TGF-β Signal Transduction and Cell Fate Decision
A/Prof. Jian-Hua Xing
University of Pittsburgh, USA

Epithelial to mesenchymal transition (EMT), a process of transforming polygon-shaped epithelial cells with tight cell-to-cell attachment to spindle-like mesenchymal cells with loose or rare cell-to-cell attachment, has been suggested to play a key role in embryo development and many pathological processes such as fibrosis and cancer metastasis. Previous studies showed that exogenesis signals such as TGF-β can induce EMT in many mammalian epithelial cell lines. According to a well established mechanism, transmembrane TGF-β receptors (TGFBR) receive the extracellular signal, pass downstream via the Smad transcription factor family, and activate multiple genes such as Snail1, a key regulator of EMT. However, our measurements and computational analysis reveal that Smad2/3 can not function as major transcriptional factors to directly induce Snail1 expression. After careful examination of the TGF-β Smad dependent and independent pathways, we hypothesize sustained Snail1 activation is achieved through a nested relay mechanism that involves Smads, Gli1/2 (a main component of the SHH pathway), and GSK3β (main components of the WNT pathway). Our combined mathematical modeling and quantitative measurements confirmed this hypothesis. Currently for live cell imaging, we are using the CRISPR-Cas9 technique to fuse fluorescence proteins to selected players in the network. Compared to the wide application of CRISPR-based gene knockout, gene knockin is more challenging and less developed. A technical obstacle lies in generating the knockin constructs, for which the Gibson assembly approach often fails. Out of a ~1045 possible sequence space, we developed a computer-aided procedure to design three DNA segment linkers to optimize the assembly fidelity. Experimental tests on multiple genes confirmed that presence of the linkers leads to high yield of DNA constructs that we failed to make otherwise.

About the Speaker


2016-07-07 10:00 AM
Room: A203 Meeting Room
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