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Published in final edited form as: Sci Signal. 2016 Dec 20;9(459):ra125. doi: 10.1126/scisignal.aaf6767

The histone deubiquitinase OTLD1 targets euchromatin to regulate plant growth

Ido Keren 1,*, Vitaly Citovsky 1
PMCID: PMC9470540  NIHMSID: NIHMS1833511  PMID: 27999174

Abstract

Histone monoubiquitination is associated with active chromatin and plays an important role in epigenetic regulation of gene expression in plants. Deubiquitinating enzymes remove the ubiquitin group from histones and thereby contribute to gene repression. The Arabidopsis thaliana genome encodes 50 deubiquitinases, yet only 2 of them—UBP26 and OTLD1, members of the USP/UBP (ubiquitin-specific protease and ubiquitin-binding protein) and OTU (ovarian tumor protease) deubiquitinase families—are known to target histones. Furthermore, UBP26 is the only plant histone deubiquitinase for which the functional role has been characterized in detail. We used gain-and loss-of-function alleles of OTLD1 to examine its role in the plant life cycle and showed that OTLD1 stimulates plant growth, increases cell size, and induces transcriptional repression of five major regulators of plant organ growth and development: GA20OX, WUS, OSR2, ARL, and ABI5. OTLD1 associated with chromatin at each of these target genes and promoted the removal of euchromatic histone acetylation, ubiquitination, and methylation marks. Thus, these data indicate that OTLD1 promotes the concerted epigenetic regulation of a set of genes that collectively limit plant growth.

INTRODUCTION

The state of plant chromatin is determined by dynamic changes in combinations of different posttranslational modifications of histones on their lysine residues, which mainly include acetylation, methylation, and monoubiquitination (1–3). Among those, histone monoubiquitination and its effectors and roles in plant growth and development are least well understood (4). Yet, this type of histone modification has been implicated in many important aspects of plant physiology and development. For example, monoubiquitination of histone H2B affects seed dormancy, root growth, leaf shape, cell cycle, flowering time, and photomorphogenesis (5–9). Generally, H2B monoubiquitination is found in the chromatin of highly expressed genes (10), and it facilitates formation of other histone modification marks and promotes transcript elongation by RNA polymerase II (4).

Histone monoubiquitination is reversed by histone deubiquitinases, which remove the ubiquitin moiety. The Arabidopsis thaliana genome encodes 50 deubiquitinating enzymes, which comprise five families—USPs/UBPs (ubiquitin-specific proteases and ubiquitin-binding proteins), UCHs (ubiquitin C-terminal hydrolases), JAMMs (JAB1/MPN/MOV34 proteases), OTUs (ovarian tumor proteases), and MJD (Machado-Joseph domain)—that play key roles in regulation of various cellular processes (11, 12). However, only two of these deubiquitinases, UBP26 and OTLD1, are known to target histones, with UBP26 being the only one for which molecular function has been characterized in detail (2, 11). UBP26, a member of the UBP family, removes ubiquitin residues from monoubiquitinated histone H2B. UBP26 can act both as a transcriptional repressor that promotes removal of euchromatic histone marks, such as trimethylation of histone H3 on Lys4 (H3K4me3) (13), and as a transcriptional activator that promotes removal of heterochromatic histone marks, such as H3K27me3 (13, 14), within the target gene promoters. Accordingly, UBP26 has been implicated in transcriptional repression of PHE1, involved in seed and embryo development (15), and in transcriptional activation of FLC, involved in flower timing (14).

OTLD1, a member of the OTU family of deubiquitinases (2, 11), also deubiquitinates monoubiquitinated histone H2B and potentiates transcriptional repression (16), but its phenotypic effects and potential role(s) in the plant life cycle are unknown. Here, we show that OTLD1 is a transcriptional repressor of GA20OX, WUS, OSR2, ARL, and ABI5, which are major regulators of plant organ growth and development. We also show that OTLD1 associates with the chromatin of these target genes and promotes removal of euchromatic histone acetylation, ubiquitination, and methylation marks at these sites.

RESULTS

Phenotypes of OTLD1 gain-of-function alleles

The Arabidopsis OTU family of deubiquitinases, which contains 12 members (11, 12), has been divided into five clades based on the biochemical properties of these enzymes, suggesting overlapping function and redundancy within each clade (17). Standard reverse genetics approaches that use loss-of-function mutants for studies of gene function, therefore, are not ideal for understanding the biological role of OTLD1. Thus, we used an alternative strategy of generating and analyzing gain-of-function alleles. Mining the Genevisible database (18) for the native OTLD1 expression pattern suggested that it is highly expressed in vascular tissues, bundle sheaths, cotyledons, and leaf epidermis. This pattern of OTLD1 expression in different organs of wild-type plants of different ages was further examined using real-time quantitative polymerase chain reaction (RT-qPCR) analysis, which showed that OTLD1 was expressed in all tissues, but in somewhat different amounts and increased as plants matured (Fig. 1A).

Fig. 1. Expression of OTLD1 in wild-type, OTLD1 OE-1, and OTLD1 OE-2 plants.

Fig. 1.

