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. 2024 Jun 17;15:5155. doi: 10.1038/s41467-024-49486-5

Fig. 2. H3R2 and H3R26 histone mutations similarly disrupt histone PTMs, PRC2 activity, and transcription.

Fig. 2

A Immunoblot analysis of total H3, HA-epitope-tagged transgenic H3, or specific histone PTMs using PTM-specific antibodies in lysates from H3WT or H3 arginine mutant expressing HEK293T (293T), B mesenchymal progenitor cells (MPC), or C murine embryonic stem cells (mESC). The epitope-tagged mutant (or wildtype control) histone is marked by an arrow, whereas the endogenous wildtype H3 histone is marked by an asterisk. The approximate molecular weight of endogenous H3 (~16 kDa) was determined by reference to a molecular weight ladder and the stereotypical core histone migration pattern by Direct Blue staining of uncropped membranes corresponding to each blot, which are available in the Source Data file along with uncropped images of the immunoblots. Blots shown in panels A–C are representative of duplicate experiments. D In vitro PRC2 methyltransferase activity for H3K27me2 nucleosome array substrates harboring H3WT versus mutant H3 using tritiated S-adenosyl-L-methionine (SAM) as a methyl donor. PRC2 activity is shown as counts per minute after background (no enzyme) counts were subtracted. Group means were determined to significantly differ (p < 0.001) using a nonparametric test (Friedman) of matched replicates (n = 5 independent assays). Dunn’s test (two-sided) was then used to compare each mutant to the H3WT control. p-values are shown. Error bars are standard error of the mean. Source data are provided as a Source Data file. E Principal component analysis of bulk RNA-seq from MPC expressing H3WT or H3R2C, H3R8C, H3R17C, or H3R26C. Three individual biologic replicates are shown for each genotype. The point labels indicate the replicate number. F H3WT or H3 arginine mutant MPC clustered based on expression values of differentially expressed genes in H3R26C vs H3WT. Only genes with at least a 2-fold change in expression and p-adj < 0.05 are shown. Three independent replicates are shown, each individually denoted by the suffix “-n”.