Figure 3.

KLF8 sumoylation is negatively regulated by histone acetyltransferases. (A) PCAF or HDAC inhibitors can inhibit KLF8 sumoylation. HEK293 cells were co-transfected with expression vectors coding for HA-KLF8, Myc-SUMO-1, or PCAF, and treated with HDAC inhibitors (trichostatin A, sodium butyrate, and nicotinamide) as indicated. Lysates were collected with NP-40 lysis buffer supplemented with 20 μM of the desumoylase inhibitor NEM followed by immunoprecipitation (IP) using an anti-HA antibody, and western blotting (IB) with either anti-Myc or anti-HA. Shown is a representative experiment where n=3. (B) Co-activators inhibit sumoylation. HEK293 cells were transfected with expression vectors coding for HA-KLF8, Myc-SUMO-1, PCAF, p300, CBP, or the HAT deficient mutants of PCAF, p300, or CBP as indicated. Lysates were collected with NP-40 lysis buffer supplemented with 20 μM of the desumoylase inhibitor NEM and immunoprecipitation experiments were performed using an anti-HA antibody where eluates were western blotted (IB) with either anti-Myc or anti-HA. Bottom, the relative expression of Myc-SUMO-1 as compared to HA-KLF8 is depicted. Results represent mean ±S.E. where n=4. *, p<0.05 compared to KLF8+SUMO-1 (column 3). Expression of the transfected constructs was verified by whole cell lysate blotting (data not shown).