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(A) OTLD1 transcript abundance in roots, seedlings, and aerial organs from 25-day-old (a) and 35-day-old (b) wild-type plants. Error bars represent SEM of independent biological replicates, n = 3. (B) Expression of OTLD1 in whole aerial parts of 25-day-old wild-type (WT; black bars) plants and two lines overexpressing OTLD1 (OTLD1 OE-1, dark gray bars, and OTLD1 OE-2, light gray bars). (C) Expression of the reference genes ACT7 and UBQ10 for the analysis shown in (B). Expression of OTLD1 was analyzed by RT-qPCR, and the transcript abundance in the wild-type plants was set to 1.0. Error bars represent SEM, n = 3 independent biological replicates, P < 0.05 for differences between wild-type plants and OTLD1-overexpressing plants in (B). Differences between all tested plants in (C) were not statistically significant. A.U., arbitrary units.

To increase the amount of OTLD1 in wild-type plants, we overexpressed OTLD1 from a general cauliflower mosaic virus 35S promoter in several independent homozygous transgenic lines, two of which, designated OTLD1 OE-1 and OTLD1 OE-2, were analyzed in detail. RT-qPCR analysis of the whole aerial parts of the T2 generation of these overexpression alleles showed that they accumulated ~2.5-fold higher amounts of OTLD1 transcripts than did the wild-type plants (Fig. 1B). Internal controls of two reference genes ACT7 and UBQ10 demonstrated similar expression in all plant lines (Fig. 1C). The moderate increase in OTLD1 transcription in the gain-of-function alleles allowed us to analyze the function of this gene while avoiding unnecessarily high expression amounts.

First, we assessed the phenotypic effects of gain of function of OTLD1. Both OTLD1 OE-1 and OTLD1 OE-2 lines developed characteristic alterations in development of their vegetative and reproductive organs. Specifically, their leaves grew larger and more numerous than did those of the wild-type plants (Fig. 2A). Quantification of these parameters showed that average rosette diameter of both transgenic lines was 24 to 40% larger than that of wild-type plants (Fig. 2B). The number of leaves per plant in both lines was also higher, 12 and ~11.7, respectively, compared to ~5.8 in the wild type (Fig. 2C). As their growth progressed, the OTLD1 OE-1 and OTLD1 OE-2 plants continued to exhibit enhanced growth of their aerial parts, specifically longer stems and larger rosettes (Fig. 2D). Quantification of plant height showed that the mature OTLD1 OE-1 and OTLD1 OE-2 plants were 25 to 40% taller than the wild-type plants (Fig. 2E). Another clear effect of OTLD1 overexpression was on the inflorescence architecture: The inflorescences of OTLD1 transgenic plants developed more numerous flowers than those of wild-type plants (Fig. 2F), with an average of 36 to 41 flowers versus 22 flowers per plant (Fig. 2G). Both OTLD1 OE-1 and OTLD1 OE-2 plant lines, however, exhibited no statistically significant difference in their flowering time relative to the wild-type plants; specifically, the onset of flowering occurred in 60% of OTLD1 OE-1 and OTLD1 OE-2 and in 50% of the wild-type plants at 25 to 26 days after germination.

Fig. 2. Morphological characterization of OTLD1 OE-1 and OTLD1 OE-2 plants.

Fig. 2.

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(A) Representative leaf rosettes of 25-day-old plants. Scale bars, 1.0 cm. (B) Leaf rosette diameter in 25-day-old plants, n = 50. (C) Number of leaves on 25-day-old plants, n = 30. (D) Representative mature 30-day-old plants. Scale bars, 2.0 cm. (E) Height of mature 30-day-old plants, n = 30. (F) Representative inflorescences of 35-day-old plants. Scale bars, 0.25 cm. (G) Number of flowers in 35-day-old plants, n = 24. (H) Cell density at the leaf epidermal blade tip (T) and mid-rib (MR), n = 6 sections per plant from three individual plants per line. Error bars represent SEM, P < 0.05 for differences between wild-type plants and OTLD1 OE-1 or OTLD1 OE-2 plants.

The enhanced growth phenotype of the OTLD1 OE-1 and OTLD1 OE-2 plants could result from an increase in cell size, cell number, or both. To assess the possible contributions of cell expansion and proliferation, we examined the size and surface density of adaxial epidermal cells of fully expanded fifth rosette leaves, which faithfully represent the characteristic features typical of rosette leaf development in Arabidopsis (19). OTLD1 OE-1 (fig. S1A) and OTLD1 OE-2 lines (fig. S1B) exhibited enlarged leaf epidermal cells as compared to the wild-type plants (fig. S1C). When cells in the blade tip and blade midrib sections of the leaf were counted, their surface density in the OTLD1 OE-1 and OTLD1 OE-2 plants was 40 to 50% lower than that observed in wild-type leaves (Fig. 2H), indicating an increase in the cell size. Thus, OTLD1 overexpression most likely promotes cell growth rather than cell proliferation.

