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. 2016 Sep 14;172(3):1746–1759. doi: 10.1104/pp.16.00193

ZRF1 Chromatin Regulators Have Polycomb Silencing and Independent Roles in Development1,[OPEN]

Jing Feng 1,2, Donghong Chen 1,2, Alexandre Berr 1,2, Wen-Hui Shen 1,2,*
PMCID: PMC5100768  PMID: 27630184

AtZRF1a/b play both PRC1-related and PRC1-unrelated functions in regulating transcription and multiple processes of plant growth and development.

Abstract

Histone H2A monoubiquitination (H2Aub1), catalyzed by Polycomb-Repressive Complex1 (PRC1), is a key epigenetic mark in Polycomb silencing. However, little is known about how H2Aub1 is read to exert downstream physiological functions. The animal ZUOTIN-RELATED FACTOR1 (ZRF1) has been reported to bind H2Aub1 to promote or repress the expression of varied target genes. Here, we show that the Arabidopsis (Arabidopsis thaliana) ZRF1 homologs, AtZRF1a and AtZRF1b, are key regulators of multiple processes during plant growth and development. Loss of function of both AtZRF1a and AtZRF1b in atzrf1a atzrf1b mutants causes seed germination delay, small plant size, abnormal meristem activity, abnormal flower development, as well as gametophyte transmission and embryogenesis defects. Some of these defects overlap with those described previously in the PRC1-defective mutants atbmi1a atbmi1b and atring1a atring1b, but others are specific to atzrf1a atzrf1b. In line with this, 4,519 genes (representing more than 14% of all genes) within the Arabidopsis genome are found differentially expressed in atzrf1a atzrf1b seedlings, and among them, 114 genes are commonly up-regulated in atring1a atring1b and atbmi1a atbmi1b. Finally, we show that in both atzrf1a atzrf1b and atbmi1a atbmi1b seedlings, the seed developmental genes ABSCISIC ACID INSENSITIVE3, CRUCIFERIN3, and CHOTTO1 are derepressed, in association with the reduced levels of H2Aub1 and histone H3 lysine-27 trimethylation (H3K27me3). Collectively, our results indicate that AtZRF1a/b play both PRC1-related and PRC1-unrelated functions in regulating plant growth and development and that AtZRF1a/b promote H2Aub1 and H3K27me3 deposition in gene suppression. Our work provides novel insight into the mechanisms of function of this family of evolutionarily conserved chromatin regulators.

Histones are the primary proteins that package genomic DNA into chromatin. They are subject to a vast array of posttranslational modifications that can occur alone or in a combinatorial fashion known as the histone code, which dictates DNA accessibility and genome function (Rothbart and Strahl, 2014). The language of histone modifications is exemplified in Polycomb (Pc) silencing, a major epigenetic mechanism conserved in both animals and plants (Simon and Kingston, 2013; de la Paz Sanchez et al., 2015; Mozgova and Hennig, 2015; Xiao and Wagner, 2015). Polycomb Group (PcG) proteins, originally identified in Drosophila spp. as repressors of homeotic genes, are broadly found in many multicellular organisms. They act in multiprotein complexes to repress the transcription of a large number of genes involved in diverse processes during animal and plant development. Classically, Polycomb-Repressive Complex2 (PRC2) catalyzes histone H3 lysine-27 trimethylation (H3K27me3), and PRC1 recognizes/reads the H3K27me3 mark and further mediates downstream H2A monoubiquitination (H2Aub1) deposition (Simon and Kingston, 2013; de la Paz Sanchez et al., 2015; Mozgova and Hennig, 2015; Xiao and Wagner, 2015).

In Arabidopsis (Arabidopsis thaliana), PRC2 components and biochemical functions are conserved and largely similar to their animal counterparts, whereas PRC1 complexes show more divergence (Molitor and Shen, 2013). The animal PRC1 core subunit Pc, which binds H3K27me3, is absent in Arabidopsis. Alternatively, LIKE HETEROCHROMATIN PROTEIN1 (LHP1), also known as TERMINAL FLOWER2, binds H3K27me3 and likely plays a Pc-like function (Turck et al., 2007; Zhang et al., 2007). The best PRC1 conservation between animals and plants can be found within RING-finger proteins (RING1 and BMI1) that act as E3 ubiquitin ligases in H2Aub1 deposition (Molitor and Shen, 2013). Functional characterization of AtRING1a/b and AtBMI1a/b/c in Arabidopsis has revealed that they are required for the suppression of a number of key regulatory genes involved in multiple processes of plant growth and development (Xu and Shen, 2008, Bratzel et al., 2010; Chen et al., 2010; Li et al., 2011; Yang et al., 2013; Molitor et al., 2014; Shen et al., 2014; Picó et al., 2015). Strikingly, in contrast to the classic hierarchical paradigm where PRC2 acts before PRC1, PRC2-mediated H3K27me3 deposition is found impaired at various gene loci in the PRC1-defective mutants lhp1 (Derkacheva et al., 2013), atbmi1a/b/c and atring1a/b (Yang et al., 2013; Molitor et al., 2014; Shen et al., 2014), and emf1 (Kim et al., 2012; Wang et al., 2014). More recently, genome-wide profiling analysis indicates that, in Arabidopsis seedlings, AtBMI1a/b and AtRING1a/b are required for H3K27me3 deposition preferentially at embryo developmental genes, whereas LHP1 is required for H3K27me3 deposition preferentially at flower developmental genes (Wang et al., 2016). Protein-protein interactions between some PRC1 and PRC2 subunits may partly explain the varied order of H3K27me3 and H2Aub1 depositions occurring within different chromatin contexts (Feng and Shen, 2014; Mozgova and Hennig, 2015). Also in animals, a large number of PRC1 variant complexes have been uncovered, and examples exist where PRC1 precedes and triggers PRC2-mediated H3K27me3 deposition in the silencing of various genes (Schwartz and Pirrotta, 2013).

In contrast to the intensive studies carried out on PRC1 and PRC2, few studies are available regarding factors that read H2Aub1 to effect downstream functions. ZUOTIN-RELATED FACTOR1 (ZRF1) proteins contain two highly recognizable types of domains, namely UBD (ubiquitin-binding domain) and SANT (Swi3, Ada2, NcoR1, and TFIIIB), with the latter one frequently found in several types of evolutionarily conserved chromatin modifiers (Chen et al., 2014; Aloia et al., 2015). The human ZRF1 protein has been reported to bind H2Aub1 via its UBD, to displace PRC1 from chromatin, and to favor H2Aub1 deubiquitination, leading to the switch from a repressive to an active chromatin state in the induction of PcG-silenced genes during teratocarcinoma cell differentiation (Richly et al., 2010). During the differentiation of mouse embryonic stem cells, ZRF1 specifically regulates the induction of master neural genes in a PcG-dependent manner and ZRF1 depletion impairs neural differentiation but not differentiation toward mesendodermal lineages (Aloia et al., 2014). Although ZRF1 homologs are found broadly present in the green lineage (Chen et al., 2014), their functions in relation to PcG silencing remain uncharacterized in higher plants so far.

