Journal: bioRxiv
Article Title: Targeting Monoallelic CREBBP / EP300 Mutations in Germinal Center-Derived B-Cell Lymphoma with a First-in-Class Histone Acetyltransferase Activator
doi: 10.1101/2025.03.13.642871
Figure Lengend Snippet: a , Ribbon diagram of p300 depicting predicted YF2 binding sites including the bromo (circled in yellow), RING (blue), and HAT (red) domains. The p300 protein is colored dark blue and the predicted binding sites are highlighted in warmer colors. b , Ribbon and surface diagram of the p300 RING domain showing predicted YF2 interactions (in cyan and circled in blue). c , Schematic representation of the domain architecture of CREBBP/p300 and proposed YF2 binding to CREBBP as assessed through thermal shift assays. The catalytic domain comprises the bromodomain (Bd; yellow), RING domain (blue), plant homeodomain (PHD; gray), and HAT domain (red). JQ1 is a known bromodomain inhibitor used as a positive control. Data are presented as mean protein melting temperatures (T m ) and box plots display the first, second, and third quartiles. Treatment conditions are compared using a one-way ANOVA followed by a Tukey’s HSD post-hoc test ( n = 3–4). d , Proposed mechanism of YF2 binding and HAT activation. YF2 binding to the bromo/RING domain causes a conformational change that reveals the previously blocked substrate-binding pocket and lysine-rich autoregulatory loop (AL), promoting CREBBP/p300 auto-acetylation that increases HAT activity. Created with BioRender.com . e , Auto-acetylation of CREBBP and p300 incubated with 50 nM YF2 for 1, 5, or 10 min and visualized via immunoblotting ( n = 3–6). f , Quantification of CREBBP auto-acetylation shown in . Data are calculated as normalized fold changes relative to respective vehicle-treated controls and presented as means ± s.e.m. Treated and control samples from the same time point are compared using a Mann-Whitney U test ( n = 5–6). g , Quantification of p300 auto-acetylation shown in . Data are calculated as normalized fold changes relative to respective vehicle-treated controls and presented as means ± s.e.m. Treated and control samples from the same time point are compared using a Mann-Whitney U test ( n = 3–4). h , Cell-free acetylation of histone H3 following exposure to increasing concentrations of YF2 over 30 min ( n = 3). i , Quantification of histone H3 cell-free acetylation shown in . Model fitting to calculate the half maximal effective concentration (EC 50 ) is performed using the drc package ( n = 3). j , Histone H3K14 and H3K27 acetylation in SU-DHL-6 cells treated with 6 µM YF2 for two days as measured by mass spectrometry. Data are calculated as fold changes relative to respective vehicle-treated controls and presented as means ± s.e.m. Treated and control samples from the same histone target are compared using an unpaired two-sided t-test ( n = 3). k , Protein histone acetylation of BCL cell lines and PBMCs isolated from a patient with DLBCL treated with YF2 in vitro over three days and assessed via immunoblotting. Data are calculated as normalized fold changes relative to respective vehicle-treated controls and presented as means ± s.e.m. Treatment conditions from the same cell line are compared using a Kruskal-Wallis test followed by a Dunn’s post-hoc test ( n = 1–3). Asterisks (*) denote statistical significance: * = P < 0.05, ** = P < 0.01, and *** = P < 0.001. n.s. = not significant.
Article Snippet: Select samples were incubated with the bromodomain inhibitor JQ1 (SelleckChem #S7110) as a positive control.
Techniques: Binding Assay, Positive Control, Activation Assay, Activity Assay, Incubation, Western Blot, Control, MANN-WHITNEY, Concentration Assay, Mass Spectrometry, Isolation, In Vitro
