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. 2019 Oct 22;182(1):555–565. doi: 10.1104/pp.19.00793

HISTONE DEACETYLASE 9 Functions with Polycomb Silencing to Repress FLOWERING LOCUS C Expression1

Xiaolin Zeng a,b,2, Zheng Gao a,b,2, Chuan Jiang c,3, Yupeng Yang a,b, Renyi Liu c,d, Yuehui He a,4,5
PMCID: PMC6945841  PMID: 31641076

Histone deacetylase, Polycomb repressive complex, and epigenome readers through a cis-element mediate histone deacetylation and methylation to repress FLOWERING LOCUS C in rapid-cycling accessions.

Abstract

Polycomb repressive complex 2 (PRC2) catalyzes repressive histone 3 Lys-27 trimethylation (H3K27me3) to mediate genome-wide transcriptional repression in plants and animals. PRC2 controls various developmental processes in plants and plays a critical role in the developmental transition to flowering. FLOWERING LOCUS C (FLC), first identified in Arabidopsis (Arabidopsis thaliana), is a potent floral repressor in crucifers and some other plants that is subjected to complex regulation. Here, we show that HISTONE DEACETYLASE 9 (HDA9)-mediated H3K27 deacetylation is required for PRC2-mediated H3K27me3 in Arabidopsis. We further demonstrate that through physical association with the epigenome readers VP1/ABI3-LIKE 1 (VAL1) and VAL2, which recognize a cis-regulatory element at the FLC locus, HDA9 and PRC2 function in concert to mediate H3K27 deacetylation and subsequent trimethylation at this residue. This leads to FLC repression in the rapid-cycling Arabidopsis accessions. Our study uncovers roles for HDA9 in PRC2-mediated H3K27me3, FLC repression, and flowering-time regulation.

Polycomb group (PcG) proteins are highly conserved in plants and animals and function as transcriptional repressors of developmental gene expression (Merini and Calonje, 2015; Wang and Shen, 2018). They encompass two distinct multiprotein complexes, namely Polycomb repressive complex1 (PRC1) and PRC2. PRC2 catalyzes repressive histone 3 Lys-27 trimethylation (H3K27me3), while PRC1 acts to maintain the repressive H3K27me3 mark and deposit histone H2A Lys 118 monoubiquitination (Pu and Sung, 2015; Förderer et al., 2016). First identified in Drosophila, the PRC2 complex is well defined and evolutionarily conserved in multicellular organisms. It is composed of four core subunits including the H3K27 methyltransferase Enhancer of Zeste [E(z)] and three structural subunits known as Esc, Su(z)12, and P55 (Pu and Sung, 2015; Wang and Shen, 2018). Compared to PRC2, PRC1 is less defined and likely exists in several complexes with varying composition (Pu and Sung, 2015; Wang and Shen, 2018).

In Arabidopsis (Arabidopsis thaliana), small gene families encode the four PRC2 core components and individual PRC2 complexes can repress unique subsets of target genes to control different developmental processes (Förderer et al., 2016; Schubert, 2019). There are three homologs of the Drosophila H3K27 methyltransferase E(z), including CURLY LEAF (CLF), SWINGER (SWN), and MEDEA (MEA), among which CLF plays a major role in vegetative growth and development and in the developmental switch from a vegetative phase to reproduction (i.e. flowering; Chanvivattana et al., 2004); in addition, Arabidopsis possesses three and five homologs of Su(z)12 and P55, respectively (Förderer et al., 2016; Schubert, 2019). Genome-wide profiling revealed that thousands of genes bear H3K27me3 in vegetative phases, and that various development-regulatory genes are repressed by PcG proteins (Zhang et al., 2007; Lafos et al., 2011; Lu et al., 2011; Wang et al., 2016); hence, Polycomb silencing plays an essential role in the control of plant growth and development. A few PRC2-associated proteins have been described as recruiting or modulating PRC2 activity in mediating dynamic H3K27me3 deposition (Yuan et al., 2016; Xiao et al., 2017; Zhou et al., 2018b; Sun et al., 2019). For instance, the plant-specific B3-domain epigenome readers VAL1 and VAL2 recognize a six-nucleotide RY motif and further engage PRC2 to deposit H3K27me3 on target gene chromatin, leading to transcriptional repression in Arabidopsis and rice (Oryza sativa; Yuan et al., 2016; Xie et al., 2018). These Polycomb partners function together with PRC1 and/or PRC2 to meditate transcriptional repression in plants.

In addition to histone methylation, the Lys residues on histones can be covalently modified by histone acetyltransferases and deacetylase (HDACs). Histone acetylation is often linked to transcriptional activation, while histone deacetylation is typically associated with gene repression (Liu et al., 2014; Shen et al., 2015). In Arabidopsis, there are 18 putative HDACs that are grouped into three types. RPD3-like type I HDACs consist of 12 members, among which are HISTONE DEACETYLASE 5 (HDA5), HDA6, HDA9, and HDA19 (Hollender and Liu, 2008; Liu et al., 2014). HDA6 is involved in transgene silencing, control of ribosomal RNA transcription, and regulation of aspects of plant development such as flowering time and plant stress responses (Earley et al., 2010; Yu et al., 2017). HDA9 plays multiple roles in plant growth and developmental processes including seed germination, flowering time, leaf senescence, and salt and drought stress responses (Kim et al., 2016; Zheng et al., 2016; Mayer et al., 2019). In addition, it has been shown that HDA9 moderately promotes flowering under noninductive day lengths (short days) in Arabidopsis, a facultative long-day plant (Kim et al., 2013; Kang et al., 2015). Recent studies revealed that the SANT-domain protein POWERDRESS directly interacts with HDA9 to promote histone H3 deacetylation at various loci (Kim et al., 2016; Mayer et al., 2019). To date, a few HDACs from plants have been characterized, but whether and how HDACs may function in concert with other repressive chromatin modifiers, such as Polycomb proteins, to mediate transcriptional repression in plants have remained elusive.