Gene targets of transcriptional repression by OTLD1

The major phenotypic hallmarks of the OTLD1 OE-1 and OTLD1 OE-2 plants, enhanced growth of their aerial organs, inform about possible identity of the target genes of OTLD1 and facilitate their rational prediction. Thus, we selected 42 genes (table S1) that include representatives of major gene families involved in the control of plant growth, cell expansion, and the transition from cell division to cell expansion. The amounts of transcripts of each of these genes were then analyzed by RT-qPCR in the OTLD1 OE-1 and OTLD1 OE-2 plants and compared to wild-type plants (Fig. 3A). Most of the tested genes showed no significant changes in their expression amounts in any of the plant lines; this group of genes is exemplified by ABI2 and GRF5, the transcripts of which accumulated to comparable amounts in the OTLD1 OE-1, OTLD1 OE-2, and wild-type plants (Fig. 3B). However, five genes—GA20OX2, WUS, OSR2, ARL, and ABI5—displayed substantial reduction in expression in both transgenic lines compared to wild type (Fig. 3A).

Fig. 3. Transcriptional repression of OTLD1 target genes in OTLD1 OE-1 and OTLD1 OE-2 plants.

Fig. 3.

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(A) RT-qPCR analyses of relative transcript abundance for OTLD1 target genes GA20OX, WUS, OSR2, ARL, and ABI5; P < 0.05 for differences between wild-type plants (black bars) and OTLD1 OE-1 (dark gray bars) or OTLD1 OE-2 (light gray bars) plants. Relative transcript abundance of nontarget genes ABI2 and GRF5 (B) and reference genes ACT7 and UBQ10 (C). Differences between wild-type plants and OTLD1 OE-1 or OTLD1 OE-2 plants were not statistically significant for (B) and (C). Error bars represent SEM, n = 3 independent biological replicates. (D and E) Hypoacetylation of chromatin in promoters of OTLD1 target genes in OTLD1 OE-1 and OTLD1 OE-2 plants. Quantitative chromatin immunoprecipitation (qChIP) analyses of relative amounts of H3 acetylation (H3Ac) are shown for ARL, GA20OX, OSR2 (D), WUS, ABI5, ABI2, and GRF5 (E) promoters. Error bars represent mean for two biological replicates, with three technical replicates for each. Locations of sequences upstream of the translation initiation site (ATG) used for qChIP analyses are indicated for each gene.

GA20OX2 is a member of the GA20OX (gibberellin 20-oxidase) family of enzymes, is involved in the biosynthesis of the plant hormone gibberellic acid (GA), and is highly abundant during vegetative and early reproductive development (20, 21). Overexpression of OTLD1 suppressed expression of GA20OX2 by 2.5- to 7-fold in the OTLD1 OE-1 and OTLD1 OE-2 plants, respectively, compared to wild type (Fig. 3A). WUSCHEL (WUS) is a homeodomain transcription factor that plays a central role in the maintenance of stem cell populations in the shoot meristem and promotes cytokinin signaling (22–25). WUS showed the strongest response to OTLD1, with its expression almost completely inhibited in both OTLD1 OE-1 and OTLD1 OE-2 lines (Fig. 3A). OSR2 is a member of the ORGAN SIZE RELATED (OSR) gene family that participates in regulation of organ growth primarily by affecting cell expansion (26). Its transcript amounts were reduced by 2- to 12-fold in OTLD1 OE-1 and OTLD1 OE-2, respectively (Fig. 3A). ARGOS-LIKE (ARL), also a member of the OSR family, promotes organ size by affecting cell expansion, and its function, at least partially, is redundant with OSR2 (26–29). ARL gene expression in OTLD1 OE-1 and OTLD1 OE-2 was reduced by 13- and 4.5-fold, respectively (Fig. 3A). Finally, overexpression of OTLD1 suppressed expression of ABI5 (ABSCISIC ACID INSENSITIVE 5), which encodes a basic zipper (bZIP) transcription factor involved in the regulation of vegetative growth and floral induction (30–32). In the OTLD1 OE-1 and OTLD1 OE-2 plants, the ABI5 transcript amounts were reduced by 6.5- and 9-fold, respectively (Fig. 3A). All RT-qPCR experiments used internal controls of two reference genes, ACT7 and UBQ10, the expression amounts of which were similar in all plant lines tested (Fig. 3C).

One of the hallmarks of transcriptional repression is histone hypoacetylation in the target chromatin (33–35). Thus, we used qChIP to examine whether the OTLD1-mediated repression of each of these potential target genes occurs through hypoacetylation of chromatin. Because regulatory elements involved in the repression of most of these genes are largely unknown, for each of them, we tested three to five regions of the putative promoter sequences that span 2.0-kb upstream of the translation initiation codon (table S1) (36). In these experiments, we observed significant amounts of histone H3 hypoacetylation in the chromatin of the ARL, WUS, GA20OX2, ABI5, and OSR2 genes in the OTLD1 OE-1 and OTLD1 OE-2 plants in comparison to the wild-type plants (Fig. 3, D and E). In each of the tested genes, histone acetylation was detected in a single specific region of chromatin within 1-kb upstream of the translation initiation codon, and overexpression of OTLD1 reduced these amounts of H3 acetylation by 1.5- to 3-fold in ARL, GA20OX2, and OSR2 (Fig. 3D), whereas H3 acetylation in WUS and in ABI5 was reduced by up to 80- and 5-fold, respectively, relative to the wild-type plants (Fig. 3E). These changes were specific because, in negative control experiments, no significant changes in H3 acetylation were observed in the chromatin of ABI2 and GRF5 (Fig. 3E), the expression of which was not affected in the OTLD1 OE-1 and OTLD1 OE-2 lines (see Fig. 3B). Collectively, our data suggest that OTLD1 may act as transcriptional repressor of five organogenesis-related ARL, WUS, GA20OX2, ABI5, and OSR2 genes.

