The transcription factor E2A activates multiple enhancers that drive Rag expression in developing T and B cells

Abstract

Cell type–specific gene expression is driven by the interplay between lineage-specific transcription factors and cis-regulatory elements to which they bind. Adaptive immunity relies on RAG-mediated assembly of T cell receptor (TCR) and immunoglobulin (Ig) genes. Although Rag1 and Rag2 expression is largely restricted to adaptive lymphoid lineage cells, it remains unclear how Rag gene expression is regulated in a cell lineage–specific manner. Here, we identified three distinct cis-regulatory elements, a T cell lineage–specific enhancer (R-TEn) and the two B cell–specific elements, R1B and R2B. By generating mice lacking either R-TEn or R1B and R2B, we demonstrate that these distinct sets of regulatory elements drive the expression of Rag genes in developing T and B cells. What these elements have in common is their ability to bind the transcription factor E2A. By generating a mouse strain that carries a mutation within the E2A binding site of R-TEn, we demonstrate that recruitment of E2A to this site is essential for orchestrating changes in chromatin conformation that drive expression of Rag genes in T cells. By mapping cis-regulatory elements and generating multiple mouse strains lacking distinct enhancer elements, we demonstrate expression of Rag genes in developing T and B cells to be driven by distinct sets of E2A-dependent cis-regulatory modules.

The Interplay Between Chromatin Architecture and Lineage-Specific Transcription Factors and the Regulation of Rag Gene Expression

“…Consistent with this finding, the Rag gene locus and its enhancers show elaborate chromatin interactions and are located in the active developing T cell-and B cell-specific sub-TAD (52). Furthermore, R-TEn deletion in mice specifically blocked thymocyte development during the b-selection of DN3 cells and positive-selection of DP cells, as seen in ASE deletion mice (52,63). In addition, mice with R1B/R2B double deletions exhibited a severe developmental block at the pro-B stage, whereas the single deletion of either R1B or R2B led to mild or moderate impairments in B cell development.…”

“…In addition, mice with R1B/R2B double deletions exhibited a severe developmental block at the pro-B stage, whereas the single deletion of either R1B or R2B led to mild or moderate impairments in B cell development. This outcome is comparable to that observed in Erag deficient mice, suggesting enhancer redundancy in R1B and R2B (7,52,58). These developmental defects in enhancer-deleted mouse lines were caused by failed Rag1/2 gene expression and TCRab or IgH recombination.…”

“…In addition to E2A, T cell-or B cell-specific TFs (T cell: Bcl11b, Tcf1, Runx1, Ikaros, and Gata3; B cell: Pax5, Ets1, Ikaros, and Irf4) bind to these enhancer regions ( Figure 3A). Furthermore, the R2B and Rag1 gene promoter region (R1pro) in pre-B cells, and R-TEn along with the Rag1/2 gene cluster in DP cells are SEs according to an analysis of histone acetylation (H3K27Ac) (52). Consistent with this finding, the Rag gene locus and its enhancers show elaborate chromatin interactions and are located in the active developing T cell-and B cell-specific sub-TAD (52).…”

“…Together, the ASE and Rag1 promoter framework functions as a chromatin hub (65). Recently, we have identified cell type-specific CREs for Rag1/2 expression in B and T cells using E2A ChIP-seq and ATAC-seq data, because E2A specifies adaptive lymphocyte and is the driver of differences in enhancer repertoires in T cells and ILCs (24,52). We mapped E2A binding to one T cell-specific Rag gene enhancer [Rag-T cell enhancer (R-TEn) included in the 8-kb ASE] and to two B cell-specific Rag gene enhancers [Rag B cell enhancer 1/2; R1B (5-kb upstream of the Rag1 promoter) and R2B (partially overlapping with Erag)] ( Figure 3A) (52).…”

“…Furthermore, the R2B and Rag1 gene promoter region (R1pro) in pre-B cells, and R-TEn along with the Rag1/2 gene cluster in DP cells are SEs according to an analysis of histone acetylation (H3K27Ac) (52). Consistent with this finding, the Rag gene locus and its enhancers show elaborate chromatin interactions and are located in the active developing T cell-and B cell-specific sub-TAD (52). Furthermore, R-TEn deletion in mice specifically blocked thymocyte development during the b-selection of DN3 cells and positive-selection of DP cells, as seen in ASE deletion mice (52,63).…”