Figure 5. A NuRD-interacting segment of the Thpok BTB domain is needed for CD4 T cell development.

(A) Schematic diagram of Thpok-SVIN (orange line box) and Thpok-RKF (cyan line box) mutations.

(B) Immunoblot analysis of anti-HA immunoprecipitates (left) or whole cell lysates (right) from HEK293T cells transfected with control vector (pcDNA3, Ctrl), HA-tagged Thpok, ΔBTB_LZ, Thpok-SVIN or Thpok-RKF. Protein blots were probed with antibodies as indicated. * indicates non-specific bands. Data are representative of three independent experiments.

(C) Venn diagram showing Thpok-RKF binding partners and Thpok 35-set as detected by mass spectrometry after streptavidin pull down in activated CD4⁺ T cells. Proteins binding Thpok but not Thpok-RKF complex are listed in the box.

(D) ChIP-qPCR was performed on Thpok and Cd4 silencer elements, and on a region of Pax5 (negative control), from in vitro activated CD4⁺ T cells from Thpok^fl/fl Ox40-Cre⁺ Rosa26^BirA+ that had been retrovirally transduced with empty pMRx (Ctrl), or pMRx expressing Thpok-Bio or Thpok-RKF-Bio. Data are expressed as percent of input DNA. Each symbol represents a separate determination, and the figure summarizes three independent experiments.

(E) Expression of CD4 and CD8 in indicated thymocyte and splenocyte subsets from indicated mice.

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

(G) Flow cytometric expression of transgenic Thpok-RKF in CD4⁺ CD8^int TCRβ^hi CD69⁺ thymocytes, shown relative to that of Thpok-expressing CD4⁺ CD8^int TCRβ^hi CD69⁺ in Thpok^fl/fl Cd4-Cre⁻ littermates (Ctrl), set as 1 in each experiment.

(H) Overlaid histograms show expression of transgenic Thpok-RKF in CD24^lo TCRβ^hi CD4⁻ CD8⁺ thymocytes from Thpok^fl/fl Cd4-Cre⁺ Thpok-RKF⁺ 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 (E-H) are representative of three independent experiments totaling n= 6 (Thpok^fl/fl Cd4-Cre⁻ littermates), 6 (Thpok^fl/fl Cd4-Cre⁺) or 8 (Thpok^fl/fl Cd4-Cre⁺ Thpok-RKF⁺) mice. One-way ANOVA followed with Tukey multiple comparison tests ****p<0.0001. Error bars indicate standard deviation.

(I) Expression of CD4 and CD8 in indicated thymocyte and splenocyte subsets from Thpok^fl/fl B2m^−/− mice expressing or not the Thpok-RKF transgene.

(J) Numbers of CD44^lo CD24^lo TCRβ^hi thymocytes, CD4⁺ CD8⁻ (top left) or CD8⁺ (including CD4⁺ and CD4⁻, top right) and of CD44^lo TCRβ⁺ CD4⁺ CD8⁻ splenocytes (bottom) in mice shown in (I).

Data (I-J) are representative three independent experiments totaling n= 6 (Cd4-Cre⁻ littermates), 8 (Cd4-Cre⁺) or 10 (Cd4-Cre⁺ Thpok-RKF⁺) mice. One-way ANOVA followed with Tukey multiple comparison tests ****p<0.0001, ns: P>0.05. Error bars indicate standard deviation.