Fig. 6: PtrVCS2 regulates the histone acetylation levels at the PtrWOX4a promoter via the PtrWOX13a–PtrVCS2–PtrGCN5-1–PtrADA2b-3 tetrameric protein complex.
a, Schematic diagram of the CAATCAC motif (the WOX13-binding motif) in the PtrWOX4a promoter. b,c, Relative enrichment fold of H3K9ac, H3K14ac and H3K27ac at the PtrWOX4a promoter in the ptrvcs2 ptrvcs2-h #1 mutants (b) and OE-PtrVCS2 #2 transgenics (c). d–k, BiFC assays in P. trichocarpa SDX protoplasts showing that PtrGCN5-1 interacts with PtrVCS2 (d) and PtrWOX13a (e) and that PtrADA2b-3 interacts with PtrWOX13a (f) but not with PtrVCS2 (g). Each BiFC pair of constructs was co-transfected with the H2A-1–mCherry nuclear marker construct. Co-transfection of each construct of interest with empty plasmid served as a control (h–k). Scale bars, 10 μm. l–n, Interactions of PtrVCS2–PtrGCN5-1 (l), PtrWOX13a–PtrGCN5-1 (m) and PtrADA2b-3–PtrWOX13a (n) dimers, as determined by pull-down assays. o–r, Interactions of PtrWOX13a–PtrVCS2–PtrGCN5-1 (o), PtrWOX13a–PtrGCN5-1–PtrADA2b-3 (p), PtrVCS2–PtrGCN5-1–PtrADA2b-3 (q) and PtrADA2b-3–PtrWOX13a–PtrVCS2 (r) trimers, as determined by pull-down assays. s, Relative expression levels of PtrWOX4a in PtrGCN5-1–RNAi transgenics and the ptrgcn5-1 ptrgcn5-2 mutants, as determined by RT–qPCR. The data are shown as mean ± s.e.m.; n = 3 biological replicates; two-tailed Student’s t-test; **P < 0.01; ***P < 0.001. t, Relative enrichment fold of H3K9ac, H3K14ac and H3K27ac at the PtrWOX4a promoter in PtrGCN5-1–RNAi transgenics and the ptrgcn5-1 ptrgcn5-2 mutants. In b, c and t, ChIP assays were performed using antibodies against H3K9ac, H3K14ac and H3K27ac, and the precipitated DNA was quantified by qPCR. Enrichment values represent the relative fold change compared with WT plants. The data are shown as mean ± s.e.m.; n = 3 biological replicates, and the asterisks indicate significant differences between each transgenic line and WT plants (two-tailed Student’s t-test; *P < 0.05; **P < 0.01). The experiments in d–r were repeated independently three times, with consistent results.
