Fig 2: 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 3: P300/CBP is required for CIC-DUX4 activity.A Western blots for P300 and CBP in NCC-CDS-X1 cells after 96 h transfection with siRNAs for P300 and CBP. B ATP assay for cell viability of NCC-CDS-X1 at 72 and 96 h of post-transfection with P300 and/or CBP siRNA. Non-treated cells and cells transfected with non-targeting siRNA (scramble) were used as negative controls. Data are presented as mean ± SEM; *p < 0.05, ***p < 0.001 by one-way ANOVA. Results are presented as relative expression to control (n = 4). C ATP viability assay in the Kitra-SRS cell line in the same experimental conditions as explained in “B”. Data are presented as mean ± SEM; ****p < 0.0001, by two-way ANOVA. Results are presented as expression relative to control (n = 4). D RT-qPCR for P300, CBP, and CIC-DUX4 target genes in NCC-CDS-X1. Analyses were performed 48 h post-transfection with P300 and/or CBP siRNA. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA. Results are presented as expression relative to B2M (n = 3).

Fig 4: Activities of 1,25(OH)2D3and FGF23 at Cyp24a1. Gene expression of Cyp24a1 after time course with (A) 10 mg/kg bw 1,25(OH)2D3 at 0, 1, 3, 6, 12, and 24 h (n = 4) and (B) 50 mg/kg bw FGF23 at 0, 3, 6, and 12 h (n = 3–4) from our previous publication (10). ChIP-Seq analysis near Cyp24a1 for (C) VDR and RXR (1,25(OH)2D3versus Veh only), (D) pCREB, (E) CBP, (F) CRTC2, (G) H3K27ac, and (H) H3K9ac from WT mice injected with 10 mg/kg bw 1,25(OH)2D3 or 50 mg/kg bw FGF23 (1 h). Additional details as for Figure 2. Genomic region displayed is chr2: 170,278,379 to 170,324,235. bw, body weight; CBP, CREB-binding protein; ChIP-Seq, chromatin immunoprecipitation sequencing; CRTC2, CREB-regulated transcription coactivator 2; FGF23, fibroblast growth factor 23; H3K9ac, histone acetylation at histone H3 lysine 9; H3K27ac, histone acetylation at histone H3 lysine 27; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; pCREB, phosphorylated (p-133) CREB; RXR, retinoid X receptor; VDR, vitamin D receptor.

Fig 5: Chromatin Accessibility Is Independent of CBP/p300 Levels in mESCs. a, c ATAC-seq levels at H3K27ac-enriched regions in mESCs expressing scramble control (Ctl) shRNA compared with a p300 shRNA (p300 KD) or c CBP shRNA (CBP KD). Blue and red dots represent regions with differential ATAC-seq signal at which read density increased or decreased by 2-fold or more (FDR < 0.05), respectively, in two independent biological replicates. b, d Fold-change ratio (log2) of ATAC-seq enrichment at promoters and enhancers in mESCs expressing scramble control (Ctl) shRNA compared with b p300 shRNA (p300 KD) or d CBP shRNA (CBP KD). Data to the right of log2(0) = 1 indicate regions with increased ATAC-seq signal after acetyltransferase knockdown, while data to the left indicate regions with reduced ATAC-seq signal