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. 2018 Nov 5;215(11):2887–2900. doi: 10.1084/jem.20172021

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© 2018 Malhotra et al.

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Figure 6. — SOX4 regulates Mir181 expression in thymocytes. (A) Mir181a-1 and Mir181b-1 transcripts in DP thymocytes of WT and CKO mice were quantified by real-time PCR. One of three experiments is shown. (B) ChIP analysis for SOX4 docking and epigenetic modifications at the Mir181 locus of DP thymocytes. Each graph shows control Ab (matching the species of origin of experimental Ab) for the protein–chromatin complex precipitation followed by the chromatin states probed with Abs to indicated markers in sorted WT and Sox4 CKO DP thymocytes. The consensus DNA binding motif for SOX4 is located ∼2.3 kb upstream of the transcription start site of Mir181a-1 and denoted on the schematic (right; not to scale), which also shows the region assessed by quantitative PCR (arrows). Active (K4me3), poised (K9me3), and suppressed (K27me3) histone modifications in the region are shown. Statistical significance based on Student’s t test denoted. (C and D) iNKT cell development from Sox4-deficient precursors can be rescued by enforced Mir181a-1 expression. (C) Representative profiles of the rescued iNKT cell development among mature (CD24^neg) thymocytes showing frequencies of iNKT cells from the Mir181a-1 retrovirus transduced (GFP⁺) and nontransduced (GFP⁻) precursors in the same mouse. Transduction studies using WT and CKO BM cells are shown. Representative plots from one of two independent experiments. (D) Summary of thymic iNKT cell frequencies from retroviral reconstitution experiments using infected WT (blank circles) and CKO (filled circles) BM cells. Only mice with >1% GFP⁺ cells among mature thymocytes were included in the calculation. Denoted significance in graphs was based on Student’s t test. Error bars denote SD.