FIGURE SUMMARY
Title

SUMOylation of zebrafish transcription factor Zbtb21 affects its transcription activity

Authors
Fang, Z., Deng, Y., Wang, H., Zhou, J.
Source
Full text @ Peer J.
Figure 1

Zbtb21 is identified as a SUMOylated substrate.

(A) Western blot analyses (anti-HA) of HA-tagged wild type (WT), Zbtb21K419R, Zbtb21K419R, Zbtb21K845R, and Zbtb21K419/845R mutant proteins expressed in HEK293T cells in the absence or presence with the SUMO conjugating enzyme UBC9 and SUMO1. TUBULIN served as internal control. (B) HA-tagged wild type (WT), Zbtb21K419R, Zbtb21K419R, Zbtb21K845R, and Zbtb21K419/845R mutant proteins were immunoprecipitated with an anti-HA antibody from HEK293T cells co-expressing SUMO1, and SUMOylated Zbtb21 protein was detected by western blot with an anti-SUMO1 antibody.
Figure 2

SUMOylation of Zbtb21 affects its transcriptional activity.

(A–C) Luciferase reporter assays in HEK293T cells expressing HA-tagged wild type (WT), Zbtb21K419R, Zbtb21K845R, and Zbtb21K419/845R 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 Zbtb21K419/845R 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 Zbtb21K419/845R 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 Zbtb21K419/845R mutant. (G) Numbers of upregulated and downregulated genes whose regulatory regions were bound by either wild type Zbtb21 or Zbtb21K419/845R mutant. (H) Co-IP experiments conducted in HEK293T cells expressing HA-tagged Zbtb21 and Zbtb21K419/845R. Lysates were immunoprecipitated using anti-HA agarose beads, and analyzed by Western blots with anti-HDAC4 and anti-BRD4 antibodies.
Figure 3

SUMOylation of Zbtb21 is irrelevant to its subcellular localization.

Immunofluorescence analyses of HEK293T cells expressing HA-tagged WT, Zbtb21K419R, Zbtb21K845R, and Zbtb21K419/845R mutant proteins. Cells were fixed, and immunofluorescent microscopy was performed using rabbit anti-HA antibody and Alexa Fluor 488 goat anti-rabbit IgG (left). Cell nuclei were stained with DAPI (middle). Digitally merged images are shown on the right side.
Figure 4

SUMOylation of Zbtb21 does not affect the hetero-dimerization with Zbtb14 or homo-dimerization with Zbtb21.

(A) Co-IP experiments conducted in HEK293T cell transfected with HA-tagged Zbtb14 and Flag-tagged Zbtb21. Lysates were immunoprecipitated using anti-Flag agarose beads, and analyzed by Western blots with anti-HA antibody. (B) Co-IP experiments conducted in HEK293T cell transfected with HA-tagged Zbtb21 and Flag-tagged Zbtb21 constructs. Flag antibody was applied for immunoprecipitation and HA antibody was used for western blot analysis.
Acknowledgments
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