Figure 3. The Thpok BTB domain binds NuRD and is needed for CD4⁺ T cell development.

(A) Schematic diagram of Thpok and mutants thereof. The Thpok BTB domain and zinc finger motifs are depicted by red and purple boxes; the Leucine Zipper sequence (LZ) by the orange box. *: R389G mutation in the Thpok^HD mutant (22).

(B) Immunoblot analysis of anti-Flag immunoprecipitates (left panel) or whole cell lysates (right panel) from HEK293T cells transfected with control vector (pcDNA3, Ctrl), Flag-tagged Thpok, Thpok-HD, ΔBTB_LZ or Thpok-ΔC. Protein blots were probed with antibodies as indicated. Data are representative of more than three independent experiments.

(C) Expression of CD4 and CD8 in indicated thymocyte and splenocyte subsets from Thpok^fl/fl Cd4-Cre⁺ mice expressing the indicated Thpok-derived transgene or no transgene (−).

(D) Numbers of CD44^lo TCRβ^hi CD24^lo CD4⁺ SP thymocytes (top) and of CD44^lo TCRβ^hi CD4⁺ splenocytes (bottom) in mice shown in (C).

(E) Expression of transgenic Thpok or ΔBTB_LZ in CD4⁺ CD8^int TCRβ^hi CD69⁺ thymocytes was assessed by intra-cellular staining and flow cytometry and is presented relative to that of Thpok-expressing CD4⁺ CD8^int TCRβ^hi CD69⁺ thymocytes in Thpok^fl/fl Cd4-Cre⁻ littermates (Ctrl), set as 1 in each experiment.

(F) Overlaid histograms show expression of transgenic ΔBTB_LZ in TCRβ^hi CD24^lo CD4⁻CD8⁺ thymocytes from Thpok^fl/fl Cd4-Cre⁺ ΔBTB_LZ⁺ mice (plain line) and of endogenous Thpok in CD4⁺ or CD8⁺ SP thymocytes from Thpok^fl/fl Cd4-Cre⁻ (Ctrl) littermates (dotted line and grey-shaded trace, respectively).

Data (C-F) are representative of 4 independent experiments totaling n= 7 (Thpok^fl/fl Cd4-Cre⁻), 12 (Thpok^fl/fl Cd4-Cre⁺), 8 (Thpok^fl/fl Cd4-Cre⁺ Thpok transgenic) or 14 (Thpok^fl/fl Cd4-Cre⁺ ΔBTB_LZ⁺) mice. One-way ANOVA followed with Tukey multiple comparison tests. ***p<0.001, ****p<0.0001. Error bars indicate standard deviation.