Molecular Mechanisms Of The Interaction Between A Novel ROR?t Gene Enhancer RORCE2 And Promoter Mediated By SOX-5 In Th17 Cells

T helper type 17(Th17)cells are a subset of the T helper(Th)lineage characterized by the production of interleukin(IL)-17A,IL-17F,IL-22,IL-21,IL-26 and tumor necrosis factor-?(TNF-?),etc.These cytokines,especially IL-17A,are the basis of various biological functions of Th17 cells.Although Th17 cells play an important role in host defense,and in autoimmune and inflammatory diseases,such as:multiple sclerosis(MS),experimental autoimmune encephalitis(EAE),rheumatoid arthritis(RA),hepatitis,systemic lupus erythematosus(SLE)and colitis.Molecular mechanism of Th17 cells in pathogenesis of various diseases need to be fully understood.The retinoic acid receptor-related orphan receptor-?(RORC)gene encodes two different isomers,ROR?1 and ROR?2(also known as ROR?t).A overwhelming evidence has suggested ROR?t is a key transcription factor(TF)in regulation of Th17 differentiation.The expression of IL-17A in ROR?t-deficient CD4⁺T cells decreased rapidly in vitro,Conversely,overexpression of ROR?t by transfection can restore the expression of IL-17A.ROR?t-deficient(ROR?t^-/-)mice have reduced Th17 differentiation and are resistant to EAE,suggesting that this transcription factor plays an important role in autoimmunity.ROR?t can regulate the transcription of many key genes in Th17 cells,ROR?t can binds to transcriptional start site(TSS)at-400?+151 bp region in target promoter,however,ROR?t targeting enhancers remain elusive.This thesis focuses on the discovery and identification of ROR?t gene enhancer and underlying mechanisms.The main conclusions are described as follows:1.RORCE2 promotes the differentiation of Th17 cells by enhancing the expression of ROR?t.Firstly,we took advantage of H3K4me2 chromatin immunoprecipitation sequencing(Ch IP-seq)data for mouse Th17 cells in the GEO database to identify potential enhancers of the ROR?t gene.Intriguingly,we found a unique H3K4me2(histone H3 Lys 4 dimethylation)peak associated with the ROR?t locus specifically in Th17 cells,named RORCE2.By comparing the chromatin signatures of the syntenic region of mouse RORCE2 between human Th17 and Th0 cells,we found that active enhancer-associated epigenetic markers,including H3K4me1 and H3K27ac,were also enriched in human Th17 cells.We next determined whether RORCE2 acts on the ROR?t promoter using a dual-luciferase assay in EL4 murine tumor T cell line,human embryonic kidney(HEK)293T cell line,and Th17-polarized cells.All of these in vitro results suggested that RORCE2 was a potent enhancer element for the ROR?t promoter in Th17 cells that might contribute to Th17differentiation and functions.To evaluate our prediction,we used a Ch IP coupled with quantitative PCR(Ch IP-q PCR)assay to examine the profiles of other epigenetic markers of an active enhancer including H3K4me1,H3K27Ac and H3Ac(H3 acetylation).To investigate the effect of RORCE2 on the regulation of ROR?t gene expression,we generated RORCE2-deficient(RORCE2^-/-)mice by CRISPR/Cas9 genome editing.We found that the m RNA and protein expression levels of ROR?t and IL-17A were decreased significantly.To further elucidate the contribution of RORCE2 to Th17 cell differentiation,we polarized na?ve RORCE2^-/-or WT CD4⁺T cells into Th17 cells,the results indicated that RORCE2 played a positive role in Th17 cell differentiation in vitro by regulating the expression of the ROR?t gene.In addition,RORCE2 deficiency impacts the development of EAE,which is a Th17-dependent inflammatory disease.The disease progression in the RORCE2-deficient mice was remarkably less than that in their WT littermates.These results suggest that RORCE2 is a novel active enhancer of the ROR?t gene in Th17 cells.2.SOX-5 is required for the looping between RORCE2 and the ROR?t promoter in Th17 cells.Since it is well known that the regulatory activity of an enhancer mainly depends on the formation of a chromatin loop between the enhancer and its target promoter.The mechanism is as follows:firstly,the transcription factors bind to the enhancer and make the chromatin free,and then recruit other transcription factors to cooperatively activate the enhancer,and finally interact with the promoter.To explore the role of the RORCE2 in Th17 cells,we firstly used a chromosome conformation capture-q PCR(3C-q PCR)assay to determine whether RORCE2 interacts with the ROR?t promoter in Th17 cells.The results showed that the AS interacted most frequently with TS3 located in the RORCE2 region among all TS sites tested in both PMA/ionomycin(PI)-simulated and unstimulated EL4 cells.Comparable results were observed in