Role And Mechanism Of LncRNA Oeblr20 In Regulating Pluripotency And Reprogramming Of Stem Cells Via Oct4 ERNA Pathway

Background:Pluripotent stem cells(PSCs)have great potential for developmental biology and regenerative medicine due to their unlimited self-renewal capacity and pluripotency,PSCs can be isolated as embryonic stem cells(ESCs)from the inner cell mass(ICM)of a mammalian blastocystorsomatic cells can be reprogrammed as induced pluripotent stem cells(iPSCs)by exposure to a cocktail of transcription factors(Oct4.Sox2.Klf-4.c-Myc)(OSKM).The reprogramming process to make iPSCs.however,is extremely inefficient and time-consuming,hindering their potential clinical applications for regenerative medicine.Recent studies have suggested that long noncoding RNAs(IncRNAs)may play a role in regulating the induction and maintenance of pluripotencyLong noncoding RNAs(IncRNAs)are non-protein-coding transcripts that are longer than 200 nucleotides.They are usually correlated well with the complexity of an organismand restricled to specific cell lineages,therefore representing a potential role in cell fate determination.IncRNAs play important roles in pluripotency.cell differentiation and during reprogramming process.Research on IncRNAs may provide novel insights into manipulating the cell fate or reprogramming somatic cells into iPSCs.Understanding the function and working mechanisms of IncRNAs could yield novel RNA-based strategies in manipulating ESCs or iPSCs to advance regenerative medicine.To map pluripotency-associated IncRNAs,we used a conventional RNA-Seq approach to isolate IncRNAs that are differentially activated in pluripotent reprogramming,and subsequently performed "RNA reverse transcription-associated trap sequencing"(RAT-seq)to profile the genome-wide targets for these IncRNAs.Using this strategy,we identified Oeblr20(Oct4enhancer binding IncRNA 20)as a IncRNA that is associated with stem cell reprogramming.Here we need to explore the potential roles and mechenisms of Oeblr20 in regulating pluripotency and reprogramming.Objectives:1.Illustrate the differential expression of Oeblr20 among different cells and tissues,its subcellular localization and gene structure.2.Confirm its roles in regulating pluripotency and reprogramming process.3.Investigate its potential mechanisms of control Oct4 enhancer RNA expression.Methods:1.Confirm differential expression of Oeblr20 among different cells and tissues by RT-qPCR,subcellular localization by RNA-FISH and gene structure by RACE.2.Functional assay----Oeblr20 knockdown in iPSCs by shRNA.3.Functional assay-Oeblr20 overexpressed in Fib by pGreen overexpression plasmid.4.Reprgraming assay----Oeblr20 overexpressed in DOX-OSKM-MEFs system and compare reprogramming efficiency.5.RAT-Seq?DNA-RNA FISH and Luciferase reporter system to confirm that Oeblr 20targets Oct4locus.6.Oeblr20 level is associated with Oct4 enhancer RNA transcripts confirmed by by RT-qPCR.7.DNA methelytion analysis of Oct4 locus.8.Confirm the relationship amongOeblr20,Oct4 locus and TETfamily by ChIP and RIP.Results:1.Identification of Oeblr20 as a pluripotent IncRNA by combining RAT-Seq and RNA-Seq.RNA-seq data showed that Oeblr20 was silenced in fibroblasts and upregulated in iPSCs.Notably,Oeblr20 bound to the regulatory elements of multiple sternness genes,including the Ocl4 enhancer.These data suggest that Oeblr20 might play important roles in pluripotency initiation and maintenance by regulating core factor genes.2.The expression of Oeblr20 was closely correlated with pluripotent status during reprogramming.as it was transcribed at very low levels in fibroblasts and un-reprogrammed cells(URCs).However,it was immediately activated once the cell was fully reprogrammed as iPSCs.As expected,it was also abundantly expressed in embryonic stem cells.The results of expressions in different mouse tissues showed that it was not transcribed in terminally differentiated tissues,which also supported that this IncRNA was potentially associated with pluripotency.3.Oeblr20 IncRNA is required for the maintenance of pluripotent state.In loss of function assay,afterOeblr20 knockdown,the expression of Oct4,Sox2,and Nanog.were also down-regulated The shOeblr20-transfected iPSCs exited from pluripotency,as demonstrated by their differentiated morphology and the loss of immunostaining staining for the pluripotent marker protein NANOG.4.Oeblr20 IncRNAcan promote expression of Oct4gene in gain of function assay.After Oebrl20 overexpressed in Fib.Oct4 expression was also improved for more than third limes.5.Oeblr20 enhances iPSCs reprogramming efficiency.On day 21,reprogramming efficiencies were examined by immunostaining for stem cell marker NANOG.The number of NANONG-positive colonies was significantly increased in the Oeblr20-overexpressing group.Andthe Oeblr20-overexpressing cellsconlained significantly higher amounts of sternness genes.6.Oeblr20 interacts with the 5'-and 3'-Oct4 enhancer elements.Using RAT-seq,we found that Oeblr20 bound to the Oct4 5'-and 3'-enhancer elements.We also performed sequential Oeblr20 lncRNA-Oct4 DNA FISH in iPSCs and Oeblr20 overexpressed Fiband found that Oeblr20 overlapped with the Oct4 DNA signals.Moreover,dual-luciferase reporter systemsshowed that Oeblr20 IncRNA significantly enhanced the activities of the Oct4enhancers.7.Oeblr20 activates Oct4 enhancer RNA expression by inducing DNA demethylation.AfterOeblr20 knockdown,we found that the eRNAs at both the Oct4 5'-and 3'-enhancer sites were significantly downregulated,while overexpression of Oeblr20 significantly increased the expression of eRNAs at the Oct4 enhancer loci.By examining the status of DNA methylation after ectopic expression of Oeblr20.we observed an extensive decrease in DNA methylation.8.Oeblr20 recruits TET2 to the Oc(t4 enhancer locus.ChIP was performed with anti-TET1,anti-TET2 and mouse IgG control antibodies in Oeblr20 overexpressed Fib.It showed that TET2.but not TET1 bound to the Oct 4 enhancer loci in Oeblr20-overexpressing fibroblasts.We then performed a RIP assay and showed that TET2 interacted primarily with the 3'-fragment of Oeblr201ncRNA.These data suggest that Oeblr20 may help recruit TET2 to the Oct4 locus,where it induces DNA demethylation and promotes eRNAs expression.Conclusions:1.The subcellular location of IncRNA Oeblr20 is nucleus.lt was abundantly expressed in iPSCs and ESCs.however,it was transcribed at very low levels in fibroblasts,un-reprogrammed cells and terminally differentiated tissues.And the expression of Oeblr20was associated with key pluripotent genes.2.Oeblr20 IncRNA is required for the maintenance of pluripotent state.After Oeblr20 knockdown iPSCs exited from pluripotency and can not keep pluripotency with key pluripotent genes down-regulated.3.Oeblr20 IncRNA can promote expression of Oct4 gene and enhances iPSCs reprogramming efficiency and pluripotency.4.RAT-Seq.RNA-DNA FISH and Luciferase assays showed that Oct4 is the target of Oeblr20 IncRNA.Oeblr20 significantly enhances the activities of the Oct4 enhancers and promotes the expression of Oct4 eRNA.5.Oeblr20 IncRNA recruits TET2 to the Oct4 enhancer locus and activates Oct4 enhancer RNA expression by inducing DNA demethylation.