Deubiquitination of chromatin by OTLD1

Increased activity of H2B-ubiquitinating enzymes at promoter regions induces gene expression and plays an important role during RNA polymerase II elongation, whereas these expression amounts are decreased when histone H2B monoubiquitination is reversed (37–40). It makes biological sense, therefore, that H2B deubiquitination by OTLD1 underlies the OTLD1-mediated reduction in expression of the ARL, WUS, OSR2, GA20OX2, ABI5, and OSR2 genes and histone hypoacetylation of these loci. Our qChIP analyses showed a significant degree of hypoubiquitination of histone H2B in the ARL, WUS, GA20OX2, ABI5, and OSR2 chromatin in the OTLD1 OE-1 and OTLD1 OE-2 plants (Fig. 4). Specifically, the ARL chromatin of both OTLD1 OE-1 and OTLD1 OE-2 contained four distinct regions located within 1.9-kb upstream of the translation initiation codon, with monoubiquitination amounts on average 7- to 24-fold lower than ARL chromatin in wild-type plants (Fig. 4A). Monoubiquitination of GA20OX2 and OSR2 was detected in three chromatin regions each, and its reduction ranged from 5.5- to 13-fold and 2.5- to 6.5-fold, respectively (Fig. 4, B and C). The WUS chromatin was hypoubiquitinated by 6- to 20-fold in two regions (Fig. 4D), one of which partly overlapped the known WUS promoter sequence involved in its transcriptional activation by the SNF2 class chromatin-remodeling adenosine triphosphatase (ATPase) SPLAYED (SYD) (41) as well as the binding site for the transcription factor ARF3 (42). Finally, the ABI5 chromatin was hypoubiquitinated in a single region by 5- to 9-fold as compared to the wild-type plants (Fig. 4D); this region partly overlapped the binding site for BRAHMA (BRM), another member of the SNF2 ATPase family, which is a known repressor of ABI5 (43). Confirming the specificity of these observations, no statistically significant changes in the degree of H2B ubiquitination were detected in the chromatin of ABI2 and GRF5 (Fig. 4E), the expression of which was not altered by overexpression of OTLD1 (see Fig. 3B).

Fig. 4. Hypoubiquitination of chromatin in OTLD1 target genes in OTLD1 OE-1 and OTLD1 OE-2 plants.

Fig. 4.

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qChIP analyses of relative amounts of H2B monoubiquitination (H2Bub) are shown for ARL (A), GA20OX (B), OSR2 (C), WUS and ABI5 (D), and ABI2 and GRF5 (E). Black bars, wild-type plants; dark gray bars, OTLD1 OE-1; light gray bars, OTLD1 OE-2. Error bars represent mean for two biological replicates, with three technical replicates for each. Locations of sequences upstream of the translation initiation site (ATG) used for qChIP analyses are indicated for each gene.

We then examined whether OTLD1 itself occupies the regions of the deubiquitinated chromatin. In the OTLD1 OE-1 and OTLD1 OE-2 lines, OTLD1 was tagged with a His6 epitope, allowing us to use qChIP to detect its presence. OTLD1 associated with exactly the same regions of the ARL, WUS, GA20OX2, ABI5, and OSR2 chromatin that displayed hypoubiquitination in the OTLD1 OE-1 and OTLD1 OE-2 plants (Figs. 4 and 5). Other putative promoter sequences tested for each gene (table S1) showed no association with OTLD1, nor did they display detectable changes in the degree of their H3 acetylation and H2B ubiquitination. Although both lines exhibited a similar extent of OTLD1 overexpression (Fig. 1B) as well as comparable amounts of hypoacetylation and hypoubiquitination of the ARL, WUS, GA20OX2, ABI5, and OSR2 chromatin (Figs. 3, D and E, and 4), the OTLD1 OE-1 line showed generally lower amounts of OTLD1 association with chromatin than did the OTLD1 OE-2 line (Fig. 5, A to D). Potentially, this difference may reflect different antigenicity of OTLD1 expressed in different plants. We detected no presence of His6-tagged OTLD1 in the chromatin of the ABI2 and GRF5 genes (Fig. 5E), the expression of which did not respond to OTLD1 overexpression (see Fig. 3B), supporting the correlation between the presence of OTLD1 at the target gene promoter and transcriptional repression of this gene.

Fig. 5. Association of OTLD1 with chromatin of target genes in OTLD1 OE-1 and OTLD1 OE-2 plants.

Fig. 5.

