Fig. 3: Involvement of HWS1/2 regulates H4Ac modification and stabilizes transcriptional reprogramming with altered histone acetylation.

a Subcellular localization of HWS1 and HWS2 fused with the yellow fluorescent protein (YFP) reporter. A nuclear localization signal marker (NLS-DsRed) was co-expressed with the HWS1/2-YFP fusion proteins in rice protoplasts. Scale bars = 10 µm. b HAT activity testing of HWS2 towards histone H4 by fluorometry. AFU, Arbitrary Fluorescence Units. c Endogenous acetylated histone H2A, H3, and H4 levels and substrate specificity determined by western blotting. The specific tested antibodies are shown on the left and the differences in visible band intensity are indicated by red arrowheads. Lane #1: NIL-hws1^WYJ7/hws2^CG14; Lane #2: NIL-HWS1^CG14/hws2^CG14; Lane #3: NIL-hws1^WYJ7/HWS2^WYJ7; and Lane #4: NIL-HWS1^CG14/HWS2^WYJ7. d The distribution profile of averaged H4Ac occupancy across the gene body in NIL-HWS1^CG14/hws2^CG14 and NIL-hws1^WYJ7/hws2^CG14 (****p < 0.0001, calculated by two-sided Welch’s t-test). e Venn diagram showing the overlap between RNA-seq differentially-regulated genes with reduced expression levels and ChIP-seq differentially-enriched genes with reduced H4Ac levels. f KEGG enrichment bubble plot of the overlapping DEG set in e. All significantly enriched (FDR < 0.05) KEGG terms are shown while the emphasized pathways are additionally indicated with a green background. g Genome browser views of mRNA and H4Ac signals of gene examples selected for validation. h qRT-RCR expression analysis of two NADH-dependent enoyl-ACP reductase (LOC_Os08g23810 and LOC_Os09g10600) and OsKASI (LOC_Os06g09630) in the young panicles of NIL-HWS1^CG14/hws2^CG14 and NIL-hws1^WYJ7/hws2^CG14 plants. The expression level of each tested gene normalized to rice Ubiquitin in NIL-HWS1^CG14/hws2^CG14 was set to 1.0. i ChIP-qPCR analysis showing the relative enrichment of indicated genes identified in h. j, k, Measurement of biotin level in NIL-hws1^WYJ7/hws2^CG14 versus NIL-HWS1^CG14/hws2^CG14 plants (j) and NIL-hws1^WYJ7/hws2^CG14 versus NIL-hws1^WYJ7/HWS2^WYJ7 plants (k). Data in b, h, i, j, and k are mean ± SEM (n = 3 (b, h, and i) or 4 (j and k) biological replicates). In b, different letters denote significant differences (p < 0.05, one-way ANOVA with two-sided Tukey’s HSD test), p values are adjusted and shown in the Source Data file. A two-sided Student’s paired t-test was used to generate the p-values in h–k. The experiments in a and c were repeated three times independently, with similar results.