In this study, we investigate the functions of the Arabidopsis ZRF1 homologs at the whole-organism level. We demonstrate that they play key roles in multiple processes affecting plant growth and development in both PRC1-like and distinct manners. We found that the Arabidopsis ZRF1 homologs are required to enhance H2Aub1 as well as H3K27me3 levels in the silencing of seed developmental genes in seedlings, uncovering a novel mechanism of function for this family of chromatin regulators.

RESULTS

Identification and Generation of Loss-of-Function Mutants of AtZRF1a and AtZRF1b

ZRF1 is evolutionarily conserved in most species (Chen et al., 2014), and the Arabidopsis genome contains two ZRF1-encoding genes, AtZRF1a (gene locus At3g11450) and AtZRF1b (gene locus At5g06110; Supplemental Fig. S1). Both AtZRF1a and AtZRF1b are broadly expressed in various plant organs/tissues (Supplemental Fig. S2). To study the biological roles of AtZRF1a and AtZRF1b, we obtained from the Arabidopsis Biological Resource Center (http://www.arabidopsis.org/) three independent T-DNA insertion mutant lines: atzrf1a-1, atzrf1a-2, and atzrf1b-1 (Fig. 1A). We also included the stock line SAIL_716_D04 (hereafter named D04) because it contains a T-DNA insertion close to the 3′ untranslated region of AtZRF1b (Fig. 1A). Both heterozygous and homozygous plants of all these different mutations show a normal phenotype, which is indistinguishable from that of wild-type Columbia-0 (Col-0) plants. Next, we generated combinatorial double mutants: atzrf1a-1 atzrf1b-1, atzrf1a-2 atzrf1b-1, atzrf1a-1 D04, and atzrf1a-2 D04. As shown in Figure 1B, both atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 display drastically reduced plant size, whereas atzrf1a-1 D04 and atzrf1a-2 D04 have a normal plant growth phenotype. Reverse transcription (RT)-PCR analysis revealed that full-length transcripts of both AtZRF1a and AtZRF1b are undetectable in either atzrf1a-1 atzrf1b-1 or atzrf1a-2 atzrf1b-1, whereas only AtZRF1a expression is impaired but AtZRF1b expression is normal in atzrf1a-1 D04 and atzrf1a-2 D04 (Fig. 1C). These data indicate that atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 are bona fide loss-of-function mutants and that simultaneous knockout of both AtZRF1a and AtZRF1b causes the reduced plant size mutant phenotype.

Figure 1.

Figure 1.

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Identification of T-DNA insertion mutants of the AtZRF1a and AtZRF1b genes. A, Schematic representation of independent T-DNA insertions within or close to the AtZRF1a and AtZRF1b genes. Promoter, untranslated region (UTR), intron, exon, and the translation start codon ATG of the gene as well as T-DNA insertion positions of the different mutants are indicated. B, Wild-type Col-0 and the double mutants atzrf1a-1 D04, atzrf1a-2 D04, atzrf1a-1 atzrf1b-1, and atzrf1a-2 atzrf1b-1 plants 35 d after germination grown on soil under medium-day (12 h of light and 12 h of light) photoperiod conditions. Bar = 3 cm. C, RT-PCR analysis of AtZRF1a and AtZRF1b expression in rosette leaves of wild-type Col-0, the single mutants atzrf1a-1, atzrf1a-2, atzrf1b-1, and D04, and the double mutants atzrf1a-1 atzrf1b-1, atzrf1a-2 atzrf1b-1, atzrf1a-1 D04, and atzrf1a-2 D04. Gene-specific primers covering the entire coding region of AtZRF1a and AtZRF1b (Supplemental Table S5) were used in RT-PCR amplification. The ACTIN gene served as an internal control.

To extend our analysis, we obtained additional T-DNA insertion mutant lines: atzrf1a-3, atzrf1b-2, atzrf1b-3, and atzrf1b-4 (Fig. 1A). Genetic cross tests show that these mutants are indeed respectively allelic of atzrf1a or atzrf1b, and all mutants having simultaneous knockout of both AtZRF1a and AtZRF1b display a similarly reduced plant size phenotype (Supplemental Fig. S3). Together, our data based on three independent allelic mutants of atzrf1a and four independent allelic mutants of atzrf1b firmly establish that AtZRF1a and AtZRF1b have redundant functions and their simultaneous loss of function has caused the mutant plant phenotype. Herein, we focused on atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 for detailed investigations.

Loss of Function of AtZRF1a and AtZRF1b Delays Seed Germination

Seed germination represents the first step for entry into postembryonic growth of the plant life cycle, and PRC1 is involved in promoting Arabidopsis seed germination (Molitor et al., 2014). Under standard growth conditions, both the atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 double mutants display retarded seed germination (Fig. 2A), whereas the single mutants atzrf1a-1, atzrf1a-2, and atzrf1b-1 show normal seed germination like the wild-type control Col-0 (Supplemental Fig. S4). Next, we tested seed germination under osmotic treatments with salt or mannitol, two stresses known to have a negative impact on seed germination. Again, the atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 mutants show impaired efficiency of seed germination under either salt (Fig. 2B) or mannitol (Fig. 2C) treatment conditions. In agreement with the previous study (Molitor et al., 2014), we found that the PRC1-deficient mutant atbmi1a atbmi1b also has a reduced efficiency of seed germination. Remarkably, seed germination defects are much more severe in atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 than in atbmi1a atbmi1b, as judged from both the germination time and the maximal germination rate (Fig. 2). The relatively moderate germination phenotype of atbmi1a atbmi1b could be explained by the partial redundancy of AtBMI1c function, which remained intact in the mutant (Li et al., 2011; Yang et al., 2013).

Figure 2.

Figure 2.

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The atzrf1a atzrf1b mutants show seed germination defects. The germination rates of wild-type Col-0 and the double mutants atbmi1a atbmi1b, atzrf1a-1 atzrf1b-1, and atzrf1a-2 atzrf1b-1 were analyzed on Murashige and Skoog (MS) medium (A), MS medium supplemented with 100 mm NaCl (B), or MS medium supplemented with 200 mm mannitol (C). Data represent average germination percentages ± sd of three biological replicates, each with more than 80 seeds, observed daily for 12 d after stratification.