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qChIP analyses of His-tagged OTLD1 associated with chromatin are shown for ARL (A), GA20OX (B), OSR2 (C), WUS and ABI5 (D), and ABI2 and GRF5 (E). Dark gray bars, OTLD1 OE-1; light gray bars, OTLD1 OE-2. Error bars represent mean for two biological replicates, with three technical replicates for each. Locations of sequences upstream of the translation initiation site (ATG) used for qChIP analyses are indicated for each gene.

OTLD1-mediated reduction in H3K4 trimethylation

Histone H2B deubiquitination during transcriptional repression often facilitates the removal of euchromatin methylation marks (7, 13, 44), a major one of which is H3K4me3 (13). Thus, we used qChIP to analyze, in the OTLD1 OE-1 and OTLD1 OE-2 plants, the chromatin of all five target genes that are suppressed by OTLD1 for possible changes in their H3K4me3 content. The chromatin of the ARL, WUS, GA20OX2, ABI5, and OSR2 genes contained reduced amounts of H3K4me3 (Fig. 6). Specifically, the H3K4me3 content of the ARL chromatin of both OTLD1 OE-1 and OTLD1 OE-2 lines was 2.5- to 2-fold lower than that of the wild-type plants (Fig. 6A). Similarly, trimethylation of H3K4 of GA20OX2 and OSR2 was reduced by 5- to 2.5-and 5- to 4-fold, respectively (Fig. 6A). The H3K4me3 content of WUS and ABI5 also was reduced by 5- to 2.5-fold and 4- to 1.7-fold, respectively (Fig. 6B). Conversely, changes in the extent of H3K4 trimethylation of the chromatin of the control, OTLD1-nonresponsive ABI2 and GRF5 genes were insignificant (Fig. 6B).

Fig. 6. Trimethylation of H3K4 in the chromatin of OTLD1 target genes in OTLD1 OE-1 and OTLD1 OE-2 plants.

Fig. 6.

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qChIP analyses of relative amounts of H3K4me3 are shown for ARL, GA20OX, and OSR2 (A) and for WUS, ABI5, ABI2, and GRF5 (B). Black bars, wild-type plants; dark gray bars, OTLD1 OE-1; light gray bars, OTLD1 OE-2. Error bars represent mean for two biological replicates, with three technical replicates for each. Locations of sequences upstream of the translation initiation site (ATG) used for qChIP analyses are indicated for each gene.

Transcriptional activation of the OSR2 gene in the OTLD1 loss-of-function alleles

Consistent with potential functional redundancy of OTLD1 with its OTU family homologs (11, 12), loss-of-function mutants of OTLD1 did not exhibit detectable phenotypes. Specifically, we examined two available Arabidopsis mutants, otld1-1 (16) and otld1-2, both of which are homozygous for transferred DNA (T-DNA) insertion into the OTLD1 gene. In both mutants, the mutagenic insert is located within the exon between the isopeptidase OTU and the UBA (ubiquitin-associated) domains, after amino acid positions 342 and 353, respectively (Fig. 7A). RT-qPCR analysis showed vanishingly low amounts of OTLD1 transcripts in the otld1-1 and otld1-2 mutants as compared to the wild-type plants (Fig. 7B), whereas the internal controls of the ACT7 and UBQ10 reference genes displayed similar expression amounts in all plant lines (Fig. 7C). Both mutants also displayed no clear and statistically significant differences in morphological or developmental parameters as compared to the wild-type plants (Fig. 7, D and E).

Fig. 7. Characterization of otld1-1 and otld1-2 mutant plants.

Fig. 7.

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(A) Schematic structure of the OTLD1 protein, with the conserved OTU and UBA domains noted. The location of the mutagenic T-DNA insertions and amino acid sequences corresponding to the coding sequence upstream of each insertion are indicated for each mutant. Numbers indicate amino acid positions within the OTLD1 sequence. (B) Relative expression of OTLD1 in wild-type (black bars), otld1-1 (dark gray bars), and otld1-2 (light gray bars) plants by RT-qPCR. P < 0.05 for statistical significance of differences between the mutant and wild-type plants. (C) Relative expression of the reference genes ACT7 and UBQ10 did not differ significantly between wild-type, otld1-1, and otld1-2 plants. Transcript abundance in wild-type plants was set to 1.0, error bars represent SEM, n = 3 independent biological replicates. (D) Representative leaf rosettes of 25-day-old plants. Scale bars, 1.0 cm. (E) Leaf rosette diameter in 25-day-old plants, n = 50 plants. Error bars represent SEM; differences between all tested plants were not statistically significant. (F) Expression of the target genes GA20OX, ARL, ABI5, OSR2, and WUS. Differences in expression of GA20OX, ARL, ABI5, and WUS between all tested plants were not statistically significant. P < 0.05 for statistical significance of differences in expression of OSR2 between the wild-type and mutant plants. (G) Expression of the nontarget genes ABI2 and GRF5. (H) Expression of the reference genes ACT7 and UBQ10 did not differ significantly between wild-type and mutant plants. Transcript abundance in the wild-type plants was set to 1.0, error bars represent SEM, n = 3 independent biological replicates.