The transition to flowering, a major developmental switch in the plant life cycle, is regulated by histone deacetylation and histone methylation (Amasino, 2010). In Arabidopsis, the MADS-box transcription factor FLOWERING LOCUS C (FLC) functions as a potent floral repressor and plays a central role in floral repression (Michaels and Amasino, 1999; Sheldon et al., 1999). FLC expression is repressed by autonomous pathway (AP) genes such as FVE, FLOWERING LOCUS D (FLD), and LUMINIDEPENDENS (LD; Amasino, 2010). FRIGIDA (FRI), encoding a plant-specific scaffold protein, activates FLC expression to inhibit flowering, whereas prolonged cold exposure or vernalization overrides FRI’s function to promote flowering, leading to the establishment of a vernalization-responsive winter-annual growth habit (Kim et al., 2009; Choi et al., 2011). FLC directly represses the expression of flowering pathway integrator genes including FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 to inhibit flowering (Helliwell et al., 2006; Searle et al., 2006). FLC repressors including FLD and FVE are known to function together with HDACs (He et al., 2003; Ausín et al., 2004; Yu et al., 2011). Several studies have shown that HDA5 and HDA6 associate with FVE and FLD to form a corepressor complex that mediates histone H3K9/H3K14 deacetylation to repress FLC expression in rapid-cycling accessions that lack a functional FRI, and thus promote the floral transition (Gu et al., 2011; Yu et al., 2011; Luo et al., 2015). Interestingly, another RPD3-type HDAC, HDA9, was reported not to be involved in FLC expression (Kang et al., 2015); HDA9 has been shown to directly repress the expression of AGAMOUS-LIKE 19 (AGL19), which represses FT expression in short days to inhibit flowering (Kang et al., 2015). Thus, discrete HDACs play different roles in flowering-time regulation.

Polycomb silencing plays a critical role in flowering-time control through direct repression of several key flowering-regulatory genes, including FLC and FLOWERING LOCUS T (FT, encoding a major mobile florigen) in Arabidopsis (Turck et al., 2007; Jiang et al., 2008). A CLF-containing PRC2 (CLF-PRC2) catalyzes H3K27 trimethylation on FLC and FT chromatin to repress both gene expression (Jiang et al., 2008). At the FLC locus, the Polycomb partners VAL1 and VAL2 bind to the RY motifs in the cis-regulatory Cold Memory Element (CME) located around the junction of the first exon and first intron. There, they recruit PRC2 to deposit H3K27me3 during prolonged cold exposure as well as under normal growth temperatures, leading to FLC repression (Qüesta et al., 2016; Yuan et al., 2016). In loss-of-function clf mutants, both FLC and FT are de-repressed, and FT upregulation overrides FLC upregulation and results in early flowering in clf mutants (Jiang et al., 2008; Doyle and Amasino, 2009). Interestingly, in an elegant genetic screen for suppressors of the late-flowering ld mutants (note that the autonomous-pathway gene LD represses FLC expression to promote flowering; Lee et al., 1994), a gain-of-function point mutant of CLF, clf-59D, was identified as a strong ld suppressor. Subsequent studies revealed that clf-59D strongly represses FLC expression through elevated H3K27me3, but with a moderate effect on FT repression, resulting in early flowering (Doyle and Amasino, 2009). Although both Polycomb silencing and histone acetylation/deacetylation have been shown to mediate FLC regulation and flowering-time control, whether (and how) they may function in concert to regulate FLC expression is unknown.

In this study, we identified an hda9 mutation that suppressed clf-59D-mediated FLC silencing and early flowering, and further found that HDA9 is required for FLC repression by CLF-PRC2. We show that HDA9 mediates global histone deacetylation preferentially at H3K27 and that HDA9-mediated H3K27 deacetylation is a prerequisite for H3K27me3 in the Arabidopsis genome. Moreover, we found that HDA9 physically interacts with the Polycomb partners VAL1 and VAL2 and that, like VAL1 and VAL2, HDA9 is enriched at the CME region to mediate H3K27 deacetylation at FLC. Together, our findings suggest that HDA9 and CLF-PRC2, through physical association with VAL1 or VAL2, function together to mediate H3K27 deacetylation and subsequent H3K27me3 at the FLC locus to repress its expression and regulate the floral transition.

