Figure 2: Glucose-induced H3 ubiquitination by NEDD4 is required for H3 K9/K14 acetylation.

(a) NEDD4 knockdown abolished glucose-induced H3 K9, K14, K27 and K56 acetylation. Control and NEDD4 knockdown Hep3B cells were glucose starved for 4 h and added-back glucose for 3 h before whole-cell extraction for western blot analysis (see experimental procedures for details). A.E., acid extraction. (b) H3.3 knockdown abolished H3 K9, K14, K27 and K56 acetylation. Control and H3.3 knockdown Hep3B cells were lysed for western blot analysis. (c) H3 K23R and K36/37R mutant abolished glucose-induced H3 ubiquitination. Hep3B cells expressing various Flag-H3.3 constructs were glucose-starved for 4 h and added-back glucose for 2 h before chromatin fractionation assay. Ubiquitination levels were normalized to input (n=5, mean±s.e.m.). NS, nonspecific band. (d) H3 K23 and K36/37R mutant abolished NEDD4 overexpression induced H3 ubiquitination. 293T cells were transfected with his-ubiquitin, NEDD4 plasmids and various Flag-H3.3 constructs for 36 h before in vivo ubiquitination assay. Ubiquitination levels were normalized to input. (e,f) H3 K23/36/37R is defect in H3 K9/K14 acetylation. WT or K23/36/37R Flag-H3.3 was stably expressed in Hep3B cells and immunoprecipitated for western blot analysis. (g) NEDD4 knockdown Hep3B cells transfected with WT, Y43/585E or Y43/585F mutant were treated with glucose and harvested for western blot analysis.