It is possible, however, that the likely functional overlap between OTLD1 and other OTU enzymes does not extend to some of the OTLD1 target genes; our identification of OTLD1 targets allowed us to examine this idea. RT-qPCR analysis of the accumulation of the ARL, WUS, GA20OX2, ABI5, and OSR2 transcripts revealed that the amount of one of them was altered, in a statistically significant fashion, in both mutants. Specifically, expression of the OSR2 gene was increased by 6- to 10-fold in the otld1-1 and otld1-2 plants, respectively (Fig. 7F). Negative control experiments detected no statistically significant changes in the expression of ABI2 and GRF5 (Fig. 7G), which also was not altered by overexpression of OTLD1 (see Fig. 3B), or of the internal reference genes ACT7 and UBQ10 (Fig. 7H). These observations are consistent with the role of OTLD1 as transcriptional repressor, the lack of which would result in enhanced expression of its target genes through chromatin modification. Previously, OSR2 has been overexpressed in Arabidopsis, but phenotypic effects have been reported only for plants that exhibited expression amounts 200- to 800-fold higher than those in the wild-type plants (26).

Histone modifications at OSR2 in OTLD1 loss-of-function alleles

The effects of OTLD1 on the chromatin of the target genes observed in the OTLD1 gain-of-function alleles were confirmed using the loss-of-function mutants. We examined H3 acetylation, H2B monoubiquitination, and H3K4 trimethylation in the chromatin of four genes—OSR2, ARL, ABI2, and GRF5—in the otld1-1 and otld1-2 mutants. In these plants, OSR2 represents the target gene derepressed in the absence of OTLD1, ARL exemplifies the target gene (the repression of which most likely can be mediated by other OTU homologs), and ABI2 and GRF5 are non-target, control genes. In both otld1-1 and otld1-2 plants, the OSR2 chromatin exhibited the hallmarks of euchromatin: H3 hyperacetylation, increased H2B monoubiquitination, and increased H3K4 trimethylation. Specifically, H3 acetylation and H2B monoubiquitination were increased 1.5- to 1.6-fold and 1.5- to 12-fold, respectively, whereas H3K4 trimethylation increased by 2- to 2.3-fold, as compared to the wild-type plants (Fig. 8). In contrast, neither ARL, ABI2, nor GRF5 chromatin underwent statistically significant changes (Fig. 8, A to C).

Fig. 8. Histone modifications at the OSR2 locus In otld1-1 and otld1-2 mutant plants.

Fig. 8.

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qChIP analyses to determine the relative amounts of (A) H3 acetylation at OSR2, ARL, ABI2, and GRF5; (B) H2B monoubiquitination at OSR2, ARL, ABI2, and GRF5; and (C) H3K4 trimethylation at OSR2, ARL, ABI2, and GRF5 in wild-type (black bars), otld1-1 (dark gray bars), and otld1-2 (light gray bars) plants. Error bars represent mean for two biological replicates, with three technical replicates for each. Locations of sequences upstream of the translation initiation site (ATG) used for qChIP analyses are indicated for each gene.

DISCUSSION

Histone deubiquitination is thought to be necessary for tighter packaging of chromatin (45) and generation of repressive histone methylation marks (46), thereby facilitating heterochromatin formation and gene repression. Biochemically, this important aspect of chromatin remodeling is affected by histone deubiquitinases (46). Most of our knowledge about histone deubiquitinases derives from animal and yeast systems (4), whereas the understanding of these enzymatic activities in plants is still in its infancy. Of 50 deubiquitinases encoded in the Arabidopsis genome, only 2 enzymes, UBP26 and OTLD1, have been shown to target histones, with just 1 of them, UBP26, playing a demonstrated role in the plant life cycle (2, 11, 12) by regulating the PHE1 and FLC genes involved in seed and embryo development (15) and flower timing (14), respectively. Thus, it was important to examine the biological role and the potential target genes of the second known plant histone deubiquitinase, OTLD1. OTLD1 is a member of the OTU family of deubiquitinases, which are linkage-specific (47), so it likely functions by a biochemical mechanism distinct from that of UBP26, a member of the USP/UBP deubiquitinase family, members of which cleave most ubiquitin chain types indiscriminately (48).

