C-MYC CONTROLLED TIP110 PROTEIN EXPRESSION REGULATES OCT4 MRNA SPLICING IN HUMAN EMBRYONIC STEM CELLS

Purpose: We hypothesize that TIP110 expression is modulated through C-MYC and herein demonstrate that the oncogenic transcription factor C-MYC up-regulates transcription of the RNA binding protein TIP110 through interaction with the TIP110 E-box in the TIP110 promoter, thus ensuring high-level Tip110 expression in proliferating hESCs. TIP110, therefor, regulates OCT4 expression through alternative splicing in hESCs. Methods: To examine direct involvement of C-MYC over TIP110 expression, we over-expressed C-MYC, or silenced C-MYC expression in stem cells to test whether C-MYC regulates Tip110 expression. To identify the elements by which C-MYC could regulate Tip110 expression, both CHIP assay and EMSA were performed. Splicing assay was used to study the alternative splicing of OCT4 by TIP110 protein. Results: There was a clear increase of Tip110 expression with overexpression C-MYC, and a reduction of TIP110 expression with knockdown of C-MYC expression at both mRNA and protein level. The results reveal the binding between TIP110 promoter E-box region and C-MYC protein. The splicing assay further shows that TIP110 alternatively splices OCT4 to produce OCT4A in stem cells. Conclusions: In this study, we demonstrate the interaction between TIP110 promoter E-box region and C-MYC protein. TIP110 expression induces OCT4 exon 1a inclusion and 1b skipping, an event required for the pluripotency of human embryonic stem cells.