Fig. 3

VIM1 and LHP1 co-regulate histone and DNA methylation at the YUCCA2 promoter. A Epigenetic landscape at the YUCCA2 (YUC2) locus. Track 1: H3K27me3 deposition by ChIP-sequencing. Track 2: LHP1 binding by ChIP-sequencing [49]. The black signal represents the input sequencing for each respective track. Gene annotation is shown in the bottom. B ChIP-qPCR analysis of H3K27me3 deposition at the YUC2 promoter in 4-day-old wild-type (WT) seedlings treated or not with heat (29 °C) for 6 h. The asterisk indicates the Student’s t test ≤ 0.05, n=3, between 23 and 29 °C. C ChIP-qPCR analysis of LHP1 binding at the YUC2 promoter in 4-day-old WT seedlings treated or not with heat (29 °C) for 6 h. D ChIP-qPCR analysis of LHP1 binding at the YUC2 gene body in WT and APOLO over-expression (OE APOLO-1) plants. The negative control corresponds to an APOLO-independent LHP1 target gene AT4G23720 [49]. E Boxplots showing hypocotyl length quantification ratio at 29 °C over 23 °C of 4-day-old lhp1 seedlings and their associated WT. Values are represented by colored points. Representative morphological phenotypes are shown on the right. Scale bars, 1 cm. FYUC2 transcript levels in 4-day-old WT and lhp1 seedlings treated or not with heat (29 °C) for 6 h. G Methylated DNA immunoprecipitation (MeDIP)-qPCR analysis at the YUC2 promoter in 4-day-old lhp1 mutant seedlings treated or not with heat (29 °C) for 6 h. H, I Chromatin immunoprecipitation (ChIP)-qPCR analyses of LHP1 binding (in H) and H3K27me3 deposition (in I) at the YUC2 promoter in 4-day-old WT and vim1-3 mutant seedlings. The negative control corresponds to an APOLO-independent LHP1 target gene AT4G23720 [49]. J Bimolecular fluorescence complementation (BiFC) assay in transiently transformed Nicotiana benthamiana leaves. CYFP was fused to VIM1 or SRA/RING2, and NYFP was fused to LHP1. In both panels, bright-field images (left), YFP fluorescence (middle), and merged images (right) are shown. A zoom-in including mCherry constitutive expression for nuclei and membrane visualization is shown in Additional file 1: Fig. S8A. Scale bars, 50 μm. In B–D and G–I, results are expressed as a percentage of the INPUT fraction. Anti-IgG antibodies were used as a negative control. Bars represent standard deviation (n = 3 biological replicates). In E, results are the mean of three biological replicates and letters indicate significant differences compared to WT, based on a Mann–Whitney test (α = 0.05; n ≥ 111). In F, transcript levels are normalized relatively to the untreated control to show fold changes. Bars represent average ± SD (n = 3 biological replicates) except for G (n = 2 closest biological replicates out of 3 performed, all showing the same trend). In J, one representative picture out of six biological replicates is shown