Extended Data Fig. 9: H4K77ac specifically regulated by the deacetylase HDAC3.
From: Acetylation profiling by Iseq-Kac reveals insights into HSC aging and lineage decision
a-j, In vitro differentiation assay of aged HSCs infected with shRNA-Hdac3 (shRNA#1) or vector control. Representative data (a,c,f,h) and cumulative mean values (b,d,e,g,i,j) of T lymphocyte (CD4-CD8-, CD4+CD8+), B lymphocyte (CD19+), myeloid cell (CD11b+) frequencies and H4K77ac abundance in CD45+mCherry+ cells derived from shRNA-Hdac3-infected HSCs or vector control infected HSCs. The cumulative mean values are presented as the fold change normalized to control (n = 14-20 wells). k-m, Representative data (k,m) and cumulative mean values (l,n) of H4K77ac, H3K27ac and H3K9/K14ac in RGFP966-treated (1/5/10 µM) or DMSO-treated HSCs (n = 3 mice). o, Pre-ranked GSEA of genes enriched by H4K77ac in RGFP966-treated versus DMSO-treated HSCs from young mice. Differential peaks (p value < 0.01) were identified using DiffBind in R. p-r, Comparisons between genes co-regulated by H4K77ac and H3K27ac. p, Venn diagrams showing the numbers of overlapping genes. q, Volcano plot displaying the genes differentially expressed between aged and young HSCs based on the 304 overlapping genes. Dotted lines indicate Log2FC = ± log2(1.5), p value adjusted ≤ 0.05. Blue indicates downregulated genes in aged HSCs, orange indicates upregulated genes in aged HSCs, and gray indicates no significant difference. r, Bubble plot showing the pathway enrichment results of the 304 genes, based on GSEA. NES, normalized enrichment score. s,t, Representative immunoblotting showing the expression of H4 (s) or H3 (t) in Lin- bone marrow cells infected with H4K77 (s) or H3K27 (t) mutant virus or vector control. u,v, Cumulative mean value showing the MFI of GFP in GFP+CD45+ cells derived from HSCs ectopically expressing H4K77R/Q (u) or H3K27R/Q (v) (n = 5-8 wells for H4K77 mutations; n = 4-6 wells for H3K27 mutations). All data are derived from three independently biological replicates (a-n and u-v). Representative blots from n = 2 or 3 independent experiments (s,t). Data are presented as the mean ± s.e.m. Statistical significance was assessed using two-side t-test (b,d,e,g,i,j), two-way ANOVA followed by Tukey’s multiple comparisons adjustments (l,n), two-side Wilcoxon Rank Sum Tests (q) or two-side Permutation test (o,r).