Loss of Function of AtZRF1a and AtZRF1b Drastically Affects Shoot Meristem Activity and Leaf Development

At the vegetative growth stage, the atzrf1a atzrf1b double mutant plants are obviously smaller (Fig. 1B). Fresh weight measurement of whole rosettes of 4-week-old plants revealed that the atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 mutants exhibit an average weight only about 25% of that of the wild-type control Col-0 (Fig. 3A). The reduced weight is due primarily to impaired leaf expansion, whereas axillary meristem formation in the rosette is enhanced in the mutant plant (Fig. 3, B and C). Scanning electron microscopy analysis of fully expanded leaves revealed that epidermal cells are significantly smaller in atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 than in Col-0 (Fig. 3D). Both cell number and cell expansion are reduced in the mutant leaves. Cell expansion can occur dependently or independently from endoreplication/endoreduplication (Inzé and De Veylder, 2006). Our analysis of ploidy levels revealed that the atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 mutant leaves contain a slightly higher proportion of polyploid cells (Fig. 3E). Thus, the reduced cell size in the mutant is independent from endoreduplication-associated cell expansion. Our observation of the expression of the SHOOT MERISTEMLESS (STM) promoter-driving GUS reporter indicates an enhanced axillary meristem activity in the atzrf1a-1 atzrf1b-1 mutant plant (Fig. 3B), and PRC1-mediated suppression of stem cell proliferation was known previously to be associated with the repression of class I KNOX genes (Xu and Shen, 2008; Chen et al., 2010). Next, we analyzed the expression levels of class I KNOX genes together with several other regulatory genes in our mutant plants. We found that the expression of the class I KNOX genes STM, BREVIPEDICELLUS/KNOTTED-LIKE FROM ARABIDOPSIS THALIANA (BP/KNAT1), and KNAT6 is up-regulated by roughly 6-, 4-, and 2-fold, respectively, in both the atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 mutants (Fig. 3F). An up-regulation by 5- to 6-fold also was detected for the expression of the organ boundary regulatory genes CUP-SHAPED COTYLEDON1 (CUC1), CUC2, and CUC3. In contrast, expression of the transcription factor genes WUSCHEL (WUS) and AGAMOUS (AG) is down-regulated in the atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 mutants (Fig. 3F). Interestingly, the expression patterns observed here are largely similar to those reported previously in the atring1a atring1b and atbmi1a atbmi1b mutants (Xu and Shen, 2008; Chen et al., 2010), suggesting that AtRING1a/b, AtBMI1a/b, and AtZRF1a/b may act on a common set of genes for the maintenance of proper SAM activity during Arabidopsis plant development.

Figure 3.

Figure 3.

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The atzrf1a atzrf1b mutants show growth defects. A, Relative fresh weights of 4-week-old plants of the double mutants atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 compared with wild-type Col-0 (set as 100). Rosettes of plants grown in soil under long-day (16 h of light and 8 h of light) photoperiod conditions were harvested and weighed. Data shown represent means ± se obtained in three independent experiments, each with 30 plants for a genotype. B, Comparison of meristem activity in shoot tips of 10-d-old plants between Col-0 and atzrf1a-1 atzrf1b-1. The pSTM:GUS reporter was introgressed from Col-0 into atzrf1a-1 atzrf1b-1 by genetic cross. Images were taken after histochemical GUS staining. Note the GUS activity detected in the primary shoot apical meristem (SAM) of both Col-0 and atzrf1a-1 atzrf1b-1 and in an axillary meristem formed specifically in the mutant (indicated by the arrow). Axillary meristem activity is detected in all examined mutant seedlings but in none of the Col-0 seedlings. Bars = 200 µm. C, Representative image of a 5-week-old plant of the atzrf1a-1 atzrf1b-1 mutant. Note the axillary/secondary shoots (indicated by red arrows) formed before any obvious bolting of the primary apical shoot (indicated by the white arrow). Bar = 1 cm. D, Scanning electron microscopy images of mature leaf adaxial epidermal cells of Col-0, atzrf1a-1 atzrf1b-1, and atzrf1a-2 atzrf1b-1 plants. Bars = 100 µm. E, Ploidy levels of cells from the first true leaf of 2-week-old plants of Col-0, atzrf1a-1 atzrf1b-1, and atzrf1a-2 atzrf1b-1. Mean values from three independent experiments are shown together with se (error bars). The differences between Col-0 and the mutants are statistically significant (P ≤ 0.05) for the 2C, 4C, and 8C levels in an ANOVA test. F, Relative expression levels of meristem and organ boundary regulatory genes in atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 compared with wild-type Col-0 (set as 1). Quantitative RT-PCR analysis was performed using 2-week-old plants. Mean values from three independent experiments are shown together with se (error bars).

Loss of Function of AtZRF1a and AtZRF1b Causes Flower Developmental Defects

In conformity with the important role of AtZRF1a/b in the maintenance of proper SAM function, multiple defects were observed during flower development in the atzrf1a-1 atzrf1b-1 mutant plants. The atzrf1a-2 atzrf1b-1 mutant has similar defects to atzrf1a-1 atzrf1b-1; here, we focus on atzrf1a-1 atzrf1b-1 for a detailed description of mutant flower developmental defects. The transition from vegetative growth to reproductive development is initiated by the cell fate change of SAM into inflorescence meristem (IM), which subsequently produces primary and secondary flowering stems. Compared with Col-0, the atzrf1a-1 atzrf1b-1 mutant shows a bushy inflorescence phenotype (Fig. 4A), implying a reduced apical dominance in the mutant plant. While absent from Col-0 plants, fasciated primary inflorescence stems (Fig. 4, B and C) are observed in atzrf1a-1 atzrf1b-1 plants, albeit at a moderate frequency (in about 15% of the total number of plants), again indicating an enhanced IM proliferation in the mutant. Floral meristem formed from the peripheral zone of the IM produces a normal wild-type flower containing four sepals, four petals, six stamens, and two fused carpels (Fig. 4D). In contrast, some mutant flowers contain reduced numbers of some of these floral organs (Fig. 4, E–G). Dissection on 100 simple flowers (10 flowers randomly sampled from 10 individual plants) revealed that the mutant flower contains fewer sepals (3.8 ± 0.7 as compared with four), fewer petals (2.4 ± 0.9 as compared with four), and fewer stamens (4.25 ± 0.6 as compared with six) but a normal number of carpels (two as compared with two). More complex flower structures (e.g. a flower forming inside of a flower; Fig. 4, G and H) also are observed in about 20% of mutant flowers. These abnormal flower phenotypes indicate that, in the absence of AtZRF1a/b function, the floral meristem fails to differentiate properly and can revert spontaneously into the IM during flower development.

Figure 4.

Figure 4.

