Cloning And Functional Study Of A Novel Gene Related To The Self-renewal Of Human Embryonic Stem Cells

Embryonic stem cells(ES cells)are totipotent cells derived from inner cell mass of the blastocyst-stage embryo and have the greatest differentiation potential,being capable of giving rise to cells found in all three germ layers of the embryo.Mouse ES cells were first established by Evans and Kaufman in 1981 and human ES cells were isolated and acquired by Thomson in 1998. Since then,the research on ES cells has aroused biomedical community's more and more interest and been a hot-spot in life science research nowadays.ES cells have broad potential application in the clinical regeneration medicine because of their pluripotency.Nevertheless,how to culture ES cells in vitro and maintain their pluripotency is the first crucial step for their clinical applications.As we all know,human ES cells easily differentiate spontaneously in vitro,therefore,they have to be cultured on feeder-layer of mouse embryonic fibroblast or in the conditional medium containing cytokines,which are troublesome and unsuitable for large scale culture required for clinical applications.In addition,such culture condition may cause contamination by exogenous proteins.In order to find an optimized and convenient way for culturing human ES cells,it is of great importance to elucidate the molecular mechanism of self-renewal and pluripotency of human ES cells.Previous reports showed that the self-renewal of human ES cells was regulated by many factors including transcription factors(Oct4,Nanog,Sox2,et al),signaling pathways(TGFβ,WNT,FGF and PI3K,et aI),cell cycle,microRNA,chromatin and DNA methylation.Currently,most of the research focuses on Oct4,Nanog and Sox2,which are expressed highly in human ES cells.These transcription factors can maintain the pluripotency of human ES cells by inhibiting their differentiation to certain germ layer derivatives.Furthermore,it has been confirmed that these transcription factors regulate each other and construct a complicated regulatory circuit in human ES cells.Recent research showed that some other known and/or unknown genes might play important roles in maintaining the self-renewal in human ES cells besides the above-mentioned three factors.In order to explore these genes and their functions and uncover the molecular mechanism of self-renewal comprehensively,we performed a large-scale screen for genes related to the self-renewal in human ES cells by microarray and successfully cloned and identified such a novel gene.Moreover, the function and transcription regulation of this novel gene were elucidated.【Preliminary Study on Gene Expression Profiling of Human Embryonic Stem Cells and Embryoid Bodies】Gene chip provides a useful tool for studying gene expression at a high throughput level.In this study,we defined the transcriptional gene expression profiles of undifferentiated human ES cells and differentiated 7-day EBs with Affymetrix Human Genome Microarray U133A and B.According to the microarray results,there were 1100 genes down-regulated and 2200 genes up-regulated in 7-day EBs.The results were verified by real-time PCR and RT-PCR.Many genes related to the self-renewal of human ES cells were included in the down-regulated gene list.For example,Oct4 was down-regulated by 14-fold and Nanog was down-regulated by 17-fold.Besides Oct4 and Nanog,TDGF1,LEFTB and DNMT3B were found to be down-regulated by 11,42 and 8 folds,respectively.Moreover,some unknown genes including hypothetical proteins and ESTs were repressed when human ES cells differentiated into EBs.Among them,two ESTs(BF 223023 and BC020935)were found to be expressed highly in human ES cells.According to our real-time PCR results,BF 223023 was expressed at a similar level to Oct4 and Nanog in human ES cells and was notably down-regulated by 19.7 folds in 7-day EBs.In addition,BF 223023 was also reported,by other groups,to be down-regulated during the differentiation of human ES cells,suggesting that this EST-representing novel gene may be involved in the maintenance of self-renewal in human ES cells.To investigate the early molecular events of embryonic development,these differentially expressed genes(≥2 folds)were analyzed with Gostat software and the representative Gene Ontology(GO)terms were obtained.According to the GO analysis of these genes,we found most of them were related to cell differentiation and embryo development.In summary,GO provided us important clues for human ES cell research. 