Our characterization of OTLD1 was complicated by the lack of detectible phenotypes of the otld1-1 (16) and otld1-2 loss-of-function mutants. This, however, is not unusual for deubiquitinases, only a small portion of which could be assigned biological roles based on phenotypic analysis of loss-of-function mutants due to functional redundancies between individual members of the same deubiquitinase family (11). One well-established approach to circumvent this problem is the use of gain-of-function alleles that allow characterization of functions of individual gene family members (49–54); for example, overexpression allowed discovery of the function of chromatin remodeling ATPase gene, AtCHR23, the knockout mutants of which did not display detectable phenotypes (49). Similarly, modest overexpression of OTLD1 allowed us to analyze the specific effects of this enzyme on plant phenotype and chromatin state. This analysis of two independent gain-of-function alleles indicated that overexpressed OTLD1 mainly increased the size of the aerial organs and the size of the cells in these organs. On the basis of this overall phenotypic effect, it became possible to predict that potential gene targets of OTLD1 likely include those involved in plant growth and cell expansion as well as in phytohormone signaling, which also affects organ growth and size (55, 56). Our analysis of expression of 42 genes that represent gene families known to have these effects on plant growth identified five genes—GA20OX2, WUS, OSR2, ARL, and ABI5—that exhibited three hallmarks of being direct targets of OTLD1. All of them were transcriptionally repressed by OTLD1, and more detailed analysis of the loss-of-function alleles revealed transcriptional activation of one of the identified OTLD1 target genes, OSR2, supporting the function of OTLD1 as transcriptional repressor. OTLD1 was found to be associated with the chromatin of the GA20OX2, WUS, OSR2, ARL, and ABI5 genes, which was also deubiquitinated when OTLD1 was overexpressed. Three of these genes—GA20OX2, WUS, and ABI5—promote plant growth through hormonal signaling, affecting the gibberellin, cytokinin, and abscisic acid pathways, respectively (21–23, 32), whereas the other two, ARL and OSR2, are themselves promoted by brassinosteroid hormones (26, 27). These observations lend support to the emerging idea of a functional relationship between plant hormones and chromatin remodeling, including histone modifications (57). Note that the goal of this study was not to understand precisely which genes produced the OTLD1 gain-of-function phenotype; we believe that it represents the result of pleiotropic and synergistic action of many genes rather than additive effects of each of the individually affected target genes. Instead, our study aims to uncover the role that OTLD1, one of only two known histone deubiquitinases in plants, may play in regulation of gene expression and the mechanism by which OTLD1 fulfills this function.

Our current knowledge about the OTLD1 indicates that it is a functional histone deubiquitinase enzyme that directly binds chromatin (16), deubiquitinates monoubiquitinated H2B (16), associates with the chromatin of promoters of specific genes, induces deubiquitination of these target chromatin regions, and represses expression of the target genes. Thus, OTLD1 likely represents a transcriptional repressor that acts by deubiquitinating its target chromatin. As an alternative to this “Occam’s razor” principle of simple theories preferable to more complex ones, we cannot rule out the possibility that OTLD1, itself an enzymatically active histone deubiquitinase, recruits yet another, unidentified, histone deubiquitinase to affect histone deubiquitination. In any case, OTLD1 obviously does not repress its target genes single-handedly; instead, it most likely functions as part of a repressor complex, the components of which may vary, depending on the specific target gene or on the cell type. Mechanistically, this OTLD1-containing repressor complex confers on the target chromatin repressive histone marks, such as H2B hypoubiquitination and H3 hypoacetylation, and removes euchromatic histone marks, such as H3K4me3. Whereas at present the composition of such OTLD1-containing repressor complexes is unknown, based on the effect of OTLD1 on its target chromatin, they should contain such common constituents as histone deacetylases, histone methyltransferases, and histone lysine demethylases. Consistent with this notion, our previous work suggests that OTLD1 can interact with the histone lysine demethylase KDM1C and co-repress at least one common target gene, At5g39160 (16); however, this gene has no known effects on plant growth, and our previous microarray analysis of genes targeted by KDM1C (16, 58) did not detect any of the OTLD1 target genes identified here. In addition, our earlier data also show that KDM1C itself can interact with the histone methyltransferase SUVR5 (58). Regardless of the exact nature of the proteins that function together with OTLD1 to repress its target genes, our data position OTLD1 as a histone-modifying enzyme and monoubiquitination as a specific histone modification that, in a concerted fashion, determine repressive chromatin structure of a spectrum of genes involved in major aspects of plant organ growth and cell expansion. These observations also afford a better insight into the fundamental role that the members of the still enigmatic family of plant OTU-type histone deubiquitinases play in growth and development.

MATERIALS AND METHODS

Plant material and growth conditions

Seeds of the wild-type A. thaliana (ecotype Col-0) plants and of the SALK_028707 and SALK_037047 lines, corresponding to the otld1-1 (16) and otld1-2 T-DNA insertion mutants of OTLD1, respectively, were obtained from the Arabidopsis Biological Resource Center (abrc.osu.edu). Seeds were surface-sterilized with 10% bleach (sodium hypochlorite) solution and 70% ethanol, and plated on Murashige and Skoog medium (59) with 0.8% (w/v) agar, containing 3% (w/v) sucrose. The plated seeds were stratified for 3 days at 4°C in the dark and then transferred to a controlled environment growth chamber and grown for 10 days at 22°C under long-day conditions (16-hour light/8-hour dark cycle at 140 microeinstein s−1 m−2 light intensity), transferred to soil, and maintained under the same growth conditions. For RT-qPCR analysis, tissue samples were taken from 21-day-old (roots), 7-day-old (seedlings), 25-day-old (aerial organs), or 35-day-old (aerial organs) plants.

Plasmid construction and production of transgenic plants

For transgenic expression of OTLD1 (At2g27350) (16), its coding sequence was amplified from Arabidopsis complementary DNA (cDNA) using primers detailed in table S1 and cloned into the Xho I–Kpn I sites of pSAT4-HIS-C1 (60). The expression cassette that produces His-tagged OTLD1 was excised and inserted into the I–Sce I site of the binary pPZP-RCS2 vector (61). The resulting construct was used to generate transgenic lines by Agrobacterium-mediated genetic transformation (62). Independent kanamycin-resistant T1 transformants were selected, and their T2 progeny was selected for morphological, molecular, and histological analyses.