RESULTS

Identification of a Suppressor of clf-59D-Mediated Early Flowering

To identify CLF partners that are involved in FLC repression and floral promotion in Arabidopsis, we performed ethyl methanesulfonate mutagenesis using an early-flowering clf-59D ld background (Doyle and Amasino, 2009). The gain-of-function clf-59D suppresses FLC de-repression in ld-3 and the late-flowering phenotype through elevated H3K27me3 at FLC (Doyle and Amasino, 2009). If a gene is required for clf-59D or CLF function, loss-of-function mutations in this locus would lead to a disruption in clf-59D-mediated FLC repression in the ld backgrounds and a noticeable late-flowering phenotype. We first introgressed clf-59D (in an Arabidopsis ecotype Wassilewskija [Ws] background) into the late-flowering ld-1 (in an Arabidopsis ecotype Columbia [Col] background; Lee et al., 1994) through multiple backcrosses, and the resulting early-flowering clf-59D ld-1 was mutagenized by ethyl methanesulfonate to create a mutant population. In the subsequent screening for genetic partners of clf-59D, a few late-flowering mutants were recovered, among which was clf-59D SUPPRESSOR 1 (clf-s1; Fig. 1, A and B). To determine whether the late-flowering phenotype in clf-s1 results from FLC de-repression, we measured the FLC transcript levels and found that FLC was strongly de-repressed in clf-s1 (Fig. 1C); thus, CLF-S1 is required for clf-59D mediated FLC repression upon functional loss of the autonomous-pathway gene LD. In addition to FLC, FLC clade members including FLOWERING LOCUS M (FLM)/MADS AFFECTING FLOWERING 1 (MAF1) to MAF5 also play a role in floral repression (Scortecci et al., 2001; Ratcliffe et al., 2003; Kim and Sung, 2010; Gu et al., 2013). We thus examined the expression of MAF1–MAF5 in clf-s1 and found that the transcript levels of these MAFs were moderately increased compared with the parental line of clf-59D ld-1 (Supplemental Fig. S1).

Figure 1.

Figure 1.

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clf-s1 suppresses FLC silencing and early flowering in clf-59D ld-1. A, clf-59D ld-1 (parental line) and clf-s1 plants grown in long days. Scale bars = 1 cm. B, Total leaf number at flowering. Twelve plants (grown in long days) per line were scored, and error bars indicate the sd. One-way ANOVA was conducted, and the lowercase letters denote distinct groups (P < 0.01). WT, wild type. C, Relative expression levels of FLC in clf-s1 and clf-59D ld-1 seedlings. FLC transcripts were quantified by RT-qPCR and normalized to the constitutively expressed TUBULIN 2 (TUB2). Bars indicate the sd of three biological replicates. One-way ANOVA was conducted, and the lowercase letters denote distinct groups (P < 0.01).

We further constructed a mapping population by crossing clf-s1 (in clf-59D ld-1) to clf-59D ld-3 (in a Ws background), and noticed that 65 out of 250 F2 plants showed late flowering (with a segregation ratio of ∼1:3 late:early flowering). Subsequent mapping delimited clf-s1 into a 700-kb interval located in the low arm of chromosome 3 (Fig. 2A; Supplemental Table S1), and further genome resequencing revealed a C-to-T mutation in HDA9 located in this interval, which resulted in the conversion of Ala-138 to Val in the conserved histone deacetylase domain (Fig. 2B; Supplemental Table S2). CLF-S1 was therefore renamed as HDA9 (the clf-s1 mutation was designated as hda9-3 accordingly). To confirm that the suppression of the clf-59D ld-1 phenotype was caused by hda9-3, we introduced a copy of native genomic HDA9 (gHDA9) into clf-s1 (clf-59D ld-1 hda9-3), and the mutant phenotypes were fully rescued by gHDA9 in multiple transgenic lines (Fig. 2C; Supplemental Fig. S2A). In addition, we introduced another hda9 allele, hda9-1 (bearing an insertional T-DNA; Kim et al., 2013), into clf-59D ld-1, and found that like clf-s1, the clf-59D ld-1 hda9-1 mutant exhibited late flowering with elevated FLC expression (Fig. 2, D and E). Taken together, these results show that HDA9 is required for clf-59D-mediated FLC silencing and early flowering.

Figure 2.

Figure 2.

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Positional cloning of the CLF-S1 gene and complementation of clf-s1 by HDA9. A, Rough mapping of clf-s1 on chromosome 3. chr3-3, chr3-5, chr3-12, and nga111 are simple sequence-length polymorphism markers. B, Amino acid sequence alignment of the HDAC domains from HDA9, HDA6, and HDA19 using ClustalW. clf-s1 bears a missense C-to-T mutation in HDA9, resulting in the mutation of the 138th Ala to Val. C, Rescue of clf-s1 by a wild-type copy of genomic HDA9. Flowering times were scored in clf-59D ld-1, clf-s1, and two independent transgenic lines of clf-s1 with HDA9. Eleven to 12 plants per line were scored; error bars represent the sd. D and E, Introduction of the hda9-1 allele into clf-59D ld-1 suppresses early-flowering (D) and FLC silencing (E). Total leaf number at flowering from 11–17 plants were scored per line, and error bars in D indicate the sd. Transcript levels were quantified by RT-qPCR and normalized to TUB2, and bars in E indicate the sd of three biological replicates. One-way ANOVA was conducted (C and D), and lowercase letters denote distinct groups (P < 0.01). A two-tailed Student's t test was conducted (E); **P < 0.01.

