Fig 2: DBC1 facilitates SE-associated histone modifications by coordinating the interplay of p300 and KMT2D. (A) Protein levels in SW480 cells depleted of KMT2D or treated with SGC-CBP30 were monitored by immunoblot using the indicated antibodies. (B) ChIP-qPCR assays. Sheared chromatin from SW480 cells infected with lentiviruses expressing either a control (NS) or KMT2D shRNA or SW480 WT cells treated with 5 µM SGC-CBP30 for 1 h was immunoprecipitated with the indicated histone modification antibodies. qPCR analyses were performed using primers specific for SEs of the PROX1, ASB4, FSCN1 and NTSR1 genes. ChIP signals were normalized with inputs and shown as fold change relative to control IgG signal, and are means ± SD (n = 3). *P < 0.05, **P < 0.01. (C, D) In vitro HMT assays using recombinant nucleosomes, immunopurified HA-KMT2D, GST–p300-Br-HAT and GST–DBC1 as indicated (C) and in vitro HAT assays using recombinant nucleosomes, immunopurified FLAG-KMT2D SET, GST–p300-Br-HAT and GST–DBC1 as indicated (D). The reactions were analyzed by immunoblot using the indicated antibodies. (E) 293T cells were transfected with the indicated constructs, and HA-p300 immunoprecipitates were analyzed by immunoblots with the indicated antibodies. (F) In vitro translated HA-KMT2D SET, HA-DBC1 or both were incubated with GST–p300-Br-HAT bound to beads as indicated. Bound proteins were analyzed by immunoblot with anti-HA antibody. (G) Proximity biotinylation assays. SW480 WT and D1KO cells transfected with 3×FLAG-TurboID-KMT2D SET or 3×FLAG-TurboID were treated with biotin as indicated. Biotinylated endogenous proteins were pulled down using streptavidin beads and analyzed by immunoblot with the indicated antibodies. (H) The PROX1 locus is schematically depicted with locations of H3K27ac peaks in SW480 WT and D1KO, sgRNAs (arrow) and ChIP-PCR primers (arrowhead). (I) SW480 D1KO cells were infected with lentiviruses encoding the dCas9–DBC1 fusion protein and sgRNAs targeting the PROX1 SE. ChIP-qPCR assays using SW480 WT, D1KO and D1KO/dCas9-DBC1 cells were performed with the indicated antibodies. **P < 0.01. (J) Relative levels of DBC1 mRNA and PROX1 seRNA in SW480 WT, D1KO and D1KO/dCas9-DBC1 cells were determined by qRT–PCR. **P < 0.001. (K) Protein levels of PROX1 and DBC1 in SW480 WT, D1KO and D1KO/dCas9-DBC1 cells were monitored by immunoblot using the indicated antibodies.

Fig 3: H3K56 acetylation requires CBP/p300 function(A) Protein levels p300, H3K56Ac and other acetylated histone H3 were examined by Western blotting in HeLa cells at indicated time periods after treatment with curcumin at 100 µM and UVR at 20 J/m2. (B) H3K56Ac and p300 levels were examined in LFS-041B+DDB2 cells after curcumin and UVR treatments. Actin blots serve as the loading controls.

Fig 4: There are two main clusters of the BCL-2 enhancer marked by H3K27Ac and bound by P300 in MLL-AF4 and E2A-PBX1 cells. (A) ChIP-seq data as indicated in SEM cells. Red boxes highlight the two enhancer ATAC/H3K27Ac clusters. The yellow arrowhead indicates a PCR primer set used for ChIP Q-PCR BCL-2 enhancer experiments in Figure 6. (B) ChIP-seq in RCH-ACV (E2A-PBX1) cells reveals binding of E2A-PBX1 and P300 at the two enhancer clusters.

Fig 5: p300 histone acetyltransferase (HAT) activity enhances mutant p53-mediated NOX4 promoter activity and cell migration(A) Hep3B cells co-transfected with NOX4 promoter pGL3 (-4760) and either vector control, p53-WT, p53-R175H, or p53-D281G; and either control, p300, or HAT-inactive p300?HAT plasmids for 24 hours. The cells were then treated with TGFß (5 ng/ml) for an additional 24 hours. Total cell lysates were collected following TGFß treatment and analyzed for luciferase activity (n = 3, in triplicate). (B) ChIP assays were performed in Hep3B cells expressing control, p53-WT, or p53-D281G plasmids and either treated with TGFß (5 ng/ml) for 24 hours or remained untreated. Histone immunoprecipitation was conducted using ChIP qualified antibodies specific for acetylated lysine-8 histone 4 (H4K8) antibodies, or IgG (negative control). ChIP-qPCR results are represented as fold enrichment of IP: a-H4K8 ChIP over input DNA relative to IgG negative control (n = 2, in triplicate). (C) PANC-1 (p53-R273H) cells were transfected with On-Target SMARTpool p53-specific siRNAs (50 nM), p300-specific siRNAs (50 nM), or non-targeting control siRNAs (50 nM) for 72 h. Fifty micrograms of total cell lysates were analyzed by immunoblotting. (D) H1299 cells transfected with vector control, p53-WT, p53-D281G, p300-WT, p300-?HAT, or NOX4-P437H plasmids and seeded into a 96-well tissue culture plate (3.5 × 104 per well) and grown to confluence. Wounds were made using the WoundMaker 96-pin tool, which creates 96 precise and reproducible wounds of 800µm. The cells were then left untreated or were treated with TGFß (5 ng/ml). Approximately 18-22 hours after wounding and TGFß treatment, the cells were fixed and stained for imaging. The images presented are representative of four experiments completed in triplicate. Significance values are indicated as ***P-value < 0.001.