Fig 2: Interaction between upstream GBSs and the KLF9 TSS in the absence and presence of glucocorticoid. Hi-C analysis (unbiased, genome-wide chromosome conformation capture) of untreated A549 cells or following 100 nM Dex treatment for 1 or 4 h is displayed as interaction matrices (upper three triangular panels). These correspond to the KLF9 locus and upstream regions containing GBS P1-4. Images were generated from publicly accessible data from Genomics of Gene Regulation (GGR) project (27). The intensity of the red color reflects the number of ligation events and is indicative of the interaction frequency between the two respective genomic loci joined by downward 45° sloping lines. Interaction hotspots (A–D) involving the four KLF9 GBSs (P1-4) and TSS are annotated and are linked with dashed lines (lower panels). Genome browser snapshot of the KLF9 gene along with ∼70 kb of the 5′ upstream region showing ChIP-seq traces for GR (black) and P300 (red) in A549 cells following treatment with 100 nM dexamethasone (Dex) for indicated times. Positions of KLF9 GBSs P1-4 are indicated. ChIP-seq traces are publicly accessible data from GGR project (24, 27).

Fig 3: p300 binds and acetylates MCL1 at K40(A) Endogenous MCL1 is targeted by acetylation in HeLa cells. Shown is immunoblot (IB) analysis of whole-cell lysates (WCLs) and anti-MCL1 immunoprecipitates derived from HeLa cells.(B) p300 promotes MCL1 acetylation in cells. Shown is IB analysis of WCLs and anti-Myc tag immunoprecipitates derived from 293T cells transfected with Myc-MCL1 along with the indicated hemagglutinin (HA)-tagged lysine acetyltransferase constructs.(C and D) Endogenous interaction between MCL1 and p300. Shown is IB analysis of WCLs (input) and anti-p300 immunoprecipitates (C) or anti-MCL1 immunoprecipitates (D) derived from HeLa cells.(E) Knockdown of p300 reduces MCL1 acetylation. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from HeLa cells stably expressing the lentiviral short hairpin RNA (shRNA) specific for GFP or p300. Cells were treated with MG132 (10 µM) for 6 h before harvesting to stabilize and accumulate MCL1 protein.(F) Treatment with the p300/CBP inhibitor A-485 reduces MCL1 acetylation. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from HeLa cells. Cells were co-treated overnight with A-485 at the indicated concentration and MG132 (5 µM) before harvesting.(G) Acetylation-deficient K40R markedly diminishes p300-mediated MCL1 acetylation. Shown is IB analysis of WCLs and anti-Myc tag immunoprecipitates derived from 293T cells transfected with the indicated Myc-MCL1 constructs.(H) K40 of endogenous MCL1 is targeted by acetylation in SKBR3 breast cancer cells. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from SKBR3 cells.(I) MCL1 and p300 are present together in cytoplasmic and nuclear compartments. Shown is IB analysis of WCLs and cytoplasmic, mitochondrial, and nuclear fractions derived from BT-20 cells. Data are representative of at least two independent experiments. See also Figure S1.

Fig 4: CBP but not P300 is dispensable for the growth and survival of AML cells, and is required for the susceptibility to peptidomimetic MYB blockade by CRYBMIM.(A) Schematic of the competitive assays to define specific genetic dependencies. AML cells expressing Cas9 and GFP are transduced with sgRNAs targeting specific genes and expressing mCherry, followed by quantitation of cell abundance by fluorescence activated cell scanning (FACS) of GFP and mCherry-expressing cells. (B) Western blots demonstrating specific depletion of CBP in MV411 (left) and MOLM13 (right) cells expressing sgCBP-1 and sgCBP-2, as compared to control sgNEG1 and sgNEG2 targeting the AAVS1 safe harbor locus. EP300 is shown for specificity, and Actin serves as the loading control. (C) Relative growth of GFP-expressing MOLM13 cells expressing mCherry and unique sgRNAs targeting AAVS1 control (sgNEG1 and sgNEG2), CBP (sgCBP-1 and sgCBP-2), and P300 (sgEP300-1 and sgEP300-2) and quantified by FACS on day 0, 3, and 6 after 2-day treatment with 10 µM CRYBMIM or PBS. Data represent biological triplicates of at least 10,000 cells per condition; *p=6.0e-10, **p=4.1e-8, t-test for day 6 versus day 0 of CRYBMIM treatment of CBP-depleted cells. (D) Analogous experiment as (C) using MV411 cells; *p=1.1e-6, **p=3.7e-6, t-test for day 6 versus day 0 of CRYBMIM treatment of CBP-depleted cells.

Fig 5: The schematic diagram of the regulatory mechanism of CHES1 acetylation on the progression of breast cancer.The dynamic acetylation of CHES1 mediated by p300 and HDAC1 regulated the tumorigenesis and invasion of breast cancer cells through modulating the stability and transcriptional activity of CHES1.