【Cloning and identification of a novel gene expressing highly and specifically in human ES cells】To clone the BF223023-representing novel gene,we performed BLAST analysis against the public human ESTs database using BF223023 as a query sequence.The matched ESTs were assembled into a contig from overlapping ESTs through manual alignment,and then a 2330bp sequence was obtained.No known genes were found to match with this sequence through searching GenBank nucleotides databases and we concluded that this sequence should represent a novel gene and named it human embryonic stem cells related gene (HESRG)(GenBank accession No.:DQ445779).HESRG mRNA sequence included the polyadenylation signal(AATAAA)and a poly(A)tail in the 3'end of cDNA sequence,demonstrating that the assembled sequence contained a complete 3' untranslated region.To clone its full-length sequence,we amplified 5'-end sequence by Smart RACE PCR kit.As a result,we obtained the complete cDNA sequence of HESRG gene consisting of consecutive 3141 nucleotides.The chromosomal localization analysis demonstrated that HESRG gene located on human chromosome 3p14.3.We did not find any homologous sequence in other species except Pan Troglodytes,indicating HESRG gene may be specifically expressed in human or primate animal cells.Northern blot analysis showed that HESRG had two transcripts different in size in human ES cells,one being about 1.2 kb and the other 3.1 kb.The HESRG mRNA sequence was predicted to contain two candidate ORFs analyzed by ORF Finder on NCBI website.ORF1 comprising 666 bp with an initiation codon at 2-4 nucleotides(nt)and a stop codon at 668-670nt encoded a 24 kilo-Dalton protein HESRP-1 composed of 222 amino acids(aa).ORF2 comprising 243 bp with an initiation codon at 2245-2247 nt and a stop codon at 2485-2487nt encoded an 8.7 kilo-Dalton protein HESRP-2 composed of 80aa. Bio-informatical analysis showed that HESRP-1 was homologous to RNR and E2F-7 partially and it may contain a leucine zipper and several protein kinase C (PKC)phosphorylation sites.Meanwhile,it was predicted to be located in nucleus with LOCtree software.Therefore,we presumed that HESRP-1 might regulate gene expression as a transcription factor.HESRP-2 contained four PKC phosphorylation sites,two casein kinaseⅡphosphorylation sites,one myristylation site,one possible hydrophobic region and two hotloops.HESRP-2 exhibited high homology with two hypothetical proteins,but it was difficult to predict its function because it was lack of any conservative domain and had no homology with any known protein.Promoter,the transcription initiator of gene,plays an important role in transcriptionally regulating gene expression.The 2000bp region upstream of the transcription start site of HESRG was analyzed by Neural Network Promoter Prediction software and the results showed that this region contained some candidate promoter sequences,indicating it may act as a functional promoter. Transcription factors regulate gene expression through binding to transcription factor binding sites(TFBS),the specific sequences located in promoter region. We screened the 2000bp upstream of transcription start site of HESRG by TESS Software and predicted many transcription factors binding sites such as Oct4, Oct1,SRY,LEF1 and TCF1 in this region.Since Oct4 expressing highly in human ES cells is a key gene in maintaining self-renewal of human ES cells,we predicted that HESRG might be a target gene of Oct4.These prediction results provided us some important clues to investigate the function and transcription regulation of HESRG.HESRG expressing highly in human ES cells was undetectable in all kinds of human fetal tissues,adult testis tissue,mutilple normal and cancerous cell lines,demonstrating a specific expression pattern.In order to confirm HESRG is a marker gene of pluripotency,we induced human ES cells to differentiation by several ways.The results from RT-PCR showed that HESRG was significantly down-regulated once differentiation of human ES cells and the mRNA level of HESRG decreased gradually with the time of differentiation. Additionally,the expression of HESRG in teratoma tissues derived from human ES cells and differentiated cells induced by retinoic acid(RA)was totally absent.The change of HESRG expression was consistent with that of Oct4 and Nanog and was even more obvious than that of Oct4 and Nanog,further supporting the idea that HESRG gene may be a new pluripotency marker for undifferentiated human ES cells. 