![RHOA G17V induces an increased p300-dependent acetylation. (A-D) The IL-2 promoter carries p300-specific marks of histone modification. In all figure panels, cells transduced with combinations of CD28 wt/RHOA wt (light gray) or CD28 wt/ RHOA G17V (red) were stimulated with anti-CD3, anti-CD28, and crosslinker at 2 μg/mL for 4 hours. Cells were then fixed, and ChIP was performed using IgG CTRL, anti-H3K4me3 (A), H3K9ac (B), H3K18ac (C), and H3K27ac (D) antibodies. The relative enrichment of 4 regions (R1 [−677; −567]; R2 [−226; −133]; R3 [+2; +100]; and R4 [+291; +386]) of the IL-2 promoter was assessed by qPCR using 4 pairs of primers. R2 and R3 regions are considered as proximal IL-2 promoter. Data are represented as mean ± SEM of 7 independent experiments. Significant differences in enrichment were determined using a 1-way ANOVA with Tukey multiple comparison test (∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001). (E) RHOA G17V expression induces increased p300-specific histone acetylation marks at whole-genome level. Sections of cell pellets (cytoblocs) were stained for H3K18ac (rabbit polyclonal ab1191; 1/800) or H3K27ac (rabbit polyclonal ab4729; 1/300) in combination with DAPI and assessed by immunofluorescence (upper). The mean intensity values of H3K18ac or H3K27ac and DAPI expression of segmented cells were quantified, and H3K18ac:DAPI and H3K27ac:DAPI ratios were calculated (lower). These ratios were compared between cells. Data are represented as mean ± SEM with the number of cells analyzed indicated in the figure. (F) TFH cells from patients with AITL carrying RHOA G17V show increased H3K18ac and H3K27ac p300-specific marks. Mean intensity ratios of H3K18ac:DAPI and H3K27ac:DAPI were compared between thousands of CD3+PD1+ (TFH) and CD3+PD1– (reactive) T cells for each of 4 patients with AITL carrying RHOA G17V alteration (1A, 1B, 1C, and 1D) and 4 patients carrying the wt RHOA (1E, 1F, 1G, and 1H). The means of each patient are plotted and compared. CD3+PD1+ cells showed increased H3K18ac:DAPI and H3K17ac:DAPI ratios compared with CD3+PD1– cells in patients with AITL carrying RHOA G17V mutation (∗P ≤ .05) but not in patients carrying wt RHOA.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/9/12/10.1182_bloodadvances.2024014671/1/blooda_adv-2024-014671-gr6.jpeg?Expires=1753352160&Signature=VsPqmn1~JHoCXR657UL5t1pd3C-FAbXY6vd4rT4EvGOMk7k62gY-4OFwSItUvMC0d-TD4FASBb~HPr5jaw2a52bjxlOdqVKtsorXYhizAV8QQyejG82Z5T1Yloo6RufvQfuU3zYtcdFAufHmTnVpK6S6xZnI70t62QUEfpxB4c~P4375vQfx-bTMmu6v3kL7tu4H~3J64dOpJw3q6eyhwenHtxGPW0WOOhX5rxxM4g3qxzWQ5IvOSya3Gb6WAS7m6zYy9JB185m63Ie2194OEJQEsez46AFUJIDOd10o3oqG4pnA6m14W~GL2yiRQ0CpFxyPkzUI-jQRO2YRem8zpg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
RHOA G17V induces an increased p300-dependent acetylation. (A-D) The IL-2 promoter carries p300-specific marks of histone modification. In all figure panels, cells transduced with combinations of CD28 wt/RHOA wt (light gray) or CD28 wt/ RHOA G17V (red) were stimulated with anti-CD3, anti-CD28, and crosslinker at 2 μg/mL for 4 hours. Cells were then fixed, and ChIP was performed using IgG CTRL, anti-H3K4me3 (A), H3K9ac (B), H3K18ac (C), and H3K27ac (D) antibodies. The relative enrichment of 4 regions (R1 [−677; −567]; R2 [−226; −133]; R3 [+2; +100]; and R4 [+291; +386]) of the IL-2 promoter was assessed by qPCR using 4 pairs of primers. R2 and R3 regions are considered as proximal IL-2 promoter. Data are represented as mean ± SEM of 7 independent experiments. Significant differences in enrichment were determined using a 1-way ANOVA with Tukey multiple comparison test (∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001). (E) RHOA G17V expression induces increased p300-specific histone acetylation marks at whole-genome level. Sections of cell pellets (cytoblocs) were stained for H3K18ac (rabbit polyclonal ab1191; 1/800) or H3K27ac (rabbit polyclonal ab4729; 1/300) in combination with DAPI and assessed by immunofluorescence (upper). The mean intensity values of H3K18ac or H3K27ac and DAPI expression of segmented cells were quantified, and H3K18ac:DAPI and H3K27ac:DAPI ratios were calculated (lower). These ratios were compared between cells. Data are represented as mean ± SEM with the number of cells analyzed indicated in the figure. (F) TFH cells from patients with AITL carrying RHOA G17V show increased H3K18ac and H3K27ac p300-specific marks. Mean intensity ratios of H3K18ac:DAPI and H3K27ac:DAPI were compared between thousands of CD3+PD1+ (TFH) and CD3+PD1– (reactive) T cells for each of 4 patients with AITL carrying RHOA G17V alteration (1A, 1B, 1C, and 1D) and 4 patients carrying the wt RHOA (1E, 1F, 1G, and 1H). The means of each patient are plotted and compared. CD3+PD1+ cells showed increased H3K18ac:DAPI and H3K17ac:DAPI ratios compared with CD3+PD1– cells in patients with AITL carrying RHOA G17V mutation (∗P ≤ .05) but not in patients carrying wt RHOA.
