Quantitative Differences In Chromatin Accessibility And The Transcriptional Regulatory Mechanises Of RAG

ObjectiveRecombination activating genes(RAGs)encode RAG1 and RAG2 protens,which play an important role in the development of T and B lymphocytes and the clonal production of lymphocyte pool.The abnormal expression of RAG will block differentiation and development of T and B lymphocytes,and then bring about fatal immunodeficiency diseases.Therefore,it is important to reveal the molecular mechanism that controls RAG-specific expression.In the multiple levels of regulatory mechanisms,transcriptional regulation plays a critical role.Previous reports are mainly about animals,and that about human-RAG are quite limited.Especially lack the study on regulatory mechanism of endogenous RAG gene at the chromatin level.Thus the purpose of this study is that discus the specific scope and openness of the chromatin at RAG2 gene 5'proximal region,so as to provide the basis for positioning cis transcriptional regulatory elements(cis-elements).The relationship between chromatin open area and transcriptional regulation functions were investigated by analysing the transcriptional activity of the specificity of chromatin open areas.Further,the binding sequences for T-cell specific transcriptional factors(TFs)were seatched,and the binding activities were verified by using both in vitro and in the chromatin of endogenous gene.At last,regulatory activities of the TF to the reporter and endogenous RAG2 gene at 5'proximal region were investigated,so as to elucidate the transcriptional regulatory michenism controlled by human RAG2 gene 5' proximal region.MethodsBy using CHART-PCR method,the DHSs in RAG2 5 'upstream region were examed and compared among RAG+ T or B cells,RAG-T or B cells,as well as non-lymphoid cell?Using deleting mutation assay,the human RAG2 5' proximal region(-1746bp to ?104bp)were deledted gradually from 5' to comstruct various mutated clones,and then linked into luciferase(Luc)reporter vector to construct reporter gene.The relative Luc activities were examed by transfectd into RAG+T lymphocytes.By using ENCODE database,the T cell specific DHS regions in human and various mammals were analyzed to identify the homologous sequences.By using JASPAR,TRANSFAC,and TESS database,the binding sites for human TFs were predicted.Using EMSA.the specific binding of the TF,existed in nuclear extracts from RAG+T cells,to the DNA sequences was confirmed.Using CHIP,the specific binding of the TF to the in situ gene was confirmed at the chromatin level.Using binding site mutation and TF over expressing assaies,the effects of TF GATA3 on RAG2 reproters were investigated.By using TF dominant negative mutation assay,investigated the direct regulation effect on RAG2 gene.ResultsThe chromatin state in the 5' proximal region of human RAG2 is RAG-expression specific,and the states in T and B cells are distinct.For T cells,the chromatin open region of RAG2 is located within-1bp to upstream-140bp or-256bp region,and that for B cell is located within-1bp to-336 bp region.In RAG+ T cells,the human RAG2 promoter core region located in-129 bp to the transcription initiation site(TSS),and the core region is coincidesd with the T cell-specific DHS.The human T cell-specific TF GATA3 can bind to a highly conserved region within the T cell-specific DHS at the chromatin level.GATA3 exibits a significant positive regulatory effect on the transcriptional activity of the human RAG2 promoter reporter gene.GTAT3 can promote the expression of endogenous RAG2 by binding to the promoter region at the chromatin level.Conclusions1.By using the approach of analyzing RAG-expressing specific chromatin structure changes,the core region of human RAG2 promoter is localized.And it is confirmed that the specific expression of human RAG2 is able to be regulated by the changes of the chromatin open state at the 5' proximal region.2.Taking use of homologous sequence analysis and TF-binding site predicting methods,it is confirmed that at the chromatin level,the human T cell-specific TF GATA3 can bind to a highly conserved sequence within the T cell-specific DHS.3.The binding of GTAT3 with the promoter region in the chromatin,can promote the expression of endogenous RAG2.