Fig 2: Acetylation of TULP3 by p300 increases its protein abundance in cells. A, western blot showing TULP3 acetylation levels in 293T cells in the presence of either empty pcDNA 3.1(+), Myc-p300, FLAG-PCAF, or FLAG-GCN5 following immunoprecipitation. Cells were harvested 48 h posttransfection. B, semiquantitative LC-MS/MS-based comparison of TULP3 acetylation corresponding to the conditions in (A). C, western blot showing total levels of endogenous TULP3 in HeLa cells following transfection with empty pcDNA 3.1(+) or a plasmid encoding HA-p300. Cells were harvested 48 h posttransfection. D, mRNA levels of endogenous TULP3 corresponding to samples in (C) examined using quantitative real-time PCR; n = 4 technical replicates, Mean ± S.D. shown. E, western blot of p300 and TULP3 protein levels following stable transduction of p300 shRNAs into HeLa cells. F, immunoblot of stably expressed FLAG-TULP3 protein in 293T cells following treatment with either DMSO, 30 μM C646, or 50 μM anacardic acid for 24 h. G, mRNA levels of FLAG-TULP3 corresponding to samples in (F) analyzed using quantitative real-time PCR; n = 4 technical replicates, Mean ± S.D. shown. H, acetylation levels of FLAG-TULP3 in stably expressing 293T cells following treatment with DMSO or 30 μM C646 for 9 h.

Fig 3: p300 and HDAC1 influence the stability of multiple tubby-like proteins.A, sequence alignment of the four human TULP proteins (TULP1-4) performed using ClustalW software. Identical, similar, and nonsimilar residues are color coded as indicated. Key acetylation sites are indicated with arrows. B, western blot showing TULP1, TULP2, and TULP4 protein levels in 293T cells transfected with either a control plasmid or a plasmid encoding HA-p300. Cells were harvested 48 h posttransfection. C, western blot showing levels of TULP1, TULP2, and TULP4 in 293T following 24 h treatment with either DMSO or 30 µM C646. D, western blot of p300, TULP1, TULP2, and TULP4 protein levels following stable transduction of p300 shRNAs into 293T cells. E, western blot showing TULP1, TULP2, and TULP4 protein levels in 293T cells transfected with either a control plasmid or a plasmid encoding Myc-HDAC1. Cells were harvested 48 h posttransfection. F, immunoblot showing p300, TULP1, TULP2, and TULP4 protein levels following stable transduction of HDAC1 shRNAs into 293T cells. G, western blot showing levels of TULP1, TULP2, and TULP4 in 293T cells following 9 h treatment with either DMSO or 10 µM MG-132.

Fig 4: ZEB1 depletion epigenetically reduces Dll4 and Notch1 expressions in bone ECs.a Schematic representation of reporter constructs of wild-type (WT) and mutated (MUT) murine Dll4 and Notch1 promoters. b Luciferase reporter analysis of cultured control and ZEB1-deleted bone ECs that were electroporated with WT or MUT Dll4 and Notch1 promoter reporter constructs (n = 3 independent experiments). c Luciferase reporter analysis of cultured bone ECs that were co-electroporated with WT or MUT Dll4 and Notch1 promoter reporter constructs in combination with pcDNA3.1 or pcDNA3.1-HA-ZEB1 (n = 3 independent experiments). d, e ChIP-PCR analysis of interactions of ZEB1-Dll4 promoter and ZEB1-Notch1 promoter within the proximal and distal regions in cultured bone ECs. Agarose gel images as shown are from three independent experiments (d) and promoter occupancy relative to corresponding input was quantified (e; n = 3 independent experiments). prox. proximal, dist. distal. f ChIP-qPCR for analyzing the enrichments of H3K4Ac, H3K14Ac, H3K18Ac, and H3K27me3 on Dll4 and Notch1 promoters in control and ZEB1-deleted bone ECs (n = 3 independent experiments). g Immunoprecipitation (IP) analysis of the physical interaction between ZEB1 and CBP or p300 in nuclear extracts of bone ECs. Images as shown are from three independent experiments. h Sequential ChIP-PCR analysis confirming the co-occupancy of ZEB1 and CBP or p300 on Dll4 and Notch1 promoters in bone ECs. Images as shown are from three independent experiments. i, j Luciferase reporter assays for analyzing Dll4 and Notch1 promoter activity in HEK293T cells that were co-transfected with the indicated constructs (i). Immunoblot analysis confirming ectopic expression of HA tagged ZEB1, p300, and CBP in nuclear extracts of HEK293T cells (j). Images as shown are from three independent experiments. Arrow marks specific bands with the expected molecular weights, respectively. All data are represented as mean ± SD. **P < 0.01, *P < 0.05; NS not significant. Differences are tested using one-way ANOVA with Tukey’s post hoc test (b, c, i) and unpaired two-tailed Student’s t-test (e, f). The source data are provided as a Source Data file. Unprocessed original scans of blots are shown in Source Data file.

Fig 5: BAF60c acts as a bridge between SRF and the SWI/SNF complex.(A) Luciferase assays in A7r5 cells transfected with MYH11, ACTA2, or TAGLN promoter-driven luciferase reporters and infected with Ad-GFP or Ad-BAF60c (10 MOI). After 48 hours, the promoter activities were determined by dual luciferase assay and normalized by Renilla activity (n = 4). (B) A7r5 cells were transfected with siControl or siBaf60c (30 nM). After 48 hours, cells were serum starved in Opti-MEM for 24 hours. ChIP assays were performed to determine SRF-binding activity to Myh11, Acta2, Cnn1, and Tagln promoters. IgG served as control. (C and D) HASMCs were transfected with siControl or siBAF60c (30 nM). After 48 hours, cells were serum starved in Opti-MEM for 24 hours and ChIP-Seq was performed using BRG1, H3K9ac, and H3K27ac Abs. (C) Histogram of ChIP-Seq reads of H3K9ac ±3 Kb surrounding the TSS of genes (left). Quantification of relative read ratio of the H3K9ac signals within the TSS ± 3 Kb is presented in box plots (right). (D) IGV image showing localization of BRG1 and H3K9ac and H3K27ac within MYH11 promoter in HASMCs. (E) BRG1 and H3K9ac binding on the predicted SRF-binding site located in the human MYH11 promoter were determined by ChIP assay in HASMCs transfected with siControl or siBAF60c (30 nM) for 48 hours, followed by serum starvation in Opti-MEM for 24 hours. (F–H) HASMCs were transfected with siControl or siBAF60c (30 nM). After 48 hours, cells were serum starved in Opti-MEM for another 24 hours, and nuclear proteins were isolated and subjected to Co-IP using BRG1, SRF, or P300 Abs. IgG was the negative control. Three independent experiments were performed for B and E–H. Data are represented as mean ± SEM. Two-way ANOVA followed by Holm-Šidák post hoc analysis (A, B, and D); Mann-Whitney U test (C); paired t test (F–H).