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The atzrf1a atzrf1b mutants show flower developmental abnormalities. A, Image of 8-week-old plants of wild-type Col-0 and the mutant atzrf1a-1 atzrf1b-1 grown in soil under long-day (16 h of light and 8 h of light) photoperiod conditions. Bar = 3 cm. B, Image of a mutant plant showing stem fasciation and termination, as indicated by the arrow. Bar = 1 mm. C, Closeup image of a stem fasciation in a mutant plant. Bar = 1 mm. D, Wild-type flower. Bar = 200 µm. E to H, Mutant flowers. Arrows indicate the formation of flowers within flowers. Bars = 200 µm.

Loss of Function of AtZRF1a and AtZRF1b Impairs Both Male and Female Transmission as Well as Embryogenesis

The atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 mutant plants are poorly fertile (e.g. a mutant plant silique contains at maximum of 10 seeds, whereas a wild-type plant silique contains 40–50 seeds). To examine whether AtZRF1a/b has a role in gametophyte function, we investigated the transmission efficiency of mutant alleles in heterozygous plants in which sporophytic growth is phenotypically normal. Analysis of the progeny produced by self-pollination of heterozygous plants revealed that the ratio of wild-type phenotype plants to double mutant phenotype plants is significantly higher than the expected ratio (e.g. 6:1–13:1 as compared with 3:1; Table I). This reduced transmission was observed with all four different homozygous/heterozygous atzrf1a and atzrf1b combinations. To further examine whether the transmission reduction is caused by male/female or both defects, reciprocal crosses of heterozygous mutant plants with the wild-type Col-0 plants were performed. Genotyping by PCR analysis revealed that the inheritance of each of the atzrf1a-1, atzrf1a-2, and atzrf1b-1 alleles in the respective combinatorial mutants is strongly reduced in both male and female gametes (e.g. 0.48–0.76 as compared with one; Table I). Together, these data indicate that, in addition to their crucial function in plant sporophytic development, AtZRF1a/b also are required for gametophyte development/function. In addition, embryogenesis also is affected in the atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 mutants. Seed abortion is observed in the mutant plant siliques (Fig. 5B) but barely in Col-0 (Fig. 5A), and about 35% of seedlings exhibit abnormal embryonic phenotypes, including single cotyledon (Fig. 5C), asymmetrical cotyledons (Fig. 5D), and fleshy narrow cotyledons (Fig. 5E).

Table I. Transmission efficiency test of the atzrf1a atzrf1b double mutant.

Differences between the observed ratio and the expected ratio are statistically significant as follows: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Parent Progeny Observed Ratio Expected Ratio
Self-pollination Wild type zrf1a
 atzrf1a-1/+ atzrf1b-1 152 22 6.90:1*** 3:1
 atzrf1a-2/+ atzrf1b-1 401 45 8.91:1*** 3:1
 atzrf1a-1 atzrf1b-1/+ 392 32 13.25:1*** 3:1
 atzrf1a-2 atzrf1b-1/+ 379 37 10.24:1*** 3:1
Reciprocal cross (♀ × ♂) AAb Aa
 atzrf1a-1/+ atzrf1b-1 × Col-0 101 77 1:0.76* 1:1
 atzrf1a-2/+ atzrf1b-1 × Col-0 185 101 1:0.55** 1:1
 Col-0 × atzrf1a-1/+ atzrf1b-1 149 71 1:0.48** 1:1
 Col-0 × atzrf1a-2/+ atzrf1b-1 241 103 1:0.43** 1:1
Reciprocal cross (♀ × ♂) BBc Bb
 atzrf1a-1 atzrf1b-1/+ × Col-0 176 125 1:0.71* 1:1
 atzrf1a-2 atzrf1b-1/+ × Col-0 153 93 1:0.61* 1:1
 Col-0 × atzrf1a-1 atzrf1b-1/+ 225 135 1:0.60* 1:1
 Col-0 × atzrf1a-2 atzrf1b-1/+ 324 169 1:0.52** 1:1
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a

zrf1 indicates the phenotype of the atzrf1a−/− atzrf1b−/− double mutant.  bWild-type AtZRF1a (A) and mutant Atzrf1a (a) alleles were determined by genotyping PCR analysis. AA, AtZRF1a+/+; Aa, atzrf1a+/−.  cWild-type AtZRF1b (B) and mutant Atzrf1b (b) alleles were determined by genotyping PCR analysis. BB, AtZRF1b+/+; Bb, atzrf1b+/−.

Figure 5.

Figure 5.

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The atzrf1a atzrf1b mutants show seed abortion and abnormal embryonic cotyledon formation. A and B, Dissected siliques from wild-type Col-0 and the mutant atzrf1a-1 atzrf1b-1 plants, respectively. Note the normal seeds that formed in Col-0 but the presence of small and/or browning (aborted) seeds before maturation in atzrf1a-1 atzrf1b-1. C, Mutant seedling showing fusion cotyledon formation. D, Mutant seedling showing asymmetric cotyledon formation. E, Mutant seedling showing fleshy and narrow cotyledons and leaves. Images were taken 2 weeks (C and D) or 4 weeks (E) after seed germination on plates. Bars = 500 µm.

AtZRF1a/b Are Required for Both the Suppression and Induction of Expression of a Large Number of Genes within the Arabidopsis Genome

To investigate the role of AtZRF1a/b in genome-wide transcription regulation, we performed transcriptome analysis using the Agilent Arabidopsis 44K oligonucleotide microarray on 6-d-old seedlings. Genes with expression that changed more than 2-fold (P ≤ 0.05) between the atzrf1a-1 atzrf1b-1 mutant and the wild-type control Col-0 are considered as differentially expressed. Thus, the mutant shows a total of 4,519 differentially expressed genes; among them, 2,111 (46.7%) genes are up-regulated (Supplemental Data Set S1) and 2,408 (53.3%) genes are down-regulated (Supplemental Data Set S2). As expected, AtZRF1a and AtZRF1b are among the down-regulated genes. Gene Ontology (GO) analysis revealed that the differentially expressed genes in atzrf1a-1 atzrf1b-1 comprise various functional classes (Supplemental Tables S1 and S2). Next, we compared our list of differentially expressed genes in atzrf1a-1 atzrf1b-1 with the lists of differentially expressed genes reported previously in atbmi1a atbmi1b and atring1a atring1b (Wang et al., 2016). A roughly similar distribution of GO classes is found for the atzrf1a-1 atzrf1b-1, atbmi1a atbmi1b, and atring1a atring1b mutants (Fig. 6A). Varied numbers of genes can be found as commonly misregulated, with different possible combinations among the up- and down-regulation lists of genes, in the atzrf1a-1 atzrf1b-1, atbmi1a atbmi1b, and/or atring1a atring1b mutants (Fig. 6, B and C). Remarkably, the degree of overlap between up-up and down-down groups is significantly larger (representation factor > 1 and P < 0.05) than expected at random, while this was not the case for the up-down pairwise comparisons (Supplemental Fig. S5), and 114 genes were found as up-regulated in all three mutants (Supplemental Table S3). These commonly up-regulated genes are overrepresented by genes involved in various processes of seed development (Supplemental Table S4), which is in line with the primary role of AtZRF1a/b, AtBMI1a/b, and AtRING1a/b observed in seed germination and seedling development. Comparisons with the previously published epigenome profiling data in Col-0 (Roudier et al., 2011; Wang et al., 2016) revealed that 75% of these commonly up-regulated gene loci are marked by H3K27me3 (Supplemental Table S3), which is in agreement with their silencing by the PcG repression pathway. Besides the commonly misregulated genes, a large number of misregulated genes are found specifically in atzrf1a-1 atzrf1b-1 but not in atbmi1a atbmi1b and/or atring1a atring1b, indicating that AtZRF1a/b largely act in the regulation of specific target genes that differ from those regulated by AtBMI1a/b and/or AtRING1a/b.

