| Backgrounds and Objectives: Pluripotent stem cells have promising application in regenerative medicine and cell replacement therapy,due to their ability to differentiate into any cell type and their indefinite self-renewal properties in vitro.Studies have shown that transcription factor regulatory networks(e.g.Oct4,Nanog and Sox2),various signaling pathways,chromatin remodeling complex and non-coding RNAs are involved in the maintenance of the pluripotent state of stem cells.Disruption of this network leads to loss of pluripotency and cellular differentiation.Therefore,in order to advance stem cell-based therapies to clinical applications in the future,it is of great significance to understand the precise mechanisms required for pluripotent stem cells to maintain a delicate balance between pluripotency and differentiation signals.In recent years,mounting studies have demonstrated that lncRNAs play critical regulatory roles in cell proliferation,apoptosis,differentiation,metabolism and other biological processes in both normal and disease states,and similarly,lncRNAs have versatile biological functions in maintaining stem cell self-renewal.However,the molecular components that coordinate pluripotent exit are poorly understood.When stem cells are cultured in medium without LIF or treated with retinoic acid,they will change the expressive levels of three core transcriptional factors(Oct4,Sox2 and Nanog),as well as the complex regulatory network formed.Ultimately,these stem cells fail to maintain pluripotency and differentiate.LncRNAs play important regulatory roles during this process of pluripotency exit.Therefore,seeking for lncRNAs associated with pluripotency exit is critical for controlling the fate of stem cells in the future.By analyzing the transcriptome sequencing data of mouse fibroblasts and ESC cells,we aimed to identify a functional lncRNA that is associated with pluripotency exit of stem cells.Its roles and epigenetic mechanism in regulating stem cell pluripotency and reprogramming were also explored.Methods: 1.Target lncRNA screening: Firstly,we identified a functional lncRNA that is associated with the pluripotency exit of stem cells by analyzing the RNA-Seq results,and we named it as Peln18(Pluripotency Exit LncRNA 18).Subsequently,quantitative real-time PCR(q PCR)was used to clarify its expression levels in different cell types and at different timepoints during embryoid body formation.The subcellular localization of Peln18 was determined by the cytoplasmic and nuclear fraction assay and RNA FISH.2.Functional studies of Peln18: We constructed Peln18 overexpression and knockdown plasmids and stably transfected them in mouse embryonic stem cells(E14)and mouse fibroblasts(NIH-3T3)by lentiviral packaging method,respectively.Their effects on the expression of core transcriptional factors(Oct4,Sox2 and Nanog)were detected by q PCR.Meanwhile,alkaline phosphatase staining,cell proliferation and colony forming assay were employed to evaluate the changes of cell morphology and biological characteristics caused by overexpression or knockdown of Peln18.The sh Peln18 lentivirus also infected mouse embryonic fibroblasts(MEFs)derived from transgenic mice that carried OSKM factors.We used immunofluorescence staining to identify NANOG-positive colonies and compared the number of positive colonies to assess reprogramming efficiency.3.Profiling the genome-wide targets of Peln18: RAT-Seq was used to seek for the genome-wide interaction target DNAs for Peln18.Meanwhile,CRIST assay was employed to further confirm the interaction between Peln18 and the specific regulatory region of the selected target gene.The dual luciferase reporter system assay was utilized to investigate the effect of Peln18 on the target gene.4.Epigenetic mechanisms underlying the role of Peln18: The methylation status of Oct4 promoter was assessed by bisulfite-sequencing assay after Peln18 overexpression.ChIP technique was used to measure the interaction between target gene and methylation modifying enzymes.At the same time,the binding of Peln18 with DNMT3A was confirmed by RIP.Finally,the chromosome conformation capture(3C)assay was used to explore the influence of Peln18 on the intrachromosomal loop structures within different regulatory regions of the target DNA.Results: 1.Peln18 was a novel lncRNA located on the mouse chromosome 9 and was 1021 bps in length.It was highly expressed in fibroblasts while it was almost completely silenced in E14 cells.The expression of Peln18 was upregulated during the process of embryoid body differentiation,showing an opposite trend compared with the stemness genes Oct4,Sox2 and Nanog.2.The cytoplasmic and nuclear fraction assay and RNA-FISH showed that Peln18 was predominantly located in the nucleus.3.The m RNA and protein levels of Oct4,Sox2 and Nanog were significantly down-regulated in E14 cell successfully overexpressing Peln18.Morphologically,alkaline phosphatase staining indicated that some cells became differentiated and were negatively stained.Besides,the capacities of proliferation and colony forming were decreased.4.After Peln18 knockdown in MEF cells,the number of NANOG-positive colonies was increased as compared with the control groups,and the upregulation of stemness genes(Oct4,Sox2 and Nanog)was observed as well.5.Peln18 knockdown in terminally differentiated fibroblasts did not affect the expression of pluripotency genes,and there were no significant change in cell proliferation and colony formation abilities.6.RAT-Seq data showed that Peln18 was interacted with numerous genes enriched in cell differentiation and stemness maintenance pathways.Especially,Peln18 could bind to both the promoter and 3’ enhancer region of Oct4.CRIST assay further verified the interaction between Peln18 and the Oct4 promoter.Dual-luciferase reporter assay showed that the promoter activity of Oct4 gene was repressed by Peln18.7.Compared with the control group,methylation levels of the Cp G island in Oct4 promoter region was increased in E14 cells when overexpressing Peln18.ChIP assay showed that Peln18 promoted the binding of DNMT3 A to the promoter region of Oct4,and RIP method further proved that Peln18 and DNMT3 A were interacted each other.8.In E14 cells,the promoter and enhancer of the Oct4 gene spatially formed intrachromosomal loops to maintain pluripotency,and the spatial enhancer-promoter architecture was disrupted when Pelnl8 was overexpressed.Conclusions: 1.Peln18 is a novel 1ncRNA located on the mouse chromosome 9,which is predominantly localized in the nucleus.It was highly expressed in fibroblasts while it became almost silenced in mouse embryonic stem cells.It is negatively correlated with the expression of stem cell core factors(Oct4,Sox2,and Nanog)during embryoid body differentiation.2.Peln18 contributes to the pluripotency exit of stem cell,Peln18-overexpressing E14 cells became differentiated and showed decreased m RNA and protein levels of core transcriptional factors.The proliferation and colony forming capabilities were reduced as well.3.Peln18 is an obstacle in the process of OSKM reprogramming,but downregulation of Peln18 alone in terminally differentiated fibroblasts is not sufficient to reactivate stemness gene expression.4.Peln18 inhibits Oct4 expression by recruiting DNMT3 A to bind to the promoter of Oct4 and further increasing the methylation status of this area.5.Peln18 regulates the expression of Oct4 by disrupting the functional intrachromosomal loops between Oct4 promoter and enhancer. |
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