Figure 5: H3 ubiquitination specifically recruits GCN5 for H3 acetylation.

(a) H3.3 knockdown impaired IL1α, IL1β and GCLM expression. qPCR was performed to analyse the mRNA level in control and H3.3 knockdown Hep3B cells (n=3, mean±s.e.m.). (b) H3 ubiquitination deficiency impaired IL1α, IL1β and GCLM expression. qPCR was performed to analyse the mRNA level in vector, H3.3 WT and H3.3 K23/36/37R mutant restored H3.3 knockdown Hep3B cells (n=3, mean±s.e.m.). (c) GCN5 is predicted as one of the potential regulator for NEDD4 target genes. Top 5,000 upregulated genes from microarray data sets in Fig. 4d were included to predict potential regulators by using iRegulon software. See experimental procedures for details. (d) GCN5 knockdown impaired IL1α, IL1β and GCLM expression. qPCR was performed to analyse the mRNA level in control and GCN5 knockdown cells (n=3, mean±s.e.m.). (e) GCN5 knockdown abolished glucose-induced H3 K9, K14, K27 and K56 acetylation. Control and GCN5 knockdown Hep3B cells were glucose-starved for 4 h and added-back glucose for 2 h before whole-cell extraction for western blot analysis. (f,g) NEDD4 knockdown impaired the interaction between GCN5 and H3.3. Transfected Flag-H3.3 or Flag-GCN5 in Hep3B cells was immunoprecipitated to analyse its co-immunoprecipitates by western blotting. (h) H3 ubiquitination deficiency impaired the interaction between GCN5 and H3.3. Hep3B cells were transfected with Flag-H3.3 WT or K23/36/37R for 36 h, then glucose starved for 4 h and added-back glucose for 2 h. Cells were then lysed for co-immunoprecipitation assay using anti-Flag antibody and subsequent western blot analysis. (i) GCN5 and ubiquitinated H3 form complex in vivo. 293T cells were transfected with Flag-GCN5 and His-Ub as indicated. Briefly, sequential purification is done by first IP with Flag antibody from whole-cell extracts in RIPA buffer. Immunoprecipitates were then released from antibody/beads by buffer A and followed by in vivo ubiquitination assay for endogenous H3. ChIP-qPCR data were all presented as relative enrichment to 5% input and 0.05 should be multiplied to calculate the actual enrichment percentage. All asterisks (*) represent P<0.05, using Student’s T-test.