The Studies On The Roles Of FoxA1Transcription Factor In The Regulation Of Stemness-related Gene Nanog

Pluripotent stem cells can self-renew indefinitely in vitro while maintaining theability to differentiate into advanced derivatives of all three germ layers. In general,these cells can be divided into three types:1. Embryonal carcinoma cells (EC Cells)derived from teratocarcinomas;2. Embryonal stem cells (ES Cells) derived from innercell mass of blastocyst;3. Embryonal germ cells (EG Cells) derived from primordialgerm cells in the gonad of embryo. The pluripotency of these cells largely depends ona core transcriptional circuitry formed by Oct4, Sox2, and Nanog.In this study, we investigated the gene expression regulation mechanism ofpluripotent stem cells differentiation during retinoic acid (RA) induction, using P19EC cells as cell model. P19cells are pluripotent and have the ability to formchimmeric animals by injecting into blastocyst, or to form teratocarcinoma with threegerm layers structure by injecting into nude mice. In addition, P19cells candifferentiate into advanced derivatives of all three germ layers in vitro. Together, P19cells are suitable model for the study in the gene expression regulation mechanism ofpluripotent stem cells differentiation.We focused on the role of transcription factor FoxA1in the regulation of stemcell differentiation. FoxA1belongs to the Forkhead Box family of transcriptionfactors that play important roles in cellular proliferation and differentiation d uringembryonic development. Typically, FoxA1is thought to act as "pioneer" transcriptionfactor, due to its ability to bind to highly compacted chromatin and facilitate thebinding of other transcription factors to DNA. We have previously discovered thatFoxAl is a critical stimulator of RA-induced P19cell neural differentiation and theexpression of FoxA1is induced immediately following the RA treatment. Theupregulation of FoxAl is ahead of the reduced expression of pluripotent transcriptionfactors, such as Nanog, and the activated expression of differentiation-related factors,such as early neurogenesis-related sonic hedgehog (Shh), neural stem cell markerNestin. Further studies showed that FoxA1activate the expression of Shh directly,while ectopic expression FoxA1alone in P19cells activated the expression of Nestin,and reduced the protein levels of Nanog. From the perspective of epigenetics, thisstudy continued to explore the role of FoxA1in the transcriptional regulation ofRA-induced neural differentiation process, and got results as follows:1. We detected the changes in gene expression levels and epigenetic states of pluripotent marker gene Nanog after RA treatment, in P19cells. Following RAtreatment, both the mRNA and protein levels of Nanog gene is reduced. We haveexamined the epigenetic states including histone H3acetylation (H3K9ac), histone H3Lys4methylation (H3K4me2), histone H3Lys27methylation (H3K27me3) and DNAmethylation. We found that in undifferentiated P19cells, the promoter sequence ofNanog is hypomethylated, and the highly acetylated histone H3is found in Nanoggene locus, where H3K4is hypermethylated. This epigenetic modification statuskeeps the high expression of Nanog in pluripotent cells. On the other hand, during thedifferentiation of P19cells, the Nanog promoter becomes hypermethylated, and thedeacetylation of H3and the demethylation of H3K4occur in Nanog gene locus area.Moreover, the hypermethylation of H3K27can be found in Nanog gene locus.2. We have found that, during RA induction, FoxA1and Grg3bound to theenhancer of Nanog gene, and repressed its expression. According to previous findings,we speculated that FoxA1repressed the expression of Nanog directly. It has beenreported that FoxA1could recruit transcriptional co-repressor Grg3. By assaying boththe mRNA and protein levels, we found that Grg3expressed in P19cells as well asduring RA-induced P19cell differentiation. By using chromatin immunoprecipitation(ChIP) experiments, we confirmed that FoxA1and Grg3bound to-2kb upsteamregion of Nanog gene two days after RA treatment. Moreover, we interfered theexpression of FoxA1and Grg3by siRNA in P19cells respectively, following RAtreatment. We found that Knockdown of FoxA1or Grg3delayed the downregulationof Nanog expression during RA-induced P19cell differentiation.3. We have investigated the role of FoxA1playing in the repression of Nanoggene. Gel mobility shift assay (EMSA) confirmed that FoxA1bound to Nanogenhancer-2025bp to-2001bp region, while Grg3alone could not bind to DNA. Byusing co-immunoprecipitation (Co-IP) and immunofluorescence colocalizationexperiments, we confirmed that FoxA1and Grg3interacted directly. Through dualluciferase activity analysis, we found that FoxA1and Grg3inhibited the activity ofNanog promoter synergistically.4. We have found that ectopic expression of FoxA1alone changed the histonemodification, but not DNA methylation status of Nanog gene locus in P19cells. Aftertwo days overexpression of FoxA1, H3K9was deacetylated, the degree of H3K4methylation levels was reduced, and H3K27was methylated.