Figure 8: GATA2 SUMOylation inhibits its DNA binding activity.

(a) SUMO conjugation reduces GATA2 DNA binding activity. GATA2-WT, GATA2-2KR, SUMO-GATA2 or control constructs were transfected into 293 T cells, and nuclear extracts were processed with EMSA using a GATA2-specific oligonucleotide probe (top). Input of GATA2 was detected by western blotting with anti-Flag antibody (bottom). (b) Overexpression of catalytic inactive form of SENP1 (SENP1-Mut) inhibits recruitment of GATA2 to the promoter of ICAM-1, VCAM-1 and E-selectin. HUVECs were infected by Ad-SENP1-Mut or Ad-LacZ and then treated with TNF. Nuclear extracts were then subjected to ChIP assay with the anti-GATA2 antibody followed by quantitative real-time PCR for the promoter sequences of ICAM-1, VCAM-1 and E-selectin containing a GATA2 binding site. Quantitative results are shown as the ratio of ChIP to input values. Data are presented as the mean±s.e.m. from three independent experiments. *P<0.05 and **P<0.01 compared with LacZ without TNF treatment (LacZ basal). ^#P<0.05 and ^##P<0.01 compared with LacZ with TNF treatment; two-way ANOVA followed by Bonferroni post-test. (c) GATA2 SUMOylation attenuates its binding activity to the promoter of ICAM-1, VCAM-1, and E-selectin. HUVECs were transfected with GATA2 or control siRNA for 48 h and then infected with various GATA2 viruses or their vector controls as indicated. Cells were treated with TNF, and the nuclear extracts were subjected to the ChIP assay as described. Quantitative results are present as the ratio of ChIP to input values. Data are shown as the mean±s.e.m. from three independent experiments. *P<0.05 compared with GATA2 knockdown. ^#P<0.05 compared with GATA2 knockdown with restored GATA2-WT; two-way ANOVA followed by Bonferroni post-test.