【Function and transcriptional regulation of HESRG】Presently RNA interference(RNAi)has been a major strategy for studying gene function because of its advantages e.g.convenience,high efficiency and specificity.In order to establish a high efficient RNAi method,we parallelly compared RNAi efficiency by transfecting siRNA into human ES cells using three transfection reagents(Lipofectamine RNAiMAX(RNAiMAX), Lipofectamine 2000 and Oligofectamine).We found that RNAiMAX showed a much higher RNAi efficiency than Oligofectamine and Lipofectamine 2000 under the same conditions and demonstrated that RNAiMAX was an excellent transfection reagent for delivering siRNA into human ES cells.Hence,we have established a high efficient and convenient RNAi method in human ES cells, providing us a useful tool to investigate HESRG function.Based on the specific expression pattern of HESRG and bio-informatic analysis results,we inferred that HESRG might play an important role in maintaining the self-renewal in human ES cells.Subsequently we investigated the function of HESRG by RNAi.The results showed that rapidly growing undifferentiated human ES cells changed into scattered,long and thin differentiated cells with a slow proliferation rate when HESRG expression was down-regulated by 75%,while non-targeting siRNA showed no obvious effect. Moreover,compared with non-targeting siRNA group,the marker genes of pluripotency including Oct4,Nanog and Sox2 were repressed significantly in the cells transfceted with siHESRG,while some ectoderm marker genes including Sox1,FGF5 and Nestin were dramatically up-regulated.No obvious change in the expression of endoderm,mesoderm and trophoblast marker genes was observed.Furthermore,we detected the expression of marker genes including Oct4,Nestin,Hand1,GATA4 and CGβat a protein level by indirect immunofluorescence before and after RNAi.The results showed that the Oct4 fluorescence signal in the cells transfected with siHESRG became weak,but the Nestin signal appeared quite strong compared to the negative control.The above results confirmed that HESRG could inhibit human ES cells to differentiate into ectoderm,thereby maintain the pluripotency of human ES cells.Transcriptional regulation is considered as the principal mechanism in modulating gene expression.The cis-acting elements determine the exact start and efficiency of transcription,so it is critical to locate and clone the promoter of target gene when studying its regulatory mechanism.According to the bio-informatic prediction,we amplified a fragment spanning from position -2049 to +11 of HESRG by PCR with human ES cells' genomic DNA as the template.Luciferase reporter assay showed this fragment exhibited a strong promoter activity.However,the core promoter of HESRG will be identified in our future work.Bio-informatic prediction revealed that there existed Oct4 transcription factor binding site in the promoter of HESRG.In order to explore the possible role of Oct4 in regulating HESRG promoter activity and mRNA expression,we generated a luciferase reporter construct containing a mutated Oct4-binding consensus sequence with two nucleotides replaced.In consequence,this mutant significantly repressed HESRG promoter activity in human ES cells.In addition, we down-regulated the expression of HESRG specifically by RNAi and then detected HESRG promoter activity.It was found that knockdown of Oct4 inhibited HESRG promoter activity.Furtherly HESRG promoter activity can be enhanced by ectogenic Oct4 in HEK293 cells.At last chromatin immunoprecipitation(ChIP)assay confirmed that Oct4 could directly bind to the Oct4-binding site in HESRG promoter.All these results demonstrated that Oct4 could positively and directly regulate the expression of HESRG.In summary,we screened the gene expression profiling of human ES cells and 7-day EBs,acquired the global biological information of genes with differential expression,cloned and identified a novel gene(HESRG)by EST assembly,explored the expression pattern of HESRG and its relation with pluripotency,accomplished the bio-informatic analysis of HESRG;and investigated the function and transcriptional regulation mechanism of HESRG. These results will enable us to clarify the function and regulatory mechanism of HESRG in human ES cells,better understand the molecular mechanisms of self-renewal in human ES cells and provide us evidence for further biological research and promising clinical application of HESRG.