Fig. 1

Binding of Bcl11b to regulatory regions in the Thpok gene. a DNA pull-down assay showing in vitro Bcl11b binding to wild-type (Wt) Thpok silencer (Sth) core sequences, but not efficiently to mutant (Mut.) sequences. One representative of two experiments. b Co-immune precipitation assay showing interaction of Bcl11b with Runx1. One representative of two experiments. c Flow cytometry showing an increase of CD8⁺ T cells de-repressing Thpok-GFP upon reduction of Bcl11b dosage in Runx mutant mice. One representative of two independent experiments. d Bcl11b ChIP-seq tracks at the Thpok, Thpok ^gfp:ΔTESPE, and Sfpi1 genes in total thymocytes along with Runx ChIP-seq (GSE90794) tracks at the Thpok gene (top) for reference. Gene structure, transcriptional orientation and conservations in mammals (Cons.) in the Thpok gene are indicated. Positions of Thpok silencer (Sth), two enhancers (TE and PE), distal P1-promoter (P1) and proximal enhancer (PE) in the Thpok gene, and upstream regulatory element (URE) in the Sfpi1 gene are indicated as arrowheads. e Co-occupancy by Runx at Bcl11b-bound genome regions. Runx recognition site (5′-ACCPuCA-3′) was listed among the top two common sequences for Bcl11b-bound regions. f ChIP-PCR assay for binding of Runx, Bcl11b, and ThPOK to Wt and Runx site mutated (RSM) Sth regions in CD4⁺ T cells from Thpok ^{+/gfp:241-401RM} mice. Lanes 1, 2, and 3 are input, control IgG and antibody-against the indicated transcription factor, respectively. One representative of two independent experiments. g ChIP-qPCR analyses showing binding of Runx and Bcl11b to the Sth and PE in CD4⁺ (white bars) and CD8⁺ (gray bars) T cells. Combined data from three independent ChIP experiments is shown. **P < 0.01 (unpaired t-test)