Fig 2: Identify the main acetylation sites of HSD17B10.a Flag-tagged HSD17B10 was co-transfected with HA-tagged CBP, and cells were treated with 10 mM NAM for 6 h. Whole-cell lysate was collected, IPed with M2 beads and eluted by Flag peptide. After SDS-PAGE gel separating and Coomassie blue staining, the specific band for HSD17B10 was analyzed by mass spectrometry. b HSD17B10 GST N-terminal (NT), middle (M), C-terminus (CT) and full-length (FL) plasmids were constructed, and in vitro acetylation assay was conducted. GST protein was used as a negative control. c HSD17B10 amino acid sequence conservation was analyzed via sequence analyzing software MEGA. d HSD17B10 KR mutant (mimicking the deacetylation status) plasmids were constructed. Flag-tagged HSD17B10 WT and KR mutants were individually transfected and cells were treated with 10 mM NAM for 6 h. Relative HSD17B10 acetylation ratios were calculated after normalizing against Flag.

Fig 3: Acute inhibition of p300/CBP activity in skeletal muscle hinders insulin sensitivity independent of Akt activity.Schematic for (A) “Pre,” (D) “Concurrent,” and (G) “Post” C646 treatment experiments; 50 μM C646 was used for all experiments. Basal 2DOGU, insulin (0.36 nmol/L) 2DOGU, and insulin-stimulated 2DOGU (calculated as insulin 2DOGU – basal 2DOGU) in isolated soleus and EDL muscles from WT mice in (B and C) “Pre,” (E and F) “Concurrent,” and (H and I) “Post” experiments. (J–L) Representative images for pAktT308, pAktS473, total Akt, pGSK3bS9, and total GSK3b in basal and insulin-stimulated (- and +, respectively) EDL muscles from DMSO and C646 treatment groups for (J) “Pre,” (K) “Concurrent,” and (L) “Post” experiments; values are presented relative to DMSO-insulin. *, P < 0.05, 2-way ANOVA with Sidak’s multiple comparison versus basal within treatment. #, P < 0.05 versus DMSO within insulin. For I-Stim, ‡, P < 0.05, 2-way ANOVA, main effect of diet. ^, P > 0.05, 1-sample t test versus “0.” DMSO/C646 n = 8/8. Data reported as mean ± SEM.

Fig 4: p300 and CBP are required for skeletal muscle insulin-stimulated glucose uptake.Male PCKO mice were assessed at 1 (D1), 3 (D3), or 5 (D5) days after initiating tamoxifen. (A–C) Blood glucose concentrations and area under the curve (AUC; inset) for male PCKO mice at D1, D3, and D5 during an oral glucose tolerance test (OGTT; 2 g/kg); D1: WT/PCKO n = 5/9, D3: WT/PCKO n = 6/10, D5: WT/PCKO n = 6/11. *, P < 0.05 2-way ANOVA repeated measures, PCKO versus WT within a time point for OGTT, and *, P < 0.05 t test for AUC. (D–F) Basal 2-deoxy-glucose uptake (2DOGU), insulin (0.36 nmol/L) 2DOGU, and (G–I) insulin-stimulated 2DOGU (I-Stim.; calculated as insulin 2DOGU – basal 2DOGU) in isolated soleus and extensor digitorum longus (EDL) muscles from male WT and PCKO mice at D1, D3, and D5; D1: WT/PCKO n = 11/10, D3: WT/PCKO n = 6/7, D5: WT/PCKO n = 7/10. *, P < 0.05 2-way ANOVA with Sidak’s multiple comparison versus basal within genotype (D–F) and #, P < 0.05 versus WT within basal or insulin (G–I). *, P < 0.05 t test for I-Stim. ^, P > 0.05 1-sample t test versus “0”. (J) Representative images for phospho-AktT308 (pAktT308), phospho-AktS473 (pAktS473), total Akt, phospho-GSK3bS9 (pGSK3bS9), and total GSK3b in basal and insulin-stimulated (- and +, respectively) EDL muscles from WT and PCKO mice at D1, D3, and D5. Quantification of (K) pAktT308, (L) pAktS473, and (M) pGSK3bS9 compared with total respective protein abundance in the EDL muscle; WT/D1/D3/D5 n = 15/5/6/11. Values are presented relative to WT-insulin. *, P < 0.05 2-way ANOVA, main effect of insulin. Data reported as mean ± SEM. For Western blots, there were no significant differences between WT mice at the different time points (D1, D3, and D5); therefore WT data were collapsed.

Fig 5: CBP-mediated DOT1L-K358 acetylation confers DOT1L stability in MLLr-AML.A, B The cells were transduced with lentivirus expressing control shRNA (shSCR) or shRNA targeting CBP (shATM) along with GFP. The cells were subjected to flow cytometry (same gating strategy as in Supplementary Fig. S1C) to analyse the proportion of GFP+ cells among the MOLM-13 (A) and MV4-11 (B) cells. The analysis was performed every two days during the culture. Three replicates for each time point for different cells were used for linear regression analysis. The difference in slopes between every two groups was compared. ***P < 0.001. Three independent experiments were performed. The total lysates of the control (shSCR) and CBP knockdown (shCBP) MOLM-13 (C) and MV4-11 (D) cells were subjected to western blotting with the indicated antibodies. Control (shSCR) and CBP knockdown (shCBP) MOLM-13 (E) and MV4-11 (F) cells were incubated with 25 μM MG132 for 4 h. The cell lysates were subjected to immunoprecipitation (IP) with DOT1L antibodies. The input and IP samples were then detected by western blotting with the indicated antibodies. G HA-DOT1L with or without Flag-CBP were cotransfected into 293T cells. The cell lysates were then subjected to western blotting with the indicated antibodies. H 293T cells were cotransfected with HA-DOT1L and a control vector (Flag-Vec) or Flag-CBP. At 36 h posttransfection, the cells were treated with 25 μM MG132 for 20 h. The cell lysates were subjected to immunoprecipitation (IP) with HA antibodies. The input and IP samples were then detected by western blotting with the indicated antibodies. I, J MOLM-13 (G) and MV4-11 (H) cell lysates were subjected to IP with CBP, DOT1L, or control IgG. The input and IP samples were then detected by western blotting with the indicated antibodies. MOLM-13 (K) and MV4-11 (L) cells were transduced with lentivirus expressing HA-DOT1L (K358Q) or not (vector). The cells were then transduced with lentivirus expressing shRNA along with green fluorescent protein (GFP). The cells were subjected to flow cytometry (See Supplementary Fig. S1C for gating) to analyse the proportion of GFP+ cells. The analysis was performed every two days during the culture. Three replicates for each time point for different cells were used for linear regression analysis. The difference in slopes between every two groups was compared. ns, not significant. *P < 0.05, ***P < 0.001. Three independent experiments were performed.