The Sequence And Function Of Alternative Splicing Variant Of Nuclear Coregulator GT198

BackgroundAlternative splicing is a very common phenomenon in the eukaryotic genome. More than 59% of the human genome is alternatively spliced, and more than 80% of alternative splicing products yield functional proteins. Alternative splicing is one of the important mechanisms to produce new protein which could serve a new function. A large number of studies confirm that alternative splicing influence gene regulatory in the stem cell differentiation. Splicing variants are frequently found in cancer. In this study, we focus on the nuclear receptor coregulator GT198 which can yield alternative splicing product GT198a and compare the sequence and function between them. Meanwhile, preliminarily study the correlation of GT198 gene with breast cancer.Part 1 Identify the sequence of GT198 variant GT198a and alternative splicing switch of GT198 gene in stem cell differentiationAim GT198 (Genomic Transcript 198) is a nuclear receptor coregulator that interacts with nuclear receptors and involves in nuclear receptor-mediated gene regulation. A GT198 variant GT198a was found in P19 and hES cells. In this part, we analyzed the full sequence of GT198a and GT198 cDNA then cloned them for further studies. And also we investigated the expression of GT198a and GT198 during stem cell differentiation. Method 5’RACE assay was used to identify the 5’sequence of GT198a and GT198. We extracted total cell RNA at different time point of P19 and hES cell differentiation for RT-PCR and quantitative real-time PCR analyses. Result Our data showed that GT198a contains the first intron of GT198. Alternative splicing at 5’of the gene produce premature stop codon with early termination of translation. The new transcript starts at the ATG in the extron 5. This transcript contains an open reading frame encoding the DNA binding domain (DBD). In addition, the expression level of GT198a decrease but GT198 increase during the P19 and hES cell differentiation. Conclusion GT198a was the alternative splicing variant of GT198 and a switched expression from GT198a transcript to GT198 transcript occurs during the EB (Embryoid Body) stages of stem cell differentiation.Part 2 Function of GT198a and GT198Aim Studies have demonstrated that alternative splicing variants often serve different and even antagonistic function with wild type. Alternative splicing switch occurs between GT198a and GT198 during P19 or hES cell differentiation, suggesting they may serve different function. In this part, we tested the transcription activity of GT198a and GT198, and also investigated GT198a and GT198 influence on DNA repair gene Rad-51 nuclear foci formation. Meanwhile we detected whether overexpression GT198a could induce the translocation of GT198 and induce tumor. Method Luciferase assay was used to test the transcription activity. Immunochemistry was performed to analysis their effect on Rad-51 nuclear foci formation and whether overexpression of GT198a could induce translocation of GT198. And a stable P19 cell line expressed GT198a was established and injected in to nude mice to investigate whether it could induce tumor. Result Our results indicated that GT198a enhanced the transcription activity and inhibited Rad51 nuclear foci formation. However, GT198 weakened the transcription activity and did not show effect on Rad51 nuclear foci formation. Overexpression of GT198a promotes GT198 cytoplasmic translocation. We found stable P19 cell line expressed GT198 or empty vector did not induce tumor three weeks later, but GT198a stable expressed P19 cell line induced small colony formation three weeks later and formed obvious tumor six weeks later. Conclusion Overexpression GT198a promotes GT198 cytoplasmic translocation, impair Rad-51 nuclear foci formation upon radiation. Meanwhile, overexpression of GT198a can induce tumor formation.Part 3 GT198 gene and breast cancerAim The human GT198 gene is 470 Kb to BRCA1 within a 17ql2-q21 locus which was linked to the early-onset of breast cancer predisposition. Alternative splicing plays an important role in BRCA1 related breast cancer since BRCA1 variants give rise to different protein variants with potentially tumorigenic properties. In the second part, we found the GT198a has striking similarities to variants of BRCA1 in function. Such as, the splice variants inhibit wild type activity and inhibit the Rad51 nuclear foci formation. So in this part, we preliminarily studied the association of GT198 with breast cancer. Method RT-PCR was used to test the expression level of GT198a in a panel of eight cancer cell lines. RT-PCR and Western Blot was used to compare the expression level of GT198a in breast or ovarian cancer to normal breast or ovarian tissue. And immunohistochemistry was used to analyze the expression position of GT198 in 114 human breast cancer tissues. Result Our results indicated that GT198a expression was higher in breast cancer and ovarian cancer than lung, prostate and pancreatic cancer. And GT198a expression level was upregulated in breast and ovarian cancer. We observed that normal breast tissue is negative in expression cytoplasmic GT198. However, in human breast cancers, cytoplasmic GT198 expression was found in 17.5% of stromal cells and in 48.2% of myoepithelial cell in 114 tumors analyzed. Conclusion Our results suggested that GT198a high expression and cytoplasmic GT198 was increased in breast cancer tissue. GT198 gene may be involved in breast cancer development.