<i>Arabidopsis</i> AL PHD-PRC1 Complexes Promote Seed Germination through H3K4me3-to-H3K27me3 Chromatin State Switch in Repression of Seed Developmental Genes

<div><p>Seed germination and subsequent seedling growth define crucial steps for entry into the plant life cycle. For those events to take place properly, seed developmental genes need to be silenced whereas vegetative growth genes are activated. Chromatin structure is generally known to play crucial roles in gene transcription control. However, the transition between active and repressive chromatin states during seed germination is still poorly characterized and the underlying molecular mechanisms remain largely unknown. Here we identified the <i>Arabidopsis</i> PHD-domain H3K4me3-binding ALFIN1-like proteins (ALs) as novel interactors of the Polycomb Repressive Complex 1 (PRC1) core components AtBMI1b and AtRING1a. The interactions were confirmed by diverse <i>in vitro</i> and <i>in vivo</i> assays and were shown to require the AL6 N-terminus containing PAL domain conserved in the AL family proteins and the AtRING1a C-terminus containing RAWUL domain conserved in animal and plant PRC1 ring-finger proteins (including AtRNIG1a/b and AtBMI1a/b). By T-DNA insertion mutant analysis, we found that simultaneous loss of AL6 and AL7 as well as loss of AtBMI1a and AtBMI1b retards seed germination and causes transcriptional derepression and a delayed chromatin state switch from H3K4me3 to H3K27me3 enrichment of several seed developmental genes (<i>e.g. ABI3</i>, <i>DOG1</i>, <i>CRU3</i>, <i>CHO1</i>). We found that AL6 and the PRC1 H3K27me3-reader component LHP1 directly bind at <i>ABI3</i> and <i>DOG1</i> loci. In light of these data, we propose that AL PHD-PRC1 complexes, built around H3K4me3, lead to a switch from the H3K4me3-associated active to the H3K27me3-associated repressive transcription state of seed developmental genes during seed germination. Our finding of physical interactions between PHD-domain proteins and PRC1 is striking and has important implications for understanding the connection between the two functionally opposite chromatin marks: H3K4me3 in activation and H3K27me3 in repression of gene transcription.</p></div

LHP1 and AL6 binding at <i>ABI3</i> and <i>DOG1</i> chromatin.

<p>Relative enrichments of LHP1-myc and GFP-AL6 proteins were analyzed at the five regions (a to e) of <i>ABI3</i> (A) and <i>DOG1</i> (B) loci. Transgenic seeds/seedlings expressing <i>LHP1-myc</i> or <i>GFP-AL6</i> were analyzed at 24 or 72 hours after stratification (HAS) by ChIP using anti-myc or anti-GFP antibodies. Samples in the absence of antibodies serve as negative controls (mock). Values were normalized to internal controls (relative to input and to <i>TUB2</i>). Data represent means ± SD of three biological replicates.</p

Functional characterization of <i>AL</i> genes.

<p>(A) Tissue-specificity of <i>AL</i> gene expression. Relative expression levels of <i>AL1</i>, <i>AL2</i>, <i>AL5</i>, <i>AL6</i>, and <i>AL7</i> were determined by quantitative RT-PCR in different plant organs. Leaves: rosette leaves from 4-week-old plants; Buds: floral buds before anthesis; Flowers: flowers at anthesis; Seeds: dry seeds. Data represent means ± SD of three biological replicates. (B) <i>AL6</i> and <i>AL7</i> genomic structure and T-DNA insertion mutants. Genes are schematically represented by black boxes for exons, black lines for introns and dashed boxes for untranslated regions. Triangles indicate T-DNA insertion sites and arrowheads indicate RT-PCR primer positions. Relative expression levels of <i>AL6</i> and <i>AL7</i> in Col-0 and in <i>al6</i> and <i>al7</i> mutants are shown as means ± SD of three biological replicates. (C) Representative seed germination images of Col-0, <i>al6 al7</i> double mutant, and the double mutant complemented by the <i>AL6</i> promoter driving <i>GFP-AL6</i> fusion gene (<i>+pAL6:GFP-AL6</i>). Images were taken five days after stratification from plates containing MS media or MS supplemented with 100 mM NaCl (MS+NaCl). (D) Germination rate of Col-0, double mutants <i>al6 al7</i> and <i>Atbmi1a Atbmi1b</i>, and the quadruple mutant <i>al6 al7 Atbmi1a Atbmi1b</i> plated on MS (top graph), MS supplemented with 200 mM mannitol (middle graph) or with 100 mM NaCl (bottom graph). Data represent average germination percentages ± SD of three biological replicates, each >60 seeds, observed daily for 12 days after stratification.</p

