The Regulatory Effect Of Foxm1on Nanog Gene Expression

Previous studies have confrmed that Foxm1plays a pivotal role in cell cycleregulation, cell-fate determination, embryonic development, adult tissue homeostasis, organregeneration and ageing. Foxm1also plays a crucial role in ensuring the fidelity of the celldivision process, as inhibition of Foxm1activity results in serious aberrancies duringmitosis. Acquisition of aberrant expression of Foxm1by normal stem cells represents thefrst step towards a multistep oncogenic evolutionary pathway. Results suggest a criticalinvolvement of Foxm1and pluripotent genes Oct4, Nanog and Sox2in maintenance of stemcell pluripotency. SUMOylation is a critical and ubiquitous post-translation modificationpathway in eukaryotes; SUMOylation modulates proteins’ subcellular location, stability andinteraction. Studies have shown that SUMOylation regulates mitosis and inhibit theexpression of Nanog in stem cells.In order to elucidate the effect of transcription factorFoxm1on Nanog, and identify whether the factor is a substrate for SUMOylation, Real timePCR, Western blot and Dual-Luciferase Reporter assay were introduced to understand themolecular mechanism of Foxm1regulating Nanog in F9carcinoma cells and fetal bovinefibroblast (FBF). The experiments’results are as follows:1. In this study, we cloned-1122-+323bp (relative to transcriptional start site) fragmentusing Nanog genomic DNA as template, and piecewise truncated the promoter (50bpwalking) from the upstream. All of these fragments were inserted into pGL4.10, andpositive recombinants were verified by sequencing. To verify the function of theseconstructs, dual-luciferase reporter assays were done in F9and fetal bovine fibroblast. Theresults demonstrated that the Nanog promoter-1122-+323bp can drive Nanog transcriptionefficiently, and the promoter is embryo-specific, because significently higher luciferaseactivity was detected in F9cells than in FBF.2. Total RNA was extracted from F9carcinoma cells, and we cloned CDS of Foxm1byreverse transcription PCR. Then the fragment was inserted into pCMV-Myc, and theeukaryotic expression plasmid of Foxm1was obtained. After enzyme digestion analysis andDNA sequencing, the construct was transfected into293FT cell and fetal bovine fibroblastrespectively. The expression of Foxm1was identified by Western Blot. The results showedthat the eukaryotic expression vector pCMV-Myc-Foxm1constructed correctly and expressed in cells, and laid the foundation for further experiments.3. We studied the molecular mechanism of Foxm1regulating Nanog in F9carcinomacells and fetal bovine fibroblast by using luciferase reporter gene vectors contained theproximal promoter of Nanog and eukaryotic expression vector of Foxm1. Thedual-luciferase reporter assay experiments in fetal bovine fibroblasts demonstrated thatoverexpression of Foxm1could enhance the activity of Nanog promoter, but there was nosignificant difference for different lengths of promoter. Combined with previous studies(Xie et al.2010), the reasons for this situation are:1, There were no specific binding sitesfor Foxm1in Nanog promoter-1122-+323bp;2, The effects of Foxm1on Nanogpromoter was indirect and may need other transcription factors to participate in. Real timePCR and Western blot experiments in F9carcinoma cells demonstrated that overexpressionof Foxm1had a significant role in promoting the Nanog gene in the level of transcriptionand translation. In summary, we have shown that the transcription factor Foxm1promotethe expression of Nanog.4According to the results of potential SUMOylated sites for Foxm1provided by theprediction software of sumoylation(SUMOsp2.0), we used overlap extension PCR (OverlapExtension PCR, OE PCR) to individually mutate the199,216,354,459,477and494lysineresidues on Foxm1, which were converted to arginine residues, and constructed6expression vectors. Each mutant and wild-type expression vector was transfected orco-transfected with pCMV-Myc-SUMO1and pCMV-Myc-UBC9into293-FT cells toidentify the SUMOylation by Western blot experiments. Then, the wild-type Foxm1wastransfected or co-transfected with pCMV-Myc-SUMO1and pCMV-Myc-UBC9into F9carcinoma cells, and Nanog expression was detected by real time PCR when F9cells are indifferent status of SUMOylation. The results showed that:1, wild-type Foxm1can beSUMOylated;2, with individually mutated199,216,354,459,477and494lysine residues,Foxm1can still be SUMOylated;3, SUMOylation can enhance Foxm1transcriptionalactivity and help raise the level of transcription in Nanog gene.