HDA9 Is Required for FLC Repression in Both Wild Type and clf-59D

A previous study indicates that HDA9 is not likely involved in FLC repression (Kang et al., 2015). We segregated hda9-3 from clf-59D and ld-1 and found that FLC expression was apparently de-repressed in hda9-3 relative to the wild-type Col-0 (a rapid-cycling accession; Fig. 3A). In addition, we found that FLC expression was de-repressed in hda9-1 seedlings (Supplemental Fig. S2B). Thus, HDA9 indeed functions to repress FLC expression.

Figure 3.

Figure 3.

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Genetic interactions of hda9 (hda9-3) with clf and clf-59D. A and B, Analysis of FLC expression in the indicated seedlings. Bars indicate the sd of three biological replicates. Analysis was conducted by one-way ANOVA (A), with lowercase letters denoting distinct groups (P < 0.01), and by two-tailed Student's t test (B). C and D, Flowering times of the indicated lines grown in long days. Ten to 16 plants per line were scored; error bars represent sd. One-way ANOVA was conducted, and letters denote distinct groups (P < 0.01). E, Relative expression of FT in the indicated seedlings (grown in long days). Bars indicate the sd of three biological replicates; a two-tailed Student's t test was conducted. ns, not significant.

We further examined FLC expression in clf-59D and hda9-3 clf-59D. The gain-of-function clf-59D strongly suppressed FLC expression in the Col background, but this suppression required HDA9, because FLC expression in hda9 clf-59D reverted back to the wild-type level (Fig. 3A). Next, to explore whether CLF requires HDA9 to repress FLC expression, we constructed a clf-29 hda9 double mutant in which clf-29 is a loss-of-function null allele (Makarevich et al., 2006). The levels of FLC transcripts in clf-29 were strongly de-repressed, and introduction of hda9 into clf-29 did not cause further de-repression of FLC expression (Fig. 3B), consistent with the notion that CLF and HDA9 function in the same genetic pathway to repress FLC expression.

To explore the role of HDA9 in flowering-time regulation, we measured the flowering times of hda9, clf-59D, clf-29, hda9 clf-59D, and hda9 clf-29 in long days. Interestingly, hda9 flowered like the wild type, and clf-29, like the gain-of-function clf-59D, flowered earlier than the wild type, inconsistent with the fact that FLC is de-repressed in both hda9 and clf-29 (Fig. 3, C and D). CLF (PRC2) plays a key role in the developmental transition to flowering through direct repression of FLC and FT expression (Jiang et al., 2008), and FT functions to promote flowering. In addition, it has been shown that HDA9 acts to repress FT expression (Kang et al., 2015). We reasoned that the unexpected flowering phenotypes in hda9 and clf-29 may be partially attributed to FT de-repression. Indeed, FT expression was apparently upregulated in both hda9 and clf-29 (Fig. 3E). Together, these results reveal that both HDA9 and CLF repress the expression of FLC, as well as that of FT, to control flowering time in Arabidopsis.

HDA9 Associates with FLC Chromatin and Mediates Histone Deacetylation

To determine whether HDA9 directly represses FLC expression, we conducted chromatin immunoprecipitation (ChIP) using a fully functional HDA9:Flag-expressing line (driven by a native HDA9 promoter region; Supplemental Fig. S3). HDA9 was enriched in the nucleation region of Polycomb silencing located near the 5′ end of the first intron of FLC, where the cis-regulatory CME is located. In addition, HDA9 was moderately enriched in regions downstream of the CME (Fig. 4A). These results show that HDA9 directly regulates FLC expression.

Figure 4.

Figure 4.

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HDA9 is required for CLF function at the FLC locus. A, ChIP analysis of HDA9:Flag enrichment at FLC in the indicated seedlings. On the left is a schematic drawing of the genomic FLC structure, and gray bars (right) denote the examined regions in ChIP. Immunoprecipitated genomic fragments were quantified by qPCR and normalized to TUB2. The fold enrichment of HDA9:Flag over that of Col (control) in each examined FLC region in the HDA9:Flag line is shown. TSS, transcription start site. B, ChIP analysis of CLF enrichment at FLC in wild-type (Col-0) and hda9-3 seedlings. Genomic fragments immunoprecipitated by a rabbit polyclonal antibody recognizing the native CLF were quantified by qPCR and normalized to TUB2. The fold enrichment of CLF over that of CK (wild type immunoprecipitated with rabbit serum) in each examined region in the wild type or hda9 is shown. C and D, ChIP analysis of H3K27ac (C) and H3K27me3 (D) on FLC chromatin in wild-type, hda9-3, and/or clf-29 seedlings. Relative fold changes in each region were calculated by normalizing first to input, then to the endogenous control TUB2. Bars indicate the SD of three biological replicates. Analysis was conducted by two-tailed Student's t test (**P < 0.01; C), and by one-way ANOVA (P < 0.05; D) with lowercase letters denoting distinct groups. ns, not significant.

Next, to address whether HDA9 mediates histone deacetylation on FLC chromatin, we carried out ChIP assays in wild-type and hda9 seedlings with an antibody to recognize acetylated Lys-27 on H3 (H3K27ac). The requirement of HDA9 for CLF-mediated FLC repression, as well as the enrichment of HDA9 at the CME-bearing nucleation region of Polycomb silencing, led us to reason that HDA9 may mediate H3K27 deacetylation to facilitate H3K27me3 by CLF at FLC. Indeed, a functional loss of HDA9 resulted in an apparent increase of H3K27ac in the CME-bearing nucleation region (Fig. 4C). Thus, HDA9 directly mediates histone deacetylation at FLC to repress its expression.