Figure 6.

Figure 6.

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The atzrf1a atzrf1b mutants show genome-wide transcription perturbation. A, Percentage of differentially expressed genes in the mutants belonging to different GO classes, as indicated. B, Venn diagram showing the number and overlap of up- and down-regulated genes found in atzrf1a-1 atzrf1b-1 compared with up-regulated genes found in atbmi1a atbmi1b and atring1a atring1b-1. C, Venn diagram showing the number and overlap of up- and down-regulated genes found in atzrf1a-1 atzrf1b-1 compared with down-regulated genes found in atbmi1a atbmi1b and atring1a atring1b-1. Differentially expressed (up- or down-regulated) genes in atzrf1a-1 atzrf1b-1 were identified in this study. The differentially expressed genes in atbmi1a atbmi1b and atring1a atring1b-1 had been reported previously by Wang et al. (2016). Details of pairwise comparisons with statistical evaluations can be found in Supplemental Figure S5.

AtZRF1a/b Are Required for H2Aub1 and H3K27me3 Deposition in the Suppression of Seed Developmental Genes in Seedlings

Our finding of a role of AtZRF1a/b in the suppression of Arabidopsis genes is in contrast to the previously described role of the animal ZRF1 in transcription activation (Richly et al., 2010). To provide insight into the molecular mechanisms of AtZRF1a/b function, we first examined their binding activity. Similar to the animal ZRF1 (Richly et al., 2010), AtZRF1b can bind ubiquitin via the evolutionarily conserved UBD and can pull down H2Aub1 and H2A from mononucleosome-enriched protein extracts (Supplemental Fig. S6). However, in contrast to the animal ZRF1, which has been shown to be involved in H2Aub1 deubiquitination in mammalian cells (Richly et al., 2010), loss of AtZRF1a/b does not cause an increase of H2Aub1 but rather results in a very slightly reduced global level of H2Aub1 in atzrf1a-1 atzrf1b-1 mutant plants (Fig. 7A). In agreement with the key role of AtBMI1a/b in H2Aub1 deposition, a drastic reduction of the global level of H2Aub1 is observed in the atbmi1a atbmi1b mutant seedlings (Fig. 7A).

Figure 7.

Figure 7.

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The atzrf1a atzrf1b mutants show derepression of seed developmental genes associated with decreases of H2Aub1 and H3K27me3 levels. A, Western-blot analysis of global levels of H3K4me3, H3K27me3, H3K36me3, and H2Aub1 in wild-type Col-0 and the mutants atbmi1a atbmi1b and atzrf1a-1 atzrf1b-1. Histone-enriched protein extracts from 2-week-old plants grown under medium-day (12 h of light and 12 h of dark) photoperiod conditions were analyzed using specific antibodies. The wild-type control and the mutant samples had been run all together on the same gel, and the images shown had been treated with identical conditions. Because the lane between the wild type and the atbmi1a atbmi1b mutant corresponds to a loading unrelated to this study, this lane is cut off, which explains the white vertical line. Arrows indicate H2Aub1 positions, and the asterisk indicates the position of H2Bub1. Histone H3 served as a loading control. The graph shows relative levels as means ± sd from quantitative densitometry measurement of three independent experiments, normalized to H3 and relative to Col-0 (set as 1). B, Relative expression levels of seed developmental genes in the mutants atzrf1a-1 atzrf1b-1, atzrf1a-2 atzrf1b-1, and atbmi1a atbmi1b compared with wild-type Col-0 (set as 1). Quantitative RT-PCR analysis was performed using 5-d-old seedlings grown on plates. Mean values from three independent experiments are shown together with se (error bars). C, Schematic representation of seed developmental gene structure indicating the regions examined for H2Aub1 and H3K27me3 enrichment. Promoter, untranslated region (UTR), intron, exon, and the translation start codon ATG are indicated. Bars above each gene structure represent regions amplified by PCR in ChIP analysis. D, Relative enrichment of H2Aub1 (top) and H3K27me3 (bottom) in wild-type Col-0 and the mutants atzrf1a-1 atzrf1b-1, atzrf1a-2 atzrf1b-1, and atbmi1a atbmi1b at different gene regions as indicated. ChIP analysis was performed using 5-d-old seedlings grown on plates. Data were normalized using ACTIN as an internal control. Mean values from three independent replicates are shown together with se (error bars).

Next, we investigated H2Aub1 and H3K27me3 levels at specific gene loci. In agreement with our transcriptome data, our quantitative RT-PCR analysis revealed that several seed developmental genes, including ABSCISIC ACID INSENSITIVE3 (ABI3), CRUCIFERIN1/CRUCIFERINA1 (CRU1/CRA1), CRU3, CHOTTO1/AINTEGUMENTA-LIKE5 (CHO1/AIL5), and CYSTEINE PEROXIREDOXIN1 (PER1), are up-regulated in the atbmi1a atbmi1b and atzrf1a-1 atzrf1b-1 as well as atzrf1a-2 atzrf1b-1 mutant seedlings (Fig. 7B). Previous studies showed that the derepression of ABI3, CRU3, and AIL5 is associated with reduced levels of H2Aub1 and H3K27me3 in the atbmi1a atbmi1b mutant (Yang et al., 2013; Molitor et al., 2014). Consistently, our chromatin immunoprecipitation (ChIP) analysis, at various regions along ABI3, CRU3, and AIL5 (Fig. 7C), revealed that H2Aub1 and H3K27me3 levels are reduced drastically in the atbmi1a atbmi1b mutant seedlings (Fig. 7D; Supplemental Fig. S7). Remarkably, we found that both H2Aub1 and H3K27me3 also are strongly reduced within the ABI3, CRU3, and AIL5 gene loci in the atzrf1a-1 atzrf1b-1 and atzrf1a-2 atzrf1b-1 mutants (Fig. 7D). Thus, similar to AtBMI1a/b, AtZRF1a/b also are required for H2Aub1 and H3K27me3 deposition to maintain the repression of seed developmental genes to allow proper postembryonic plant growth and development.

