The Mechanism Underlying The Role Of Oct4-interacting LncRNA Osclr8 In Cell Reprogramming

Background and objective:Terminally differentiated cells can be reprogrammed into a pluripotent stage to become induced pluripotent stem cells(iPSCs).The iPSC technology is a major breakthrough in the history of science.iPSCs are similar to embryonic stem cells,have the potential of self-renewal and multi-differentiation,and there are no immune rejection and ethical problems.However,there are still technical bottlenecks in the clinical application of iPSCs,such as induction efficiency,safety issues,and the mechanism of reprogramming is still unclear.Therefore,the research focusing on the molecular mechanisms of cell reprogramming is meaningful.This may help explore the key factors controlling the fate of cells,and may contribute to a more efficient and safe induction system,being useful for the clinical application of iPSCs in the future.Long noncoding RNA(lncRNA)is involved in the regulation of protein-coding genes in epigenetic regulation and transcriptional regulation.It has been considered as a new molecular mechanism to maintain the pluripotency and differentiation ability of pluripotent stem cells,and lncRNA controls the fate of cells with its unique epigenetic mechanism.In this study,we used the combined “chromatin lncRNA in situ reverse transcription-associated sequencing”(CRIST-seq)and RNA-seq approach to identify a pluripotency-assocaited lncRNAs.Among them,Osclr8(Oct4-Sox2 coating long non-coding RNA 8)binds to the Oct4 promoter and is associated with reprogramming.It is expressed only in iPSCs,but not in fibroblasts.After knockdown of Osclr8,stem cells become differentiated and exit from pluripotency.This suggests that Osclr8 is closely related to reprogramming.In this study,the structure,intracellular localization,function and epigenetic mechanism of Osclr8 in the regulation of reprogramming and the fate of stem cells will be researched.The study may provide theoretical basis for clinical transformation of iPSCs.Methods:1.The full length sequence of Osclr8 was identified by 5'-3' RACE.The cellular localization of Osclr8 was detected by RT-PCR of cytoplasmic and nuclear RNAs,respectively.In addition,the enrichment of Osclr8 in CRIST Cas9-g RNA mediated immunoprecipitated products was verified by PCR.2.The expression of Osclr8 was detected by RT-PCR during reprogramming.The expression of Osclr8,Oct4 and Sox2 at different time during embryonic body differentiation are detected by RT-PCR.The overexpression of Osclr8 facilitates reprogramming.The function of Osclr8 was determined by knocking down Oslcr8 lncRNA by sh RNA lentivirus in inducing pluripotent stem cells.3.RAT-seq(RNA reverse transcription-associated trap sequencing)was used to identify the gene targets that are interacted with Osclr8.The binding of Osclr8 lncRNA at the Oct4 locus was detected by PCR in Osclr8 RAT products.4.Changes in the three-dimensional structure of chromatin were detected using 3C(chromosome conformation capture)technique in reprogrammed and Osclr8 knockout cells.5.The protective effect of Osclr8 on Oct4 promoter methylation was detected by promoter methylation analysis.In addition,the expression of three TETs during reprogramming was detected by RT-PCR,and the binding of TET protein to Osclr8 was detected by lncRNA chromatin immunoprecipitation method.We used overlap primers of Osclr8 to identify the fragments that bind to the TET1.Results:1.Using CRIST-seq and RNA-seq,we identified a noval pluripotency-associated lncRNA Osclr8.It was differentially expressed between mouse fibroblasts and iPSCs.It was located at Chr3:75647440-75655731.Its 5'-and 3'-terminal sequences and its isomers were determined by 3'-and 5'-RACE.We analyzed lncRNA Osclr8 by RTPCR in cytoplasmic and nuclear extracts,and found that Osclr8 was mainly located in the nucleus.Osclr8 was enriched in CRIST immunoprecipitated complexes comapared with the g CT controls.2.Osclr8 was detected by RT-PCR in the reprogramming process,which verified the results of RNA sequencing.Osclr8 was expressed in iPSCs and embryonic stem cells,but not in fibroblasts and cells expressing OSKM factors but without reprogramming.The expression of Osclr8,Oct4 and Sox2 decreased significantly after embryoid differentiation.The overexpression of Osclr8 facilitates reprogramming.After knocking down Osclr8 from iPSCs,iPSCs became differentiated.3.Osclr8 was found to bind to Oct4 promoter region and Sox2 using a gene coating mechanism.We examined the binding of Osclr8 lncRNA at the Oct4 locus by RAT-seq.We found that most of the binding sites were located in the promoter and enhancer regions of the Oct4 gene.Osclr8 coated broadly at both the Oct4 and Sox2 loci.4.Using 3C method,we found that Oct4 promoter-enhancer intrachromosomal interaction loop was formed in iPSCs,but not in fibroblasts.When Osclr8 was knocked out,the intrachromosomal loop disappeared.5.By DNA methylation analysis,we found that the Oct4 promoter was not methylated by Sss1 after Osclr8 lncRNA binding with Oct4 promoter.We also found that TET1 was highly expressed after reprogramming,but not in differentiated cells and cells expressing OSKM without reprogramming by RT-PCR.Lnc RNA immunoprecipitation assay revealed that the TET-Osclr8 chromatin complex was formation in reprogramming.And the fragment binds to both TET1 and Oct4.Conclusions:1.In this study,we find that lncRNA-Osclr8,transcribed from Chr3:75647440-75655731,interacts with Oct4,a multipotent stem gene.It is expressed in stem cells,but not in fibroblasts.2.Lnc RNA-Osclr8 is required for the multipotent maintenance of iPSCs.Its expression is closely related to that of Oct4 and Sox2.3.Osclr8 binds to the Oct4 and Sox2 promoters using a gene coating mechanism,and regulates cell reprogramming and controls the fate of stem cells.4.The overexpression of Osclr8 facilitates reprogramming.5.After the knockdown of Osclr8 in iPSCs,the structure of the intrachromosomal loop formed between the Oct4 promoter and enhancer was destroyed,suggesting that Osclr8 is required for the formation of the intrachromosomal loop,which is critical to regulate reprogramming and maintain the pluripotency of stem cells.6.After binding with the Oct4 promoter,Osclr8 protects it from de novo DNA methylation.Lnc RNA chromatin immunoprecipitation reveals that TET1 binds to Osclr8,suggesting that Osclr8 may maintain the pluripotency of stem cells by recruiting demethylase.