A proposed model for AL PHD-PRC1 complexes in silencing seed developmental genes during seed germination.

<p>ALs, <i>via</i> their highly conserved PHD domains, bind H3K4me3 of chromatin, triggering the recruitment of PRC1 components BMI1 and RING1 <i>via</i> AL-AtBMI1, AL-AtRING1, and AtBMI1-AtRING1 physical interactions. Next, two possible pathways (1 and 2) can lead to stable repressive chromatin state formation. In the first case (1), PRC2 is recruited <i>via</i> its subunit CLF interaction with AtRING1 and deposits H3K27me3, favoring further LHP1 recruitment <i>via</i> H3K27me3-LHP1 binding. In the second case (2), LHP1 is first recruited <i>via</i> its interaction with AtRING1 or AtBMI1, and then PRC2 is recruited <i>via</i> its subunit MSI1 interaction with LHP1 and deposits H3K27me3. In both cases, H3K27me3-LHP1 and PRC2 MSI1-LHP1 interactions form a positive loop in H3K27me3 enrichment. This hypothetic model can explain how seed developmental genes (<i>e.g. ABI3</i>, <i>DOG1</i>) are switched from active transcription to a stably repressed state, which is necessary for timely seed germination and proper seedling growth and development.</p

Relative expression levels of seed developmental genes in Col-0, <i>al6 al7</i> and <i>Atbmi1a Atbmi1b</i>.

<p>Relative expression levels of <i>ABI3</i>, <i>DOG1</i>, <i>CRU1</i>, <i>CRU3</i>, <i>PER1</i> and <i>CHO1</i> were analyzed by quantitative RT-PCR using seeds/seedlings at 0, 24 and 72 hours after stratification. Data represent means ± SD of three biological replicates.</p

Interactions of ALs and PRC1 ring-finger proteins in yeast two-hybrid assay.

<p>(A) Schematic representation of full-length and truncated AtRING1a and AL6 proteins. The conserved domains PAL, PHD, RING and RAWUL are indicated. (B) Yeast two-hybrid assays. Yeast cultures co-expressing the indicated protein combinations from pGADT7 and pGBKT7 were plated as a 1∶10 dilution from left to right onto SD-LTA selective media. Growth of yeast cells indicates positive protein-protein interaction.</p

Regulation of <i>Arabidopsis</i> Flowering by the Histone Mark Readers MRG1/2 via Interaction with CONSTANS to Modulate <i>FT</i> Expression

