SATB1 Condensates Reinforce Super-Enhancer Architecture to Lower the Threshold of Thymocyte Positive Selection

The generation of mature CD4? helper and CD8? cytotoxic T cells from double-positive (DP) thymocytes is a central step in building an effective adaptive immune system. This process relies on positive selection, whereby aßTCR? thymocytes are tested for recognition of self-peptide–MHC complexes presented by cortical thymic epithelial cells. Thymocytes with insufficient signaling die by neglect, while those receiving appropriately tuned signals survive and differentiate. Positive selection thus plays a dual role: establishing MHC restriction and directing lineage commitment into the CD4 or CD8 fate. After TCR rearrangement is completed, T cells can interact with APCs to transmit TCR signals and produce CD69 protein. SO, CD69+DP cells is undergoing positive selection. Here, we explored the mechanism of SATB1 regulating thymocyte positive selection. Firstly, We analysis the function of SATB1 in the transtion of CD69-DP to CD69+DP by CUT&Tag, ATAC and HiC assays. Then, Single-cell RNA sequencing assay of SATB1-deficient thymocytes showed that the cell identity of CD69+DP thymocytes was changed, and the genes differently expressed in CD69+DP cells. Smart-seq2 showed the similar tendency. CUT&Tag , ATAC-seq and HiC data show the mechanism for SATB1 in T cell positive selection or before this stage. ACC-seq confirms that SATB1 forms phase separation at these sites and regulates gene expression, These data helps us to understand the role of SATB1 in thymocyte positive selection. Overall design: scRNA-seq analysis of Satb1+/+ and Satb1-/- CD69+DP thymocytes(2 replicates); Bulk Smart-seq2 analysis of Satb1+/+ and Satb1-/- for CD69-DP and CD69+DP thymocytes(4 replicates); SATB1, CTCF, RAD21, H3K4me3 and H3K27ac CUT&Tag analysis of Satb1+/+ and Satb1-/- for CD69-DP and CD69+DP thymocytes(3 replicates); ATAC-seq analysis of Satb1+/+ and Satb1-/- for CD69-DP and CD69+DP thymocytes(4 replicates);Hi-C analysis of Satb1+/+ and Satb1-/- CD69-DP and CD69+DP for thymocytes(4 replicates); ACC-seq analysis of Satb1+/+ and Satb1-/- for thymocytes(2 replicates).