HDA9 Is Partly Required for CLF Enrichment on FLC Chromatin

To explore the functional coordination of HDA9 with CLF-PRC2, we first explored whether HDA9 is required for CLF enrichment on FLC chromatin using ChIP with an antibody that recognizes the native CLF protein (Tao et al., 2019). We found that the level of CLF bound to the CME region was reduced in hda9 relative to the wild type (Fig. 4B). Thus, HDA9 is partly required for CLF enrichment on FLC chromatin. Notably, the HDA9 homolog HDA19 is linked with PRC2-mediated FLC repression (Supplemental Fig. S4A; Qüesta et al., 2016); consistently, in the hda9 hda19 double mutant the enrichment of CLF at the CME region was strongly reduced (Supplemental Fig. S4C).

Next, using ChIP with anti-H3K27me3, we examined whether H3K27me3 on FLC chromatin is disrupted in hda9. Consistent with the role of CME in Polycomb silencing, H3K27 residues were trimethylated at a higher level around the CME region compared to other regions (Fig. 4D). Loss of HDA9 function led to an apparent reduction in H3K27me3 at CME (Fig. 4D). Thus, HDA9 is partly required for CLF-mediated H3K27me3 at FLC.

We further explored whether CLF might be required for HDA9 enrichment at FLC. HDA9:Flag was introduced into clf-29 by genetic crossing, followed by ChIP assays with anti-Flag. The levels of HDA9:Flag bound to the CME region, as well as other regions, remained nearly unchanged upon functional loss of CLF (Supplemental Fig. S4D). Thus, CLF is not required for HDA9 to bind FLC chromatin, consistent with the idea that HDA9-mediated H3K27 deacetylation is a prerequisite for PRC2-mediated H3K27me3, but not vice versa.

HDA9 Interacts with Polycomb Partners Including VAL1 and VAL2

The cis-regulatory CME is recognized by transacting VAL1 and VAL2, which physically associate with the PRC2 subunit MSI1 and the H3K27me3 reader LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) to mediate Polycomb silencing at the FLC locus (Yuan et al., 2016; Chen et al., 2018). We have observed that HDA9 is enriched at the CME region and required for FLC repression by CLF-PRC2. This prompted us to explore whether HDA9 physically associates with VAL1 and/or VAL2 to mediate FLC repression. Yeast two-hybrid assays revealed that the full-length HDA9 interacted with the full-length VAL1 and an N-terminal VAL2 fragment (amino acids 1–393) in yeast cells (Fig. 5, A and B). Next, we constructed a fully functional HDA9:HA line (Supplemental Fig. S3) and crossed this line to a functional VAL1:Flag line (Yuan et al., 2016); in subsequent coimmunoprecipitation assays using the F1 seedlings expressing HDA9:HA and VAL1:Flag, HDA9:HA was pulled down by an antibody recognizing VAL1:Flag (Fig. 5C). These results together show that HDA9 physically associates with VAL1 in Arabidopsis, providing a molecular link between HDA9 and CLF-PRC2, namely HDA9-VAL1-PRC2.

Figure 5.

Figure 5.

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HDA9 physically associates with VAL1 and VAL2. A and B, HDA9 interacted with VAL1 (A) and VAL2 (B) in yeast cells. The full-length HDA9 was fused with GAL4-AD, whereas the full-length VAL1 or N-terminal VAL2 (amino acids 1–393) was fused with the GAL4-BD. Yeast cells were grown on selective synthetic dropout (SD)media lacking Trp (W), Leu (L), His (H), and/or adenine (A); yeast cells on synthetic dropout media lacking W and L serve as growth control. C, Coimmunoprecipitation assay of HDA9 with VAL1. HDA9-HA was immunoprecipitated by anti-Flag recognizing VAL1-Flag from the F1 seedlings expressing both fusion proteins (double hemizygotes). Asterisks indicate a degradation product of VAL1-Flag (Yuan et al., 2016).

HDA9 Mediates Global Histone Deacetylation Preferentially at H3K27

Previous studies have revealed that loss of HDA9 function leads to moderate elevation of acetylation at several Lys residues on total histones extracted from Arabidopsis (Kim et al., 2013, 2016; Mayer et al., 2019), but the preferential histone Lys residues for HDA9, if any, remain to be determined. Given the functional coordination of HDA9 with CLF-PRC2, we further explored whether HDA9 mediates histone deacetylation preferentially at H3K27. Total histones were extracted from hda9 seedlings, followed by quantitative immunoblotting using antibodies that recognize specific acetylated Lys residues on H3 or H4. Among the examined acetylated H3 or H4 residues, we found that loss of hda9 function had no effect on H3K23 or H4K5 acetylation, and a moderate effect on H3K9, H3K18, and H3K14 acetylation, but gave rise to a great increase in acetylated H3K27 (Fig. 6A). These results reveal that HDA9 preferentially deacetylates H3K27 in the Arabidopsis genome.

Figure 6.

Figure 6.

