| Dppa2 (Developmental pluripotency-associated 2) is a recently identified gene specifically expressed in pluripotent cells. Current information on DPPA2 (human)/Dppa2 (mouse) were collected from the expression analysis of DPPA2/Dppa2 as a pluripotent marker and DPPA2/Dppa2 expression in cancer cells as a cancer/testis gene. Despite the current information, a further study in DPPA2/Dppa2 function as well as the mechanisms is needed. Our study is aimed at exploring the role of Dppa2 in maintaining the mESCs undifferentiation and the possible mechanisms.Firstly, we used Real-time PCR to analyze the change of Dppa2 expression during mESCs differentiation, and compared the change with those of Oct4, Sox2 and Nanog. We adapted two methods to induce mESCs differentiation:suspension culture to form embryoid bodies (EBs) and all trans-retinoic acid (atRA) induction. Our results showed that, under the two differentiation conditions, Dppa2 displayed a similar trend of expression change to Oct4, Sox2 and Nanog:The expression was upregulated in earlier time then significantly downregulated to a very low level in later period during mESCs differentiation.To achieve ideal transfection efficiency on mESCs and a successful RNA interference (RNAi) on Dppa2, we investigated and optimized the conditions of mESCs transfection with LipofectamineTM2000. Our results suggest that a low confluence of mESCs (30-50%) is prone to result in a relatively high transfection efficiency (≥50%).For the first time, we used RNAi to inhibit Dppa2 expression in mESCs. The results of RT-PCR and Real-time PCR demonstrated that the RNAi on Dppa2 is successful, where the interference efficiencies in two inference groups shl and sh2 respectively reached 85% and 78%. After Dppa2 RNAi, mESCs showed considerable trends of differentiation, which indicated as a decreased AKP activity, a weak stain; slight downregulation (-20%) of Oct4 and Nanog expression as well as differently levels of upregulation on parts of germ layer marker genes (such as Fst of endoderm and Psx1 of Trophoblastic ectoderm). In addition, we detected the change of mESCs proliferation capability using BrdU assay. The results indicated that the inhibition of Dppa2 expression caused a significant decline on mESCs'proliferation capability.We located the transcriptional start site (TSS) of Dppa2 at-30bp upstream its start codon ATG using 5'-RACE method. In addition, we used a dual luciferase reporter system to analyze and detect the activities of different candidate regions of Dppa2 promoter. The results suggested that-176bp-+293bp region is the basal promoter region of Dppa2. Dppa2 promoter has a high activity in mESCs and a certain activity in P19 (mouse embryo carcinoma cells, mECCs) while nearly no activity in other cell lines we have detected, suggesting the promoter has a specific activity in pluripotent cells.We performed bioinformatics analysis and found that multiple recognition sites (ATGC(A/T)(A/T)-(A/T)) of Oct4 transcription factor exist in upstream region of Dppa2 gene, suggesting Dppa2 might be under transcriptional regulation of Oct4. Further chromatin immunoprecipitation (ChIP) assay and electrophoretic gel mobility shift assay (EMSA) assay confirmed that we for the first time determined-1964--1950bp region downstream of Dppa2 is the recognition site of Oct4 transcription factor.In summary, our study indicated that Dppa2 play a role in maintaining mESCs undifferentiation. Besides, we for the first time analyzed the TSS of Dppa2, isolated Dppa2 promoter, confirmed Dppa2 receives a (positive) transcriptional regulation from Oct4 and it might play as a downstream gene of Oct4 in maintaining mESCs undifferentiation condition. Our study laid a foundation for a further clarification of molecular regulation mechanisms in maintaining ESCs undifferentiation.
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