Extended Data Fig. 7: VIL1 and phyB directly regulate the expression of HFR1 and PIL1 by inducing H3K27me3 on their loci.
a and c, Hypocotyl length phenotype of VIL1 complementation lines (pVIL1:VIL1-myc/vil1-1 and pVIL1:VIL1-Flag/vil1-1) and another allele of vil1 mutant (vil1-2). The seedlings were grown for 7 days at 22 °C in SD. A scale bar = 5 mm. b and d, Quantification data of hypocotyl length shown in a, n = 25 seedlings. The box plot boundaries reflect the interquartile range, the horizontal line is the median and the whiskers represent 1.5X the interquartile range from the lower and upper quartiles. The letters above each box indicate statistical difference between the genotypes determined by One-way ANOVA followed by Tukey HSD test for multiple comparisons (P < 0.05). e, ATHB2, HFR1 and PIL1 loci, G-box elements, ChIP-qPCR amplicons. The red vertical bars indicate G-box regions. RE1, RE2, RE, P1, and P2 indicate ChIP-qPCR amplicons. f, ChIP assay showing the relative enrichments of VIL1-myc in phyB-9 on ATHB2, HFR1, and PIL1 loci by comparing to enrichments of VIL1-myc at ZT0 and at ZT6. The ChIP assay used anti-myc antibody on seven days old seedlings grown at 22 °C in SD. The immunoprecipitated DNA relative to input was further normalized to that of 5 S rDNA. Error bars: ± s.d., n = 3, biological replicates. The asterisks indicate statistical difference in a two-tailed student t-test (P < 0.05). g, H3K27me3 levels in Col-0, vil1-1, phyB-9, vil1-1phyB-9, and pif4-2 at ZT6. This ChIP assay used anti-H3K27me3 antibody and anti-H3 antibody. The y axis indicates H3K27me3 enrichment relative to H3 enrichment (normalized by that of 5 S rDNA). Error bars: ± s.d., n = 2, biological replicates (each biological replicate is an average value of four technical replicates). The asterisks indicate statistical differences in a two-tailed student t-test (P < 0.05). n.s. indicates not significant.
