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. 2020 May 11;48(11):5873–5890. doi: 10.1093/nar/gkaa321

Figure 6.

Figure 6.

TCF1 directly regulates the SATB1 expression in CD4SP thymocytes during T-cell development. (A) A TCF1/LEF1 canonical DNA-binding motif was identified in the P2 promoter region of Satb1 using MEME-Suite (Version 5.1.1, http://meme-suite.org/tools/meme). (B) TCF1 ChIP was performed in sorted DP and CD4SP thymocytes, followed by qRT-PCR analysis using primers spanning the TCF1/LEF1 binding site on P2 promoter. Two different antibody clones were used (depicted as #1 and #2 in the figure axis) to assess the antibody-factor specificity. Different control regions were used to ascertain whether TCF1 specifically binds to the Satb1 promoter regions, labeled C1 and C2, at 1000 and 1500 bp upstream of P2, respectively. (C) qRT-PCR analysis of expression profile of total Satb1, P1, P2, P3 and P4 variants in CD4SP thymocytes along with other sorted T-cell developmental stages from wild-type and TCF1 KO mice. Hprt gene expression was used as endogenous control. The represented data are from three independent experiments and is shown as means of ± SEM. P-values were calculated using one-way ANOVA or. *P < 0.05; **P < 0.01; ***P < 0.001. (D) Total thymocytes were isolated from either WT or TCF1 KO mice and stained with anti-CD4, anti-CD8 and anti-SATB1 antibodies. FACS analyses were performed using BD FACS canto II with the indicated gating. (E) Flow cytometry analysis shows the expression of SATB1 in thymocyte developmental stages; DN, DP, CDSP and CD8SP from wild-type and TCF1 KO mice as indicated by black and red color curved lines respectively. Solid gray peaks indicate the staining controls. (F) Satb1 P2 promoter was cloned in pGL3 basic vector and subjected to dual luciferase activity assay in the background of TCF1 overexpression in HEK293 cells. Renilla was used as an endogenous control. The represented data are from three independent experiments and is shown as means of ± SEM. P-values were calculated using the unpaired two-tailed Student's t-test. *P < 0.05. (G) A schematic representation depicting how TCF1 acts upstream of SATB1 during differentiation of CD4SP from DP in thymus.