Fig. 5: Functional analysis of non-phosphorylatable mutant CARM1.
From: Tyrosine phosphorylation of CARM1 promotes its enzymatic activity and alters its target specificity
A Cell proliferation assays of non-phosphorylatable CARM1 mutant knock-in HEL cells, where cell numbers were measured using a cell-counting apparatus. Y149F/Y334F (n = 3) vs. WT type (n = 3); day 3 (p < 0.001), day 5 (p = 0.003), day 7 (p = 0.001). B The flow cytometry analysis of BrdU-stained HEL cells expressing non-phosphorylatable CARM1 mutants. Mean fractions ± s.d. in sub G1, G0/G1, S, and G2/M populations. n = 3. S population (p = 0.015) in Y149F vs. WT; S (p = 0.011) and G2/M populations (p = 0.009) in Y334F vs. WT; S (p = 0.008) and G2/M populations (p = 0.002) in Y149F/Y334F vs. WT. C Heatmap shows the differentially expressed coding genes at 2-fold cut-off, representing replicates of HEL cells expressing CARM1 WT or two independent cells expressing CAMR1-Y149F/Y334F double mutation (Y149F/Y334F-1 and Y149F/Y334F-2). D Gene ontology analysis of significant downregulated genes in HEL cells expressing CARM1-Y149F/Y334F compared to CARM1-WT. E Heatmaps of FDR (q < 0.25) values from GSEA of hallmark gene set collections. F Representative GSEA plot depicting the downregulation of G2/M checkpoint and apoptosis/anti-apoptosis pathways. G Volcano plot representing gene expression changes triggered by CARM1-Y149F/Y334F mutation knock-in in HEL cells. Genes associated with apoptosis/anti-apoptosis, G2/M checkpoints, stemness in hematopoietic stem cells, and RUNX1-target are shown in red, yellow, blue, and violet, respectively. The red dots indicate upregulated genes in HEL cells expressing CARM1-Y149F/Y334F, whereas the blue dots indicate downregulated genes. P values correspond to a two-sided Wilcoxon rank-sum test with Bonferroni correction. H Representative GSEA plot depicting the downregulation of “hematopoietic stem cell up” signature. I qRT-PCR analysis showing BMI-1 and CD34 in HEL cells expressing CARM1 WT (n = 3), Y149F (n = 6), Y334F (n = 6), and Y149F/Y334F mutation (n = 6). Mean and SD are expressed as a percentage of HPRT-1 expression. J qRT-PCR analysis showing ID2 and MIR144 in HEL cells expressing CARM1 WT (n = 3), Y149F (n = 6), Y334F (n = 6), and Y149F/Y334F mutation (n = 6). Mean and SD are expressed as a percentage of HPRT-1 expression. n = 3. K Heat map of total R319-RUNX1 or asymmetrically dimethylated R319-RUNX1 binding tag intensity by ChIP-seq analysis for HEL cells expressing CARM1 WT, Y149F, Y334F, or Y149F/Y334F mutant proteins. L ChIP-seq analyses were performed to assess total RUNX1 and asymmetrically dimethylated R319-RUNX1 chromatin binding. Target occupancies at the ID2 gene are shown in IGV genome browser tracks. All error bars represent the mean ± SD. P values were determined by two-tailed Student’s t-test (A, B) and one-way ANOVA followed by Dunnett’s post hoc test (I, J). *p < 0.05, **p < 0.01, ***p < 0.001.