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HDA9-mediated H3K27 deacetylation and PRC2-catalyzed H3K27 trimethylation are coordinated for transcriptional repression in Arabidopsis. A, Levels of histone acetylation at the indicated residues on total histones were measured by immunoblotting. Total histones were extracted from wild-type (WT), hda9-1, and clf seedlings, followed by immunoblotting, and the blotting signals were scanned by ECL Plus ChemiDocTM Touch Imaging System. On the left are representative western blots. B, Analysis of the levels of H3K27me3 on total histones from the indicated seedlings. On the bottom is one set of western blots. C, Overlap between the upregulated genes in hda9 and the H3K27me3-bearing loci in seedlings. The list of genes upregulated in hda9-1 (relative to the wild type) and the genes bearing H3K27me3 at a seedling stage were extracted from published data (Wang et al., 2016; Zheng et al., 2016). P value, Fisher’s exact test. Values in A and B are means ± sd of three biological replicates; one-way ANOVA was conducted, and lowercase letters denote distinct groups (P < 0.05).

HDA9-Mediated H3K27 Deacetylation and PRC2-Catalyzed H3K27me3 Are Coordinated for Transcriptional Repression

Given the functional coordination between HDA9 and CLF-PRC2 in FLC repression, we reasoned that an antagonistic switch between H3K27ac and H3K27me3 may exist in the Arabidopsis genome. We further examined global H3K27ac and H3K27me3 in clf-29 and the gain-of-function clf-59D mutant, and found that gain of function of CLF caused a marked reduction in H3K27ac, whereas loss of CLF function in clf-29 led to a great increase in H3K27ac on total histones (Fig. 6A), which may be related to the competitive nature between CLF-PRC2 and histone acetyltransferases for the H3K27 residue at certain loci. In addition, loss of HDA9 function gave rise to a reduction in H3K27me3 on total histones (Fig. 6B), consistent with the idea that HDA9-mediated H3K27 deacetylation is a prerequisite for H3K27me3 by PRC2 at genomic sites bearing H3K27ac. Taken together, these results reveal an antagonistic switch between H3K27ac and H3K27me3 in the Arabidopsis genome, and the coordination of H3K27 deacetylation with H3K27me3.

To further explore whether HDA9 functions in concert with PRC2 for transcriptional repression at loci other than FLC, we examined the overlap between HDA9-repressed loci and H3K27me3-marked loci in Arabidopsis seedlings using published data (Wang et al., 2016; Zheng et al., 2016). Of the 877 genes de-repressed in hda9, 211 are PRC2 targets, as they bear the H3K27me3 mark (Fig. 6C). Collectively, these results suggest that HDA9 and PRC2 may function synergistically to mediate H3K27 deacetylation and subsequent H3K27me3 for transcriptional repression at various loci.

DISCUSSION

In this study, we found that HDA9 is required for CLF (PRC2)-mediated H3K27me3 and Polycomb silencing. Both HDA9 and CLF-PRC2 physically associate with VAL1 and VAL2, which recognize the cis-regulatory CME at the FLC locus and function in concert to mediate H3K27 deacetylation and subsequent trimethylation at this residue, leading to FLC repression in the rapid-cycling accessions. Thus, our study uncovers a previously undescribed role of HDA9 in PRC2-mediated H3K27me3 and FLC repression.

Polycomb silencing regulates the expression of various developmental genes in the Arabidopsis genome (Lafos et al., 2011; Wang and Shen, 2018). H3K27me3 is a dynamic modification and balanced by methylation and demethylation (Lu et al., 2011). PcG proteins are known to switch off the expression of various genes in response to developmental and/or environmental inputs such as developmental transitions, biotic and abiotic stresses, and prolonged cold exposure (vernalization; Kim et al., 2009; Molitor et al., 2014, 2016; He and Li, 2018). Acetylation of H3K27 and other residues on core histone tails is associated with active gene expression (Liu et al., 2014; Shen et al., 2015). H3K27 deacetylation is a prerequisite for PRC2-mediated H3K27me3 and transcriptional repression if a locus has been marked by H3K27ac. Furthermore, about a quarter of the HDA9-repressed genes are PRC2 targets (Fig. 6C). In addition to FLC, we confirmed that a development-regulatory MADS-box transcription factor, AGL24 (involved in the floral transition; Yu et al., 2002; Michaels et al., 2003), and a biotic stress-responsive transcription factor WRKY48 (Xing et al., 2008) are corepressed by HDA9 and CLF-PRC2 (Supplemental Fig. S5, A and B). These findings suggest that histone deacetylation and Polycomb silencing function together to repress gene expression in the Arabidopsis genome.

In several studies, Arabidopsis homologs of several core subunits in the yeast Rpd3 histone deacetylase complex have been copurified with MSI1 and other PcG proteins (Derkacheva et al., 2013; Chhun et al., 2016; Zhou et al., 2018a), indicating an association of HDAC activity with PRC2 activity. We found that the DNA-binding proteins VAL1 and VAL2 associate not only with PcG proteins (Yuan et al., 2016) but also with HDA9 (Fig. 5). In addition, the HDA9 homolog HDA19 also physically associates with VAL1 (Qüesta et al., 2016) and, like HDA9, functions to repress FLC expression (Supplemental Fig. S4A). These findings suggest that HDA9, HDA19, and PRC2 are connected through VAL proteins for transcriptional repression. Consistent with this notion, we found that HDA9 and HDA19 are required for CLF-PRC2 enrichment on FLC chromatin (Supplemental Fig. S4C). Taken together, our findings suggest a model whereby HDA9-mediated H3K27 deacetylation and PRC2-mediated H3K27me3 function in concert to repress (or switch off) target gene expression (Fig. 7).