DISCUSSION

In this study, we have shown that AtZRF1a/b play key roles in multiple processes of plant growth and development. Consistently, over 16% of the analyzed genes within the whole Arabidopsis genome are found to be misregulated in the loss-of-function atzrf1a atzrf1b mutant. Very interestingly, AtZRF1a/b are required to enhance H2Aub1 and H3K27me3 levels in the silencing of some seed developmental genes in seedlings, uncovering a novel molecular mechanism of function for this family of evolutionarily conserved chromatin regulators.

The atzrf1a atzrf1b mutant plants are obviously reduced in body size. Epidermal cells are smaller, but polyploidy levels are increased slightly in the atzrf1a atzrf1b mutant plant leaves. Polyploidy is the result of endoreplication, which represents the characteristic switch from the mitotic cell division cycle to the endocycle in cellular differentiation. It is believed that endoreplication may enhance metabolic capacity and support cell growth by maintaining an optimal balance between cell volume and nuclear DNA content (for review, see Inzé and De Veylder, 2006). A positive correlation between cell size and endoreplication is observed frequently in studying various cell cycle regulators and specific transcription factors (Cebolla et al., 1999; De Veylder et al., 2002; Yu et al., 2003). In sharp contrast, the slightly elevated polyploid level does not lead to cell size increase in the atzrf1a atzrf1b mutant. The reduced cell size observed in the atzrf1a atzrf1b mutant might be explained by the drastic reprogramming of the genome function, which affects the expression of several thousands of genes involved in multiple molecular, physiological, and cellular activities (Fig. 6). Nonetheless, it is also worthy to note that the cyclin gene CYCD4;1 is up-regulated and the cyclin-dependent kinase inhibitor gene KRP6 is down-regulated in the atzrf1a atzrf1b mutant (Supplemental Data Sets S1 and S2). Both CYCD4;1 and KRP6 had been demonstrated as key regulators of mitotic cell cycle and endocycle progression (Kono et al., 2007; Guérinier et al., 2013; Vieira et al., 2014). It is thus reasonable to speculate that their perturbed expression may affect cell division and expansion in the atzrf1a atzrf1b mutant.

The growth and development of the atzrf1a atzrf1b mutant plants are severely impaired because of abnormal stem cell activities. During postembryonic plant growth, SAM activity needs to be tightly controlled for proper organ initiation and development. It is found that loss of AtZRF1a/b function causes elevated expression of the class I KNOX genes STM, BP/KNAT1, and KNAT6 and the organ boundary genes CUC1, CUC2, and CUC3. STM is activated early during embryogenesis, concomitant with the initiation of cotyledons and SAM formation, and is required for the maintenance of the indeterminate cell fate and for the prevention of cell differentiation in the meristem throughout plant development (Long et al., 1996; Lenhard et al., 2002). The other class I KNOX genes are expressed and perform redundant, albeit weaker, functions compared with STM in the SAM, with BP/KNAT1 and KNAT6 playing remarkable roles during flower gynoecium development (Arnaud and Pautot, 2014). Because the induction of class I KNOX genes can cause the up-regulation of CUC genes (Spinelli et al., 2011), future investigation is necessary to determine whether the elevated expression of CUC1, CUC2, and CUC3 is a secondary effect in the atzrf1a atzrf1b mutant. Within the SAM, WUS is expressed specifically in the stem cell organizing center and acts both independently and complementarily with STM in the maintenance of stem cell activity (Laux et al., 1996; Mayer et al., 1998). During flower development, AG terminates stem cell maintenance by repressing WUS by directly or indirectly recruiting PRC2 and LHP1 (Liu et al., 2011; Sun et al., 2014). Interestingly, AtZRF1a/b are not required for the repression of AG and WUS, because expression levels of both AG and WUS are down-regulated in the atzrf1a atzrf1b mutant. Similarly, AtRING1a/b and AtBMI1a/b are required for the suppression of class I KNOX genes but not AG and WUS (Xu and Shen, 2008; Bratzel et al., 2010; Chen et al., 2010; Yang et al., 2013). It is likely that the shoot fasciation and abnormal flower development observed in the atzrf1a atzrf1b, atring1a atring1b, and atbmi1a atbmi1b mutants are largely associated with the ectopic expression of class I KNOX genes.

Seed production is reduced drastically in the atzrf1a atzrf1b mutant. Defects at both sporophytic (e.g. abnormal floral organ/tissue formation) and gametophytic (as evidenced from low gamete transmission efficiency) generations have impaired the mutant plant fertility. Furthermore, embryogenesis also is affected, as indicated by seed abortion and abnormal embryonic organ/tissue formation observed in the atzrf1a atzrf1b mutant. Interestingly, seed developmental genes are overrepresented in the list of commonly up-regulated genes in the atzrf1a atzrf1b, atring1a atring1b, and atbmi1a atbmi1b mutants. Seed developmental genes are essential for seed formation, but they need to be silenced upon seed germination, and the silenced state needs to be stably maintained during subsequent plant growth and development. In line with this notion, the performance of seed germination is impaired in the atzrf1a atzrf1b and atbmi1a atbmi1b mutants. Consistently, the atzrf1a atzrf1b and atbmi1a atbmi1b mutants show derepression of ABI3, CRU1/CRA1, CRU3, CHO1/AIL5, and PER1. These seed developmental genes have been demonstrated to negatively regulate seed germination (Parcy et al., 1994; Haslekås et al., 2003; Yamagishi et al., 2009). The AtBMI1b and AtRING1a proteins interact physically with the plant-specific PHD domain proteins of the ALFIN1-like (AL) family (Molitor et al., 2014). AL proteins bind H3K4me2/me3 (Lee et al., 2009) and may subsequently recruit PRC1 to switch the active chromatin state into a repressive state at seed developmental genes. Consistently, AL6/7 and AtBMI1a/b promote the progressive replacement of H3K4me3 by H3K27me3 within the chromatin of seed developmental genes during seed germination (Molitor et al., 2014). Interestingly, our study here indicates that AtZRF1a/b also are required for the suppression of seed developmental genes in seedlings. Both H2Aub1 and H3K27me3 levels are reduced at various regions of the ABI3, CRU3, and CHO1/AIL5 loci in the atzrf1a atzrf1b mutant. Mutual reinforcing mechanisms through AtZRF1a/b-H2Aub1 and AtZRF1a/b-PRC1/PRC2 interactions probably are involved in the spreading of H2Aub1 and H3K27me3 deposition for an efficient silencing of seed developmental genes for proper vegetative plant growth. Future studies are necessary to test this assumption and to further insight into the detailed molecular mechanisms of PcG silencing of seed developmental genes.

