1 Inst. for Life and Med. Sci., Kyoto Univ.Two E-boxes in the Rag1-promoter is essential for the V(D)J recombination of Tcr and Igh genes.37Adaptive immune cells, such as T and B cells, evoke antigen (Ag)-specific responses by recognizing specific antigens. This Ag-specific recognition relies on the V(D)J recombination of T cell receptor (TCR) and immunoglobulin (Ig) genes, which is mediated by recombination-activating gene (Rag) 1 and 2 (Rag1/2) protein complex. The Rag1/2 gene expression is restricted to the developing T and B cell progenitors/precursors and is a hallmark of adaptive lymphocyte lineages. We previously identified several T- or B-cell-specific enhancers for Rag1/2 gene expression, which are controlled by the transcription factor (TF) E2A. Recruitment of E2A to the enhancer induces super-enhancer formation in the Rag gene locus through the regulation of histone acetylation and chromatin accessibility in a cell-type-specific manner. E2A also binds to the Rag1 promoter (R1pro) region to initiate Rag1 expression, however, it remains to be elucidated how E2A regulates the enhancer-promoter interaction through its binding to both regions. To address this, we generated E-box mutant mice, in which seven E-box motifs in R1pro were mutated to disrupt E2A binding (R1pro-mut). Surprisingly, R1pro-mut mice exhibited severely reduced Rag1 expression and developmental blocks at the pro-B and pro-T cell stages, due to failure in V(D)J recombination of TCR and Ig genes. Unlike enhancer deletions, mutations in R1pro did not alter chromatin accessibility across the Rag gene locus except the R1pro, and the three-dimensional (3D) structure in the entire Rag1/2 gene locus was maintained. These findings demonstrate that E2A differentially regulates enhancer and promoter functions: E2A orchestrates chromatin interactions and maintains their accessibility via enhancers, while initiating Rag1 transcription via the promoter. Finally, we identified two highly conserved E-box motifs among the seven sites in mammalian species, and reintroducing these two conserved E-boxes restored T and B cell development in the R1pro-mut mice, highlighting their essential role in Rag1 promoter activity. Overall our results reveal distinct yet complementary roles of enhancer and promoter elements in Rag gene regulation, both controlled by the same lineage-specific TF. ○Reiko Hidaka1 Kazuko Miyazaki1 Hiroshi Kawamoto1 Masaki Miyazaki1P 007
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