(A) Part of the genomic sequence of wild-type LHP1; exons are marked in bold. The EMS allele lhp1-7 has a G to A transition in the second intron (position marked in red). (B) The point mutation in lhp1-7 creates a new splice site. The critical region of the alignment of wild-type and mutant LHP1 cDNAs is shown. (C) The lhp1-7 mutant protein has a defect in the chromodomain. The critical region of the alignment of the wild-type and mutant LHP1 proteins is shown. The chromodomain is shown in bold; the aromatic cage residues are marked in green and the cysteine that substitutes one of them in the mutant protein is marked in red. (D) The structure of wild-type and mutant LHP1 transcripts and primers for PCR amplicons. Black boxes, grey boxes and lines represent exons, untranslated regions and introns, respectively. The asterisk marks the position of the point mutation in lhp1-7 and the additional exon inclusion is shown in dark grey. The arrow marks the position of the T-DNA insertion in lhp1-6. Note that amplicon 1 is not specific for either splice variant while amplicons 2 and 3 are specific for the wild-type and mutant splice variants, respectively. For primer sequences see Table 3. (E) lhp1-6 is a transcriptional null mutant. (F) lhp1-7 expresses the mutant splice variant at the same level as the wild-type expresses LHP1. RNA in (E, F) was isolated from seedlings grown under long day conditions for 9d or 16d.

(A) Structural model of the LHP1 chromodomain based on homology modeling using the coordinates of the Drosophila Pc chromodomain complexed with an H3K27me3 peptide [3]. (B) Structural model of the LHP1-CD* chromodomain, which is encoded by lhp1-7(CD*). The arrow indicates the mutated region in LHP1-CD*. The position of the trimethylated lysine side chain (arrow heads) in (A) and (B) was derived from the template crystal structure. (C) Peptide-binding pull-down assay for wild-type LHP1 and LHP1-CD* (left) and quantification (right).

(A) Wild-type (Col, left) and lhp1-7(CD*) (right) after five weeks of growth under long day photoperiod. (B) LHP1 35S::LHP1-GFP (left) and lhp1-7(CD*) 35S::LHP1-GFP (right) plants. Plants are in the msi1-tap1 background. (C-K) 35S::LHP1-GFP (C-F) and 35S::lhp1-7(CD*)-GFP plants (G-K) were used to analyze protein localization in leaf nuclei. Protein localization was detected by confocal laser scanning microscopy of GFP-fluorescence (C, G) or by immuno-localization (D, H). (E, I) DAPI-staining of the nuclei in D and H; merged images of D and E (F) and of H and I (K). (L) ChIP assays for binding of LHP1-GFP and LHP1-CD*-GFP to the AG and SEP3 loci. Top: Genomic structure of AG and SEP3. Lines represent introns, narrow bars 3′ and 5′ UTRs and wide bars represent coding exons. Black lines represent regions probed by qPCR. Values are recovery as percent of input; IgG served as negative control.

(A) Rosette leaves produced until bolting in long days (LD). (B) Phase transition in LD. (C) Rosette leaves formed until bolting in SD. Values in (A-C) are mean±S.E. (n≥7). (D) FT expression at ZT = 4h (ZT, zeitgeber time; ZT = 0 is lights on) in 12 days old seedlings from LD and at ZT = 6h in 14 days old seedlings from SD. Samples were taken at times when FT expression in wild-type is low [43]. Values in D are mean±S.E. (n = 4). Note that expression values for LD and SD were independently normalized to the corresponding wild-type. For all panels: White, grey, and dark-grey bars represent wild-type, lhp1-6(null) and lhp1-7(CD*), respectively.

(A-C) Adaxial epidermis of Col (A), lhp1-6(null) (B) and lhp1-7(CD*) (C) leaves. (D) Area of first and second rosette leaf after bolting (n≥11). (E) Cell size in the adaxial epidermis of the first and second rosette leaves (n≥244). (F) Estimated cell number in the adaxial epidermis of the first and second rosette leaf. For all panels: White, grey, and dark-grey bars represent wild-type, lhp1-6(null) and lhp1-7(CD*), respectively. Values in (D-F) are mean±S.E.

(A) The total number of reproductive organs (siliques, flowers and flower buds) on the primary shoots of five weeks old plants from LD. Values are mean±S.E. (n≥8). (B) Example of the growth curve of a wild-type plant's primary shoot. (C) Length of linear growth phase. (D) Growth rates during linear growth phase of primary shoots. Values in (C, D) are averages over two experiments with n≥9 per experiment. For all panels: White, grey, and dark-grey bars represent wild-type, lhp1-6(null) and lhp1-7(CD*), respectively.

(A–C) Flowers of wild-type Col (A), lhp1-6(null) (B) and lhp1-7(CD*) (C) produced late during development. (D) Expression of PcG targets in seedlings at ZT = 5h after 16 days in LD. (E) Expression of heterochromatic loci in rosette leaves at ZT = 5h after 25 days in LD. RNA from ddm1-2 was used as positive control.

(A) Segment of the alignment that contains the chromodomain. The arrow heads highlight the aromatic cage residues that form the binding cavity for histone methyl groups. (B) Phylogenetic tree of 15 LHP1 and HP1 homologs (Arabidopsis thaliana: At_LHP1 GI:15625407. Populus trichocarpa: Pt_LHP1A, estExt_Genewise1_v1.C_LG_XIX1329; Pt_LHP1B, eugene3.00130688. Oryza sativa: Os_LHP1, GI:110810411. Zea mays: Zm_LHP1 GI:22135459. Physcomitrella patens: Pp_LHP1, jgi|Phypa1_1|169812|estExt_fgenesh1_pg.C_2200058. Selaginella moellendorffii: Sm_LHP1, jgi|Selmo1|407083|fgenesh2_pg.C_scaffold_6000334. Homo sapiens: Hs_HP1alpha, GI:6912292; Hs_HP1beta, GI:48428808; Hs_HP1gamma, GI:5732187. Drosophila melanogaster: Dm_HP1a, GI:17136528; Dm_HP1b, GI:24640713. Caenorhabditis elegans: Ce_HP1-1, GI:17568757; Ce_HP1-2, GI:71987888. Schizosaccharomyces pombe: Sp_SWI6, GI:510930.). The evolutionary history was inferred using the Maximum Parsimony method; the most parsimonious tree with length = 2089 is shown. Support for each node, assessed with bootstrap analysis (1000 replicates) is given when higher than 60%. Note that the tree is displayed as circular cladogram with all branches of the same length.