The requirement of AtZRF1a/b in maintaining H2Aub1 and H3K27me3 in Arabidopsis strongly contrasts with the known function of ZRF1 in H2Aub1 removal during the transcriptional induction of cell differentiation genes in mammalian embryonic cell lines (Richly et al., 2010; Aloia et al., 2014). It is generally known that the loss of PRC1/PRC2 function causes embryonic stem cells to differentiate into specific functional cell types in mammals but leads to the dedifferentiation of seedlings to form embryo-like tissues in Arabidopsis (Köhler and Villar, 2008; Chen et al., 2010). However, the mechanisms underlying this difference between mammals and plants seem to be based essentially on different cellular functions of PRC1/PRC2-targeted genes (e.g. genes required for cell differentiation in mammals versus genes required to maintain stem cell activity in Arabidopsis). The biochemical functions of PRC1 and PRC2 in H2Aub1 and H3K27me3 deposition are similar between mammals and plants. It is remarkable, therefore, to find that ZRF1 proteins are required for H2Aub1 deposition in Arabidopsis but for H2Aub1 removal in mammals. Nevertheless, at this stage, one needs to remain cautious, particularly because the function of ZRF1 in regulating H2Aub1 homeostasis might be gene chromatin context dependent. Transcriptome analysis in Arabidopsis (this study) as well as in mammals (Demajo et al., 2014) reveals that the loss of ZRF1 function affects the expression of a large number of genes, comprising roughly half up-regulated and half down-regulated ones. It cannot be excluded that AtZRF1a/b may be involved in H2Aub1 removal at some genes in Arabidopsis and, vice versa, that the mammalian ZRF1 may be involved in H2Aub1 deposition at some yet unexamined genes. Lastly, it is crucial to note that many genes perturbed in expression do not overlap between the atzrf1a atzrf1b mutant and the PRC1-defective mutants, pointing toward the existence of PRC1-unrelated function(s) for AtZRF1a/b. Future studies will be necessary to shed further light on the molecular mechanisms underlying these different regulations of gene transcription by ZRF1, which will undoubtedly stimulate additional interest about this family of evolutionarily conserved proteins that play crucial roles in plant and animal development.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) pSTM:GUS reporter line and the atring1a atring1b and atbmi1a atbmi1b double mutant lines had been described previously (Xu and Shen, 2008; Chen et al., 2010). The other mutant lines were obtained from the Arabidopsis Biological Resource Center (http://www.arabidopsis.org) and the European Arabidopsis Stock Center (http://arabidopsis.info), with the corresponding stock numbers SAIL_786_F09 (N876841) for atzrf1a-1, SALK_070956.55.25.X (N570956) for atzrf1a-2, SALK_070965.50.20.x (N570965) for atzrf1a-3, FLAG_110A05 for atzrf1b-1, FLAG-099c10 for atzrf1b-2, SAIL_625_B03.v2 for atzrf1b-3, SAIL_629_F09.v1 for atzrf1b-4, and SAIL_716_D04 for D04. Higher order combinations of mutants and introgression of the pSTM:GUS reporter were obtained in this study by genetic crosses and PCR-based molecular genotyping. The PCR primers used in genotyping are listed in Supplemental Table S5.

Plants were grown on soil at 21°C to 22°C under either long-day (16 h of light and 8 h of dark) or medium-day (12 h of light and 12 h of light) photoperiod conditions. For in vitro plant growth, seeds were surface sterilized in 96% ethanol for 5 min and then in 70% ethanol for another 5 min, then plated on MS medium (MS salts, 1% Suc, pH 5.8, and 0.8% bacto agar). The plates were incubated for 2 d at 4°C to synchronize seed germination and then transferred to a growth chamber at 22°C under long-day photoperiod conditions.

Seed Germination Tests

Seed germination tests were performed as described previously (Molitor et al., 2014). Briefly, surface-sterilized seeds were plated on MS medium with or without the addition of 100 mm NaCl or 200 mm mannitol. Germination rates were scored daily for 12 d following stratification. Seeds were considered as germinated when radicle emergence out of the seed coat was visible using a dissecting microscope. Typically, three biological replicates, each with more than 80 seeds, were performed for each genotype in comparative analysis of the wild-type Col-0, the single mutants atzrf1a-1, atzrf1a-2, and atzrf1b-1, and the double mutants atzrf1a-1 atzrf1b-1, atzrf1a-2 atzrf1b-1, and atbmi1a atbmi1b.

GUS Histochemical Staining

For GUS activity assays, Col-0 and atzrf1a atzrf1b mutant plants containing the pSTM:GUS reporter were fixed in ice-cold 90% acetone for 30 min, washed in 50 mm sodium phosphate buffer (pH 7.2) for 5 min at room temperature, and incubated for 4 h at 37°C in the GUS staining solution, which contains 0.1 m sodium phosphate buffer (pH 7.2), 0.5 mm Fe(CN)2, 0.5 mm Fe(CN)3, 0.1% Tween 20, and 2 mm 5-bromo-4-chloro-3-indolyl β-d-glucuronide. The plants were washed subsequently in 70% ethanol overnight at 4°C to remove chlorophyll. Observation and imaging were performed using a Nikon E800 microscope. A similar GUS staining pattern was observed with over 30 plants, and the experiments were repeated three times.

RT-PCR Analysis

Total RNA was isolated from plant materials using the TRIzol kit according to the manufacturer’s recommendations (Invitrogen; http://www.invitrogen.com). The synthesis of first-strand cDNA was performed using the ImProm-II reverse transcriptase system (Promega; http://www.promega.com). Quantitative RT-PCR was carried out in a total volume of 10 μL of SYBR Green Master Mix (Roche) on a LightCycler LC480 instrument (Roche) according to the manufacturer’s instructions. Data are normalized using internal reference genes as reported previously (Molitor et al., 2014). Gene-specific PCR primers are listed in Supplemental Table S5. In all experiments, three biological replicates and three technical replicates on each sample were performed.

Flow Cytometry Analysis

Nuclei were prepared from leaves of 2-week-old plants and stained with propidium iodide as described previously (Galbraith et al., 1983) and analyzed on a FACStarPLUS flow cytometer (BD Biosciences) equipped with an INNOVA 90-C argon laser (Coherent). A total of 10 to 16 plants were used for each sample, and typically, 10,000 nuclei per sample were analyzed. Three replicates were performed for each sample.

