Figure 2

SUMOylation of Zbtb21 affects its transcriptional activity.

(A–C) Luciferase reporter assays in HEK293T cells expressing HA-tagged wild type (WT), Zbtb21^K419R, Zbtb21^K845R, and Zbtb21^K419/845R mutant plasmids on CDC6, zbtb14, and pu.1 reporters. Bars showed the relative luciferase activity. Luciferase activity was measured after 48 h and normalized to Renilla luciferase (one-way ANOVA, N = 5–6. Error bars represent mean ± SEM. ns: not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001). Specific P-values are provided in Table 2. (D) Western blot analysis showed equal amount of wild type Zbtb21 and Zbtb21^K419/845R mutant was expressed in HEK293T cells. TUBULIN serves as internal control. (E) Comparison of the differentially expressed genes (DEGs) in HEK293T cells transfected with control, wild type Zbtb21 and Zbtb21^K419/845R mutant plasmids, respectively. (F) ChIP analysis showed that while the regulatory regions of 2041 genes were bound by wild type Zbtb21, those of 845 genes were bound by Zbtb21^K419/845R mutant. (G) Numbers of upregulated and downregulated genes whose regulatory regions were bound by either wild type Zbtb21 or Zbtb21^K419/845R mutant. (H) Co-IP experiments conducted in HEK293T cells expressing HA-tagged Zbtb21 and Zbtb21^K419/845R. Lysates were immunoprecipitated using anti-HA agarose beads, and analyzed by Western blots with anti-HDAC4 and anti-BRD4 antibodies.