Figure 7.

Figure 7.

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A working model for the recruitment of HDA9 and CLF-PRC2 by VAL1 and/or VAL2 bound to CME at the FLC locus. Through their physical association with VAL1/VAL2, HDA9 and CLF-PRC2 function in concert to repress (or switch off) target gene (e.g. FLC) expression. HDA9 deacetylates H3K27 to enable subsequent H3K27me3 by CLF-PRC2, leading to FLC repression in Arabidopsis.

There are 18 histone deacetylases in the Arabidopsis genome (Hollender and Liu, 2008), and discrete HDACs may have substrate preference. Previous studies revealed that HDA5 and HDA6 mediate H3K9/H3K14 deacetylation to repress the expression of several examined target loci, including FLC (Yu et al., 2011; Luo et al., 2015). In this study, we found that HDA9 has a preference for acetylated H3K27, consistent with its role in CLF-mediated H3K27me3. PRC2s are known to regulate thousands of genes in the Arabidopsis genome (Lafos et al., 2011; Lu et al., 2011; Wang et al., 2016), and loss of HDA9 function leads to de-repression of only hundreds of PRC2 targets; it is very likely that other HDACs (e.g. HDA19) may participate in H3K27 deacetylation for subsequent H3K27me3 by PRC2s.

Multiple HDACs are involved in the repression of the central floral repressor FLC. It has been shown that HDA5 and HDA6 mediate H3K9/H3K14 deacetylation to repress FLC expression in the rapid-cycling accessions, and that both associate with the histone H3K4 demethylase FLD and the histone binding protein FVE to form a corepressor complex that possesses H3K4 demethylation activity in addition to histone deacetylation (Gu et al., 2011; Yu et al., 2011; Luo et al., 2015). On the other hand, HDA9 mediates H3K27 deacetylation as well as CLF-PRC2-mediated Polycomb silencing at FLC. Interestingly, the Polycomb partners VAL1 and VAL2 physically associate not only with HDA9, but also with HDA6 (Chhun et al., 2016). This raises a possibility that through their association with VALs bound to CME, the HDA5/HDA6-FVE-FLD corepressor complex, HDA9, and CLF-PRC2 function in concert to establish a repressive chromatin environment at the FLC locus. In addition, the FLD-mediated repressive chromatin modifications have been shown to be required for FLC repression by autonomous-pathway genes including FPA, FCA, and FY (encoding RNA-processing factors; Liu et al., 2010); hence, it is likely that at the FLC locus, histone deacetylation, Polycomb silencing, and cotranscriptional RNA processing function together to repress its expression, resulting in early flowering in the rapid-cycling Arabidopsis accessions.

HDA9 plays a complex role in flowering-time regulation. A few flowering-regulatory genes are regulated (or repressed) by HDA9. In addition to FLC, FT, an FT relative (BROTHER OF FT [BFT]), and AGL19 are all repressed by HDA9 (Supplemental Fig. S5C; Kim et al., 2013; Kang et al., 2015). FLC and BFT act to repress the floral transition, whereas AGL19 and FT promote flowering (Schönrock et al., 2006; Amasino, 2010; Yoo et al., 2010). Under noninductive short days, HDA9 mediates histone deacetylation at AGL19 (a major gene that activates FT expression in short days) to repress its expression and inhibit flowering (Kang et al., 2015; Kim et al., 2013). In long days, FLC is apparently de-repressed upon loss of HDA9 function, but hda9 flowers like the wild type, possibly because the misexpression of other flowering-regulatory genes (e.g. FT) may prevent hda9 from flowering later. Similarly, loss of HDA19 function leads to upregulation of both FLC and FT (Supplemental Fig. S4A; Ning et al., 2019), and the early-flowering phenotype in hda9 hda19 (Supplemental Fig. S4B) may be partly attributed to FT upregulation.

In summary, we have uncovered a previously undescribed role for HDA9 in the repression of the central floral repressor FLC in rapid-cycling accessions. Furthermore, we provide evidence that HDA9, through H3K27 deacetylation, functions in collaboration with PRC2 to mediate H3K27me3 for transcriptional repression in Arabidopsis.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) clf-59D, ld-1, clf-29, hda9-1, hda19, and clf-59D ld-3 mutants have been described previously (Lee et al., 1994; Makarevich et al., 2006; Kim et al., 2008, 2013; Doyle and Amasino, 2009). Plants were grown under cool-white fluorescent light at ∼22°C in long days (16 h light/8 h dark), unless noted otherwise in figure legends.

Map-Based Cloning of CLF-S1 (HDA9) by High-Throughput Genome Resequencing

clf-s1 (in clf-59D ld-1) was crossed to clf-59D ld-3 (in the Ws-2 background), and 63 late-flowering mutants in an F2 population were selected to map the clf-s1 mutation with simple sequence-length polymorphism and derived cleaved amplified polymorphic sequence markers (Supplemental Table S1). For the whole genome sequencing, 30 late-flowering mutants in the F2 population were pooled for genomic DNA extraction using the DNeasy Plant Mini Kit (Qiagen), followed by high-throughput sequencing. Genome resequencing reads were preprocessed to remove low-quality ends and adaptors and then mapped onto the TAIR10 reference genome sequence using Burrows-Wheeler Aligner (Algorithm BWA-SW) with default parameters (Li and Durbin, 2010). Genome-wide single-nucleotide polymorphisms were called using Samtools (Li, 2011) and filtered by VCFtools with default parameters (Danecek et al., 2011). The allele frequency distributions of single-nucleotide polymorphisms along all chromosomes were displayed using SHOREmap (version 2.0; Schneeberger et al., 2009). The candidate region that might contain suspected mutations is typically indicated by the highest regional allele-frequency peak.