Microarray Analysis

Six-day-old seedlings of atzrf1a-1 atzrf1b-1 and Col-0 grown on plates were harvested separately. Three independently derived sets of seedlings (30–40 plants per set) were pooled for each genotype. Total RNA was isolated from each sample and analyzed for gene expression using the Agilent Arabidopsis 4x44K oligonucleotide array containing 43,603 probes (G2519F; Agilent Technologies; http://www.agilent.com) via the custom service of the Shanghai Biotechnology Corporation (http://www.shbiotech.org). Raw signal intensities were extracted from scanned microarray images using the Agilent Feature Extraction software. Normalization between the set of arrays was performed using the quantile method. The raw and normalized data of microarrays were deposited in the public National Center for Biotechnology Information-Gene Expression Omnibus database (accession no. GSE76244). Differential gene expression analysis was performed by the online information analysis platform based on the statistics package R (http://sas.ebioservice.com). Genes with expression levels that changed more than 2-fold together with the Bonferroni P < 0.05 between the wild-type control and the mutant from three biological replicates were considered as differentially expressed. Lists of genes differentially expressed in the different mutants were compared in Venn diagrams generated using VENNY 2.1 (http://bioinfogp.cnb.csic.es/tools/venny). The representation factor and statistical significance of the observed overlap were measured with a hypergeometric test using the Web-based calculator developed by Jim Lund at the University of Kentucky (http://nemates.org/MA/progs/overlap_stats.html). A representation factor > 1 together with P < 0.05 indicate more overlap than expected between two groups at random.

Glutathione S-Transferase Pull-Down Assay

Two AtZRF1b cDNA fragments encoding the UBD (amino acids 175–300) and the SANT domain (amino acids 472–663), namely AtZRF1bUBD and AtZRF1bSANT, were amplified using specific PCR primers (Supplemental Table S5) and cloned into the bacterial expression vector pGEX-4T-1 (Sigma-Aldrich) for the production of the N-terminal glutathione S-transferase (GST) fusion proteins GST-AtZRF1bUBD and GST-AtZRF1bSANT, respectively. To obtain the FLAG-H2A production transgenic plant line, H2A.1 cDNA was amplified using specific PCR primers (Supplemental Table S5), cloned into the binary vector pCAMBIA1300-3×FLAG (Li et al., 2012), and transformed into Arabidopsis by the floral dip method (Clough and Bent, 1998). Mononucleosome-enriched protein extract (Richly et al., 2010) was prepared from 2-week-old 35Spro:3xFLAG-H2A transgenic plants. GST pull-down experiments were performed as described previously (Yu et al., 2004). The pull-down fractions were analyzed on western blots using the monoclonal anti-FLAG antibody (F1804; Sigma-Aldrich). For binding to ubiquitin, recombinant His tag fusion proteins of AtZRF1b were tested in pull down using GST or GST-ubiquitin in combination with western-blot detection using the anti-His antibody (H1029; Sigma-Aldrich).

Western-Blot Analysis of Histone Modifications

Histone-enriched nuclear protein extracts were prepared from 2-week-old plants of the wild type and the atbmi1a atbmi1b and atzrf1a-1 atzrf1b-1 mutants according to a previously described protocol (Yu et al., 2004). Proteins were analyzed on SDS-PAGE gels and detected using different antibodies specifically recognizing histone H3 (catalog no. 05-499; Millipore), H3K4me3 (catalog no. 07-473; Millipore), H3K27me3 (catalog no. 07-449; Millipore), H3K36me3 (catalog no. Ab9050; Abcam), H2Aub1 (catalog no. 8240; Cell Signaling), or ubiquitin (catalog no. FK-2; Upstate Millipore).

ChIP Analysis

ChIP was performed as described previously (Berr et al., 2010) with some minor modifications. The fixation time of seedlings was extended to 1 h, and chromatin was precleared with protein A beads. Immunoprecipitation was performed overnight using antibodies against H3K27me3 (catalog no. 07-449; Millipore), H2Aub1 (catalog no. 8240; Cell Signaling), or without antibodies as a negative control. The low- and high-salt wash buffers were supplemented with 0.01% SDS and 0.1% Triton X-100, and the LiCl wash buffer was supplemented with 1% sodium deoxycholate. DNA recovered after immunoprecipitation was analyzed by quantitative real-time PCR using gene-specific primers (Supplemental Table S5). Enrichment of H2Aub1 and H3K27me3 in the wild-type Col-0 and the mutants atzrf1a-1 atzrf1b-1, atzrf1a-2 atzrf1b-1, and atbmi1a atbmi1b was normalized using ACTIN as an internal control, as reported previously (Yang et al., 2013). Three independent experiments were performed, and similar results were obtained.

Accession Numbers

Microarray data have been deposited in the National Center for Biotechnology Information-Gene Expression Omnibus public database with accession number GSE76244.

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_172_3_1746__index.html (1.9KB, html)

Acknowledgments

We thank Zhongyuan Bu, Yu Yu, and Valérie Cognat for assistance in microarray analysis.

Glossary

Pc

Polycomb

PcG

Polycomb Group

Col-0

Columbia-0

RT

reverse transcription

SAM

shoot apical meristem

IM

inflorescence meristem

GO

Gene Ontology

ChIP

chromatin immunoprecipitation

MS

Murashige and Skoog

Footnotes

1

This work was supported by the Centre National de la Recherche Scientifique, the Agence Nationale de la Recherche (grant no. ANR–12–BSV2–0013–02), the European Commission (FP7–PEOPLE–2013–ITN; grant no. 607880), and the China Scholarship Council (fellowship to J.F.).

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Supplementary Materials

Supplemental Data
supp_172_3_1746__index.html (1.9KB, html)
supp_pp.16.00193_PP2016-00193R3_Figure_S1_to_Figure_S7.pdf (2.2MB, pdf)
supp_pp.16.00193_PP2016-00193R3_Supplemental_Dataset_1.pdf (678.8KB, pdf)
supp_pp.16.00193_PP2016-00193R3_Supplemental_Dataset2.pdf (876.9KB, pdf)
supp_pp.16.00193_PP2016-00193R3_Supplemental_Table_S1.pdf (61.6KB, pdf)
supp_pp.16.00193_PP2016-00193R3_Supplemental_Table_S2.pdf (56.4KB, pdf)
supp_pp.16.00193_PP2016-00193R3_Supplemental_Table_S3.pdf (57.6KB, pdf)
supp_pp.16.00193_PP2016-00193R3_Supplemental_Table_S4.pdf (42.3KB, pdf)
supp_pp.16.00193_PP2016-00193R3_Supplemental_Table_S5.pdf (112.3KB, pdf)

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