Figure 1. p300 Catalyzes Histone Khib.
(A) p300 deficiency impairs both Khib and Kac of histones in cells. Histone Khib and Kac levels were analyzed in WT and p300 KO HCT116 cells by immuno-blotting with indicated antibodies.
(B) Overexpression of WT but not a mutant p300 increases both Khib and Kac levels on histones. HEK293T cells were transfected with either empty vector (V), or constructs expressing Flag-p300 (WT) or Flag-p300D1399Y (Mut) for 48 hours. The indicated histone modification marks were analyzed by immuno-blotting.
(C) Overexpression of WT p300 enhances both Khib and Kac of histones in U2OS cells. U2OS cells transfected with WT p300 were immuno-stained with anti-H4K8hib and anti-H3K9ac antibodies, respectively. Please note that cells overexpressing FLAG-p300 have higher levels of H4K8hib (left) and H3K9ac (right) than untransfected cells. Bars, 50 μm.
(D) Inhibition of p300 dose-dependently reduces Khib and Kac levels of histones in cells. HCT116 and HEK293T cells were treated with a p300 inhibitor A485 for 24 hours, and the Khib and Kac levels of histones were analyzed by immuno-blotting with indicated antibodies.
(E) p300 2-hydroxyisobutyrates histone octamers in actively transcribed chromatin in vitro. p300-dependent in vitro transcription system was set up as described in STAR Methods, and the Khib and Kac levels of histones were analyzed by immuno-blotting with indicated antibodies.
(F) p300-mediated histone 2-hydroxyisobutyration activates p53-dependent transcription in vitro. p300-dependent in vitro transcription system was set up using WT histones and K-R mutant histones. RNA products were visualized by autoradiography.
See also Figure S1.