FACS-purified Th17 cells and Th17-polarized cells.In addition,we investigated which TF(s)was involved in the interaction between TS3 in RORCE2 and the AS in the ROR?t gene promoter in Th17 cells.Based on the JASPAR database and previous study,SOX-5 emerged as the best candidate.We speculated that the transcription factor SOX-5 may be a key transcription factor that mediates the RORCE2 interacts with the ROR?t gene promotor,and SOX-5-binding site(SOX-5-b S)is located about 50bp upstream of TS3.Using Ch IP-q PCR,we found that SOX-5 bound to both RORCE and the ROR?t promoter in PI-stimulated EL4 cells or WT Th17-polarized cells,respectively.Furthermore,a Ch IP-loop assay demonstrated that SOX-5 was involved in the process of chromatin loop formation between TS3 and the ROR?t promoter in both PI-stimulated EL4 cells and Th17-polarized cells.To further characterize the role of SOX-5 in the chromatin loop in vivo,we generated SOX-5-BS-deficient(SOX-5-BS^-/-)mice by CRISPR/Cas9-mediated genome editing.We found that SOX-5-BS deficiency in RORCE resulted in a significant decrease in SOX-5enrichment.Intriguingly,3C-q PCR and Ch IP-loop assays showed that the degree of TS3-ROR?t promoter interaction was also significantly reduced by deleting the SOX-5-BS in the promoter region.Furthermore,both the m RNA and protein levels of ROR?t and IL-17A were decreased in SOX-5-BS-deficient mice.SOX-5-BS deficiency resulted in a significantly decreased frequency of CD4⁺ROR?t⁺Th17 or CD4⁺IL-17A⁺Th17 cells under the Th17-polarizing conditions.We further investigated whether SOX-5-BS deficiency affects EAE severity,the results indicated that SOX-5-BS deficiency phenocopied RORCE2deficiency,including significant decreases in disease progression,cell infiltration and spinal cord demyelization.These results suggested that SOX-5 mediated interaction between RORCE2 and ROR?t gene promoter in Th17 cells.3.SOX-5 and STAT3 cooperatively induce chromatin opening of ROR?t gene RORCE2 in Th17 cell.The transcription factors of SOX family can open chromatin by binding to DNA to regulate gene transcription,but they cannot recruit chromatin remodeling complex to the open the chromatin structure alone,which must depend on the cooperation of partner transcription factor(Partner-TF)to recruit chromatin remodeling complex.Firstly,we detected chromatin accessibility by Ch IP-q PCR and chromatin accessibility test based on WT and SOX-5-BS^-/-mice and found that the level of chromatin openging in the RORCE2region was significantly decreased after SOX-5-BS deletion.Interestingly,we found a putative DNA binding site for STAT3(STAT3-BS),a well-known transcriptional activator,close to the SOX-5-BS.Due to the critical role of STAT3 in the induction of ROR?t expression and Th17 differentiation,we investigated the contribution of STAT3 to RORCE2enhancer function.Ch IP-q PCR results showed that STAT3 more strongly interacted with RORCE2 in PI-stimulated EL4 cells and Th17-polarized cells than in B16 cells and na?ve CD4⁺T cells,respectively.Moreover,Stat3 overexpression led to the increased STAT3-RORCE2 interaction and up-regulation of ROR?t and IL-17A expressions in WT Th17-polarized cells as evidence by RT-q PCR and/or ELISA;accordingly,Stat3 knockdown caused the reverse results.However,this binding was significantly disrupted upon SOX-5-BS deficiency in Th17-polarized cells,suggesting that SOX-5 was required for the binding of STAT3 to RORCE2.When RORCE2 lacked the STAT3-BS upstream of the ROR?t promoter in the p GL3 vector,the transcriptional activity of RORCE2 was lower than that of normal RORCE2 in EL4 cells.Furthermore,Co-Immunoprecipitation(Co-IP)assay showed that STAT3 interacted with SOX-5,suggesting that the SOX-5 might affect STAT3 recruitment to RORCE2.In addition,when knockdown of STAT3 expression in Th17 cells,we found that the level of chromatin opening in the RORCE2 region was significantly reduced.These results suggest that SOX-5 and STAT3 cooperatively induce chromatin opening of ROR?t gene RORCE2 in Th17 cell.In this study,we identify a novel enhancer,RORCE2,for promoting ROR?t gene expression in Th17 cells in vivo and in vitro.Further investigation shows that the interaction between RORCE2 and the ROR?t gene promoter is mediated by SOX-5 binding.Knocking out the BS for SOX-5 in RORCE markedly decreases ROR?t expression and Th17differentiation,resulting in decreased EAE severity.Finally,we demonstrate that STAT3cooperated with SOX-5 to induce hromatin opening of ROR?t gene RORCE2 in Th17 cell.The present study will provide not only a new mechanism underlying Th17 differentiation but also a potential clue for intervention in Th17-related diseases in the future.