AbstractsBiology & Animal Science

Transcriptional regulation in heart and skeletal muscle is controlled by the combined action of 3 major families of transcription factors namely the the bHLH, MADs box transcription factors and the Transcriptional enhancer factor (TEFs) which play important roles for the development of muscle tissues and regulated expression of muscle specific genes. MCAT element (5´-CATTCCT-3´) has been found in a number of cardiac, smooth, and skeletal muscle-specific genes, including cardiac troponin T, beta-myosin heavy chain (beta-MHC), smooth and skeletal muscle -actin. The proteins that bind to the MCAT element belong to the TEF family of transcription factors. VITO-1 was identified as an essential co-factor of TEF1 and activates TEF1 through its SID domain, which results in the transcription of genes involved in muscle cell differentiation. In this thesis, I have analyzed various molecular characteristics of VITO-1 and studied its role in different cell types. To analyze and compare the subcellular localization in different cell types, VITO-1 was over-expressed in HEK 293, C2C12 and CH10T1/2 cell line. VITO-1 displayed a predominant localization in the nucleus of C2C12 and CH10T1/2 cells whereas a cytoplasmic distribution in HEK 293 cells which might be attributed to the absence of endogenous TEFs in this cell line. Co-expression of TEFs with VITO-1 in HEK 293, C2C12 CH10T1/2 cell lines resulted in a complete translocation of VITO-1 into the nucleus. Expression of VITO-1 deletion mutant constructs in C2C12 cells proved the requirement of SID domain to translocate into the nucleus. In the presence of VITO-1, differentiated C2C12 cells formed large myotubes which supports its role in muscle cell formation. To identify additional binding partners of VITO-1, a yeast two hybrid screen was performed with a skeletal muscle cDNA library using the GAL4 system. From a total of 48 positive clones we focused on Telethonin (T-cap) and Myozenin1 (MYOZ1, FATZ) as they were isolated as independent overlapping clones. Telethonin (T-cap) is one of the most abundant transcripts expressed in striated muscle at the Z-discs. MYOZ1 is also a sarcomeric protein that is known to interact with T-cap. The interactions of Tcap and MYOZ1 clones with VITO-1 were reconfirmed using the Y2H assay. An ONPG beta-galactosidase reporter gene assay revealed that VITO-1 binds TEFs with greater efficiency than T-cap and MYOZ1. A failure of Co-IP using the in vitro transcripted / translated proteins to substantiate the interaction between these proteins might be due to the absence of native cellular environment. Since VITO-1 is known to be up regulated in differentiated C2C12 cells and T-cap and MYOZ1 are Z-disc proteins, we used differentiated C2C12 myotubes and chicken primary myocytes for the Co-IP experiments. Indeed VITO-1 containing the SID domain co-immunoprecipitated with Tcap and Myozenin1 in differentiated C2C12 myotubes and primary chicken myocytes thus demonstrating efficient physical interaction. This interaction is mediated through the SID domain as…