<div><p>Day-length is important for regulating the transition to reproductive development (flowering) in plants. In the model plant <i>Arabidopsis thaliana</i>, the transcription factor CONSTANS (CO) promotes expression of the florigen <i>FLOWERING LOCUS T</i> (<i>FT</i>), constituting a key flowering pathway under long-day photoperiods. Recent studies have revealed that <i>FT</i> expression is regulated by changes of histone modification marks of the <i>FT</i> chromatin, but the epigenetic regulators that directly interact with the CO protein have not been identified. Here, we show that the <i>Arabidopsis</i> Morf Related Gene (MRG) group proteins MRG1 and MRG2 act as H3K4me3/H3K36me3 readers and physically interact with CO to activate <i>FT</i> expression. <i>In vitro</i> binding analyses indicated that the chromodomains of MRG1 and MRG2 preferentially bind H3K4me3/H3K36me3 peptides. The <i>mrg1 mrg2</i> double mutant exhibits reduced mRNA levels of <i>FT</i>, but not of <i>CO</i>, and shows a late-flowering phenotype under the long-day but not short-day photoperiod growth conditions. MRG2 associates with the chromatin of <i>FT</i> promoter in a way dependent of both CO and H3K4me3/H3K36me3. <i>Vice versa</i>, loss of <i>MRG1</i> and <i>MRG2</i> also impairs CO binding at the <i>FT</i> promoter. Crystal structure analyses of MRG2 bound with H3K4me3/H3K36me3 peptides together with mutagenesis analysis <i>in planta</i> further demonstrated that MRG2 function relies on its H3K4me3/H3K36me3-binding activity. Collectively, our results unravel a novel chromatin regulatory mechanism, linking functions of MRG1 and MRG2 proteins, H3K4/H3K36 methylations, and CO in <i>FT</i> activation in the photoperiodic regulation of flowering time in plants.</p></div

MRG2 and CO enhance each other's binding to the <i>FT</i> promoter.

<p>A. Enrichment of MRG2 at the <i>FT</i> promoter at ZT16 in wild-type and <i>co</i> mutant at ZT16. Error bars show standard deviation from three biological replicates. Asterisks indicate statistically significant differences between <i>co</i> and the wild-type (P<0.01). B. Enrichment of MYC-CO at the <i>FT</i> promoter at ZT16 in indicated genotypes at ZT16. Error bars show standard deviation from three biological replicates. Asterisks indicate statistically significant differences between <i>35S::MYC-CO/mrg1 mrg2</i> plants and <i>35S::MYC-CO</i> plants (P<0.01). C. Protein levels of MYC-tagged CO in indicated genotypes at ZT16.</p

MRG1 and MRG2 act redundantly in flowering time control of <i>Arabidopsis</i> in the photoperiodic flowering pathway.

<p>A. Gene structure of <i>mrg1</i> and <i>mrg2</i> mutant alleles. Dark boxes represent exons; lines represent introns; red triangles indicate T-DNA insertions. B. RT-PCR analysis of <i>MRG1</i> and <i>MRG2</i> expression in leaves of <i>mrg1</i>, <i>mrg2</i>, and <i>mrg1 mrg2</i> plants. <i>ACTIN2</i> was used as the internal control. C. Phenotypes of the wild-type (WT), the single mutants <i>mrg1</i> and <i>mrg2</i>, and the double mutant <i>mrg1 mrg2</i> grown under long-day photoperiods (LD; 16 h light: 8 h dark). D. Phenotypes of WT and <i>mrg1 mrg2</i> plants grown under short-day photoperiods (SD; 8 h light: 16 h dark). E. Flowering time, as measured by rosette leaf number at bolting, in plants grown under LD and SD conditions. The mean value from 20 plants is shown. Error bars represent standard deviations. F. Tissue expression pattern analyses of <i>MRG1</i> and <i>MRG2</i> by histochemical GUS staining in <i>P_MRG1::MRG1-GUS</i> and <i>P_MRG2::MRG2-GUS</i> transgenic plants. Staining was performed at different times after seed germination (day 5 or 12), and in inflorescences.</p

Chromodomains of MRG1 and MRG2 specifically bind to tri-methylated H3K4 and H3K36 <i>in vitro</i>.

<p>A. Binding assays of N-terminal His-tagged chromodomains of MRG1 (His-MRG1N) and MRG2 (His-MRG2N) with H3 peptides containing varying degrees of methylation at K4, K9, K27, or K36. B. ITC measurements of binding between the MRG2 chromodomain and histone peptides. Top panel, MRG2 and H3K4me3 peptide; bottom panel, MRG2 and H3K36me3 peptide.</p