Fig. 5: SAT1 directly acetylated H3K27 to regulate H3K27ac.
a Total polyamine content in the indicated detached cells (n = 5, each). b Relative polyamine uptake into the indicated detached cells determined via 3H-putrescine labeling (n = 5, each). c Schematic representation of polyamine metabolism. Heatmap showing the changes in the mRNA levels of the enzymes in detached SAT1-depleted cells compared with control pLKO cells via RNA-seq analysis (n = 3, each). GEO accession: GSE267614. d Relative 3H-arginine uptake into detached pLKO and SAT1-knockdown cells (n = 5, each). e Cellular location of SAT1 in detached cells analyzed via confocal microscopy. Nuclei were counterstained with DAPI. Scale bars represent 30 μm. f Analysis of interaction between SAT1 and H3 in detached cells. g Molecular docking of H3 with SAT1. SAT1 and H3 proteins are shown as cartoons and colored silver and cyan, respectively. Residues mediating the interaction of H3 and SAT1 are highlighted and shown in stick representation. h Analysis of H3 acetylation in the in vitro acetylation assay using 3H-ac-CoA. Recombinant His-H3 protein was incubated with recombinant flag-SAT1 WT or flag-SAT1 K166A mutant protein in the presence of ac-CoA (n = 10, each). i Analysis of enzyme activity of recombinant SAT1 WT or SAT1 K166A mutant protein using spermine as a substrate (n = 5, each). j Analysis of the interaction between wild-type (WT) or K166A mutant (K166A) SAT1 and His-tagged H3 in detached cells. k, l γH2AX levels (k, timepoint=18 h) and apoptosis (l, timepoint=24 h) in SAT1-depleted cells rescued with the recombinant rSAT1 WT or the rSAT1 K166A mutant upon detachment (n = 5, each). The data are presented as the means ± SDs of biological replicates for (a), (b), (d), (h), (i), (k) and (l) and are representative of two independent biological experiments for (e), (f) and (j). Statistical analysis was performed via two-tailed Student’s t test for (a), (b), and (d) and via 1-way ANOVA for (h, (i), (k) and (l). The samples derive from the same experiment but different gels for all indicated antibodies were processed in parallel for (f) and (j). See also supplementary Figs. S7–S10.
