate. Identifying these recurring modifications in aneuploid cancer genomes will offer insights into tumorigenesis and potential drug targets. We have realized the limitations of regular 2D cell culture in studying cancer so are exploiting different 3D cell culture systems and animal models for cancer research. We currently focus on dissecting the functions of chromosomal instability (CIN) signature genes in breast cancers.
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Major Scientific Achievements of the Lab:1. Searching for novel mitosis regulators: Compiled a database of >200 known centromere/kinetochore proteins in human cells; Identified novel mitosis regulators based on data mining: TRIP13, KIAA1377/Cep126 etc.
2. Activation of the mitotic checkpoint: In establishing the connection between signaling transducer and effector, we first reported that intracellular C-MAD2 concentration is the major determinant for MCC assembly; first reported and characterized direct interaction between BUBR1 and C-MAD2; demonstrated that a fully assembled MCC is critical in inhibiting mitotic APC/C.
3. Silencing of the mitotic checkpoint: By inquiring the energy requirement during mitotic checkpoint silencing, we first identified and characterized TRIP13 AAA-ATPase as a mitotic checkpoint silencing protein; first proposed the functional importance of C to O-MAD2 conversion in silencing the mitotic checkpoint and the role of TRIP13 in the conversion.
1. The centromere/kinetochore complex in human cells:
The centromere/kinetochore complex connects chromosomes with spindle microtubules during mitosis. Not only chromosome movement is mainly regulated by proteins located at the centromere/kinetochore complex, the spindle assembly checkpoint (SAC) signals that control the fidelity of chromosome segregation also originate from the complex. We have compiled a comprehensive list of 199 centromere/kinetochore proteins in human cells. Data mining of genomic-scale co-transcription profiling and proteomic-scale protein-protein interaction (PPI) analyses have led to the discovery of TRIP13 as a novel centromere/kinetochore protein. We are searching for additional components of the centromere/kinetochore complex and are also interested in dissecting how the complex is assembled and disassembled dynamically to meet functional requirements.
2. Activation of the spindle assembly checkpoint (SAC): BUBR1:C-MAD2 interaction in MCC assembly and function
The spindle assembly checkpoint (or simply the mitotic checkpoint) monitors kinetochore-spindle connections to control the timing of chromosome segregation. Aberrant functioning of the checkpoint has been linked to aneuploidy, which is observed in >90% of solid tumors. We are studying how the Mitotic Checkpoint Complex (MCC) is assembled from BUBR1, BUB3, MAD2 and CDC20 to form a potent effector for the SAC in human cells. Biochemical purification and reconstitution, together with analyses using mitotic cell lysates and live cell imaging, are major research approaches. We have provided evidence that only C-MAD2 is incorporated into the MCC. We were also the first to report direct interaction between BUBR1 and C-MAD2, which was confirmed later in the crystal structure of the S. pombe MCC (naturally lacking BUB3). How C-MAD2 is produced, how MCC is assembled and how MCC inhibits the Anaphase Promoting Complex/Cyclosome (APC/C) are being investigated.
3. Silencing of the SAC: How C-MAD2 is converted back to O-MAD2 and how MCC is disassembled
Silencing of the SAC is essential for proper completion of cell division. The SAC silencing involves events at two venues: at attached kinetochores and in the cytoplasm. We focus on the cytoplasmic events during SAC silencing, particularly on MCC disassembly. We also study SAC silencing by examining proteins that interact with p31comet, a known protein that is dedicated to SAC silencing. TRIP13 turns out to be a p31comet interacting protein and is involved in silencing the SAC by disassembling MCC [link]. Cep126 is a novel centrosome protein that also interacts with p31comet.
4. Characterization of two novel centrosomal proteins: KIAA1377(Cep126) and DDX39
6. Functional characterization of cancer "signature" genes: Surprisingly the biological functions of quite some genes in commonly used cancer gene signatures are still unclear. Focusing on chromosomal instability (CIN) signature genes in breast cancers.
The research techniques a trainee can expect to learn:
Molecular cloning, Gateway Technology, mammalian and insect cell culture, DNA transfection, siRNA and shRNA, CRISPR, immunofluorescence, live cell imaging with fluorescent proteins, in vitro translation, PCR, Western blot, immunoprecipitation/co-immunoprecipitation, pull-down assay, protein chemical cross-linking, Far-Western blot, Yeast two-hybrid, recombinant protein expression in E. coli, yeast, insect cells and mammalian cells, affinity chromatography, ion exchange chromatography, size-exclusion chromatography, column chromatography using the AKTA FPLC system, in vitro binding assay, in vitro kinase assay, in vitro ubiquitylation assay, protein degradation assay using mitotic cell extracts, two-dimensional (2-D) protein electrophoresis, 3D cell culture, xenograft mouse, transgenic mouse, etc.