Study On Mechanisms Of Grg3-mediated OCT4Transcription Repression

Stem cells (SC) possess the capacity of self-renewal and potential ofdifferentiation. Stem cell research provides new ideas for treatment of diseases andpromoted the development of basic medical research dramatically in recent years. Theprocesses between maintainance of pluripotency and the differentiation in stem cellsinvolve the switch on and off expression of specific gene, which rely on thecomplicated and precise transcription regulation network. In this study， wepreliminary explored the repression mechanism of the key pluripotency gene OCT4during the early stage of P19embryonic carcinoma (EC) cells differentiation.Transcription corepressor Grg3(Groucho-related gene3) belongs to the Grouchocorepressor family and plays important role in the physiology process of embryonicdevelopment and organ formation. Previous studies have shown that, Grg3is usuallyrecruited to the regulation regions of its target gene by transcription factors due to thelack of DNA binding domain in Grg3, and thereby plays its role in gene repressionpossibly though changing the modification state of chromatin. We use variousmethods to try to figure out the mechanism that Grg3participates in the transcriptionrepression of pluripotency gene OCT4. The levels of Grg3remain unchanged duringretinoic acid (RA)-induced neural differentiation of pluripotent P19EC cells. Byusing the specific Grg3siRNA and Grg3-V5fusion protein expression tools, we foundthat the expression levels of OCT4are regulated by the protein levels of Grg3in P19EC cells. The over expression of Grg3inhibits the OCT4expression, while the knockdown of Grg3brings a rise of OCT4protein levels. Further results achieved byDual-Luciferase Reporter assay and CHIP assay show that Grg3represses OCT4bybinding specificlly to the－3500bp~－3345bp region of OCT4promoter.Transcription factor FoxA1(Forkhead box A1), also known as HNF3α, belongsto the fork head protein family and involves in the regulation of various physiologicalprocesses. Studies show that FoxA1is a key factor during early stage of P19neuraldifferentiation and a direct protein-protein interaction between FoxA1and Grg3isdetected in vitro. We also performed immunofluorescence assay to confirm theinteraction between FoxA1and Grg3in vivo. The－3500bp~－3345bp region ofOCT4promoter contains two FoxA1binding sites, Dual-Luciferase Reporter assayshow that point mutation of these two sites impaired the repression of promotor activity by Grg3. Thus, FoxA1may be the key factor that recruits Grg3inGrg3-mediated repression of OCT4.