Yeast Two-Hybrid Assay

The Matchmaker GAL4 Two-Hybrid System 3 (Clontech) was adapted for this assay. HDA9 (full length), VAL1 (full length), and VAL2 (N-terminal) were cloned into the pGADT7 and/or pGBKT7 vectors and subsequently were introduced into the yeast strain AH109 according to the manufacturer’s instructions (Clontech). For protein–protein interaction assays, yeast cells bearing paired plasmids were spotted on a stringent selective medium without adenine, His, Trp, and Leu, or a selective medium without Leu, His, and Ala.

Plasmid Construction

For complementation of the hda9-3 mutation, a 4.6-kb genomic HDA9 fragment (2.0 kb promoters + 2.3 kb genomic coding sequence + 0.3 kb 3′ untranslated region) was cloned into the pBGW vector via Gateway technology (Invitrogen). To create FLAG or hemagglutinin (HA)-tagged HDA9 fusions, a 4.3-kb genomic HDA9 fragment (2.0-kb promoters + 2.3 kb genomic coding sequence without the stop codons) was first fused with 3xFlag or 3xHA and cloned into pBGW.

RNA Extraction and Reverse-Transcription Quantitative PCR

Total RNA was extracted from aerial parts of 10-d-old seedlings grown in long days using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. Approximately 2 μg RNA was reverse-transcribed into complementary DNAs, followed by reverse-transcription quantitative PCR (RT-qPCR) assay with a SYBR Green master mix using a QuantStudio6 Flex real-time PCR system (Applied Biosystems) as described previously (Gu et al., 2013). Relative levels of each transcript to TUB2 were calculated with 2-∆Ct [∆Ct = Ct(gene of interest) − CtTUB2]. Detailed primer information can be found in Supplemental Table S3.

Coimmunoprecipitation

Coimmunoprecipitation assays were conducted as previously described (Yuan et al., 2016). Briefly, total proteins were extracted from about 1.0-g, 10-d-old seedlings expressing HDA9:HA and/or VAL1:FLAG and subsequently precipitated with an anti-FLAG M2 affinity gel (Sigma, F2426). HDA9:HA and VAL1:FLAG were detected by western blotting using anti-HA (Roche, 12013819001) and anti-FLAG (Sigma, A8592), respectively.

ChIP Immunoprecipitation

ChIP assays were carried out as described previously with minor modifications (Wang et al., 2014). Briefly, total chromatin was extracted from 10-d-old seedlings grown in long days and subsequently immunoprecipitated with rabbit polyclonal anti-CLF (Tao et al., 2019) or anti-FLAG (Sigma). qPCR was conducted to measure the levels of FLC genomic fragments and the internal control TUB2 using a SYBR Green master mix on QuantStudio6 Flex real-time PCR system (Applied Biosystems). The primer sequences can be found in Supplemental Table S3.

Histone Extraction and Immunoblotting

Total histones were extracted from ∼10-d-old seedlings grown in long days as previously described (Jiang et al., 2008). Briefly, nuclei were extracted, followed by total histone extraction. Total histones were resolved on a 15% (w/v) SDS-PAGE gel and transferred onto a nitrocellulose membrane (Bio-Rad). Protein blotting was conducted with anti-H3 (Millipore, 07-690), anti-H3K27ac (Abcam, ab 177178), anti-H3K14ac (Millipore, 07-353), anti-H3K9/18ac (Millipore, 07-593), anti-H3K23ac (Millipore, 07-355), or anti-H4K5ac (Millipore, 07-327), and western blotting signals were scanned using the ECL Plus ChemiDoc Touch Imaging System (Bio-Rad). H3K27me3 was examined with anti-H3K27me3 (Millipore, 07-449) using ECL Plus films (Carestream). The blotting signals were further quantified with Image J.

Statistical Analyses

Flowering-time data were log-transformed for statistical analysis. One-way ANOVA analyses were conducted using GraphPad Prism7.00 (GraphPad), and two-tailed Student's t tests were carried out using Excel.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Information Resource (TAIR) database under the following accession numbers: AT2G23380 (CLF); AT3G44680 (HDA9); AT5G10140 (FLC); AT4G02560 (LD); AT1G77080 (MAF1); AT5G65050 (MAF2); AT5G65060 (MAF3); AT5G65070 (MAF4); AT5G65080 (MAF5); AT1G65480 (FT); AT4G38130 (HDA19); AT2G30470 (VAL1); AT1G60860 (VAL2); AT4G24540 (AGL24); AT5G49520 (WRKY48); and AT5G62040 (BFT).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We are very grateful to Richard M. Amasino for providing the clf-59D ld-3 and ld-1 seeds. We thank the in-house genomics facility for next-generation sequencing.

Footnotes

1

This work was supported by the National Natural Science Foundation of China (grant no. 31830049 to Y. H.) and the Chinese Academy of Sciences (grant no. XDB27030202).

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