Chromatin organizer SATB1 controls the cell identity of CD4+ CD8+ double-positive thymocytes by compacting super-enhancers

CD4+ and CD8+ double-positive (DP) thymocytes are at a critical stage during the T cell development in thymus. DP cells rearrange the T cell receptor gene Tcra to generate T cell receptors with TCR. Then DP cells differentiate into CD4 or CD8 single-positive (SP) thymocytes, Regulatory T cells, or invariant nature kill T cells (iNKT) according to the TCR signal. Chromatin organizer SATB1 is highly expressed in DP cells and plays an essential role in regulating Tcra rearrangement and differentiation of DP cells. Here we explored the mechanism of SATB1 orchestrating gene expression in DP cells. Single-cell RNA sequencing assay of SATB1-deficient thymocytes showed that the cell identity of DP thymocytes was changed, and the genes specifically highly expressed in DP cells were down-regulated. ChIP-seq and ATAC-seq data showed the similar tendency. The super-enhancers regulate the expressions of the DP-specific genes, and the SATB1 deficiency reduced the super-enhancer activity. Hi-C data showed that interactions in super-enhancers and between super-enhancers and promoters decreased in SATB1 deficient thymocytes. We further explored the regulation mechanism of two SATB1-regulating genes, ETS2 and Bcl6, in DP cells and found that the knockout of the super-enhancers of these two genes impaired the development of DP cells. Our research reveals that SATB1 globally regulates super-enhancers of DP cells and promotes the establishment of DP cell identity, which helps understand the role of SATB1 in thymocyte development. scRNA-seq analysis of Satb1+/+ and Satb1-/- thymus;Bulk RNA-seq analysis of Satb1+/+ and Satb1-/- DP thymocytes(triplicates);H3K4me3 and H3K27me3 ChIP-seq analysis of Satb1+/+ and Satb1-/- DP thymocytes(duplicates);ATAC-seq analysis of Satb1+/+ and Satb1-/- DP thymocytes(duplicates);Hi-C analysis of Satb1+/+ and Satb1-/- DP thymocytes(duplicates);3C-HTGTS analysis of Satb1+/+ and Satb1-/- DP thymocytes (duplicates)using different baites(promoters of Bcl6 or Ets2);3C-HTGTS analysis of Bcl6SE +/+ and Bcl6SE-/- thymus at the baits of promoters from Bcl6;4C-seq analysis of Ets2SE +/+ and Ets2SE-/- thymus at the baits of promoters from Ets2(duplicates);5'RACE analysis of TCRA repertoire in Bcl6SE +/+ and Bcl6SE-/- thymus;5'RACE analysis of TCRA repertoire in Ets2SE +/+ and Ets2SE-/- thymus (duplicates).