Real-time quantitative PCR

Total RNA (1 μg), extracted from frozen plant tissues using the NucleoSpin RNA Plant Kit (Macherey-Nagel GmbH & Co.), was reverse-transcribed using oligo(dT)18 primer (Thermo Fisher Scientific Inc.) and ProtoScript II reverse transcriptase (New England Biolabs) according to the manufacturers’ instructions. The resulting cDNA preparations (2 μl) were used as template for RT-qPCR using Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific Inc.), specific primers described in table S1, and a StepOnePlus real-time PCR system (Applied Biosystems) with the following conditions: 1 cycle at 95°C for 5 min and 40 cycles each at 95°C for 10 s, 57°C for 10 s, and 72°C for 15 s. Each sample was analyzed in two biological repeats and in three technical replicates. Two validated stably expressed reference genes, UBQ10 (At4g05320) and ACT7 (At5g09810) (table S1), were used for normalization of RT-qPCR data by the comparative cycle threshold (Ct) method, using ΔCt, which represents the value obtained by subtracting the Ct value of the tested transcript from the Ct value of UBQ10 and ACT7 transcripts in each sample. Then, the relative gene expression amounts were calculated using the cycle threshold (CT) 2−ΔΔCt method (63). All quantitative data were analyzed by the Student’s t test; P values of <0.05, corresponding to the statistical probability of greater than 95%, were considered statistically significant. SEM and t test calculations were performed using Excel 2010 (Microsoft Inc.).

Quantitative chromatin immunoprecipitation

ChIP was performed as described with slight modifications (64). Briefly, a combination of 2 g of flower tissue, 4 g of leaf tissue, and 4 g of stem tissue was harvested from 25-day-old plants and cross-linked using 1% (v/v) formaldehyde. Nuclei were isolated and disrupted using a Sonic Power Sonifier-185 (Branson Ultrasonics Power Co.), and the sheared chromatin was preincubated at 4°C for 1 hour with pre-equilibrated protein A agarose beads (16-157, Millipore), followed by centrifugation at 3800g for 2 min. The resulting supernatant was incubated at 4°C overnight with gentle shaking with 1:100 dilution of the appropriate antibody [anti-acetyl-histone H3 (06-599, Millipore), anti–monoubiquityl–histone H2B (Lys120) (5546S, Cell Signaling Technology Inc.), anti–trimethyl H3K4 (8580, Abcam), or anti-penta-His (34660, Qiagen)], followed by addition of protein A agarose beads (75 μl) and further incubation at 4°C for 2 hours. Note that core histones and their epigenetic marks are remarkably conserved in all eukaryotes, including plants (13, 65), allowing the use of the corresponding antibodies across both plant and animal species [for example, (66–68)]. The immunocomplexes attached to the protein A agarose beads were washed sequentially with low- and high-salt wash buffers [20 mM tris-HCl (pH 8.0), 2 mM EDTA, 0.1% SDS, 1.0% Triton X-100 supplemented with 0.15 M NaCl (low salt), and 0.5 M NaCl (high salt)], with LiCl wash buffer [250 mM LiCl, 10 mM tris-HCl (pH 8.0), 1.0 mM EDTA, 1% NP-40, 1.0% deoxycholate], and twice with TE buffer [10 mM tris-HCl (pH 8.0), 1.0 mM EDTA], and eluted from the beads by incubation at room temperature for 15 min in elution buffer (0.1 M NaHCO3, 0.5% SDS). Cross-linking was reversed by incubation in 200 mM NaCl at 65°C overnight, and the immunoprecipitated DNA was recovered by treatment with proteinase K (20 mg/ml) at 45°C for 90 min, followed by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. Finally, the amounts of specific DNA sequences were analyzed by qPCR with 10 ng of recovered DNA per reaction and appropriate primers (table S1). To control for nonspecific background signal, we used protein A agarose beads incubated with chromatin samples in the absence of antibody.

Light microscopy and cell size measurements

Differential interference contrast (DIC) images were acquired using charge-coupled device digital camera (AxioCam MRm, Carl Zeiss) mounted on a microscope (AxioImager, Carl Zeiss) with a Plan Neofluar 20×/0.5 DIC objective controlled by Zen 2012 (Carl Zeiss). The acquired images were analyzed using ImageJ (Fiji Life-Line version, 2014) and Paint.NET software (version 4.0.6, dot PDN LLC). Cell size and surface density were determined in six different images, recorded with identical magnification, of two transverse sections of the middle region of the fifth leaf blade at about 1.0 mm from the middle vein and at the blade tip. Inflorescences were imaged in 21-day-old plants.

Supplementary Material

supplementary Fig. 1

Fig. S1. Increased size of leaf epidermal cells in OTLD1 OE-1 and OTLD1 OE-2 plants.

supplementary Table 1

Table S1. List of tested genes and corresponding PCR primers.

Acknowledgments:

We thank S. Wu (Department of Applied Mathematics and Statistics, Stony Brook University) for the statistical discussion of the data.

Funding:

The work in the laboratory of V.C. was supported by grants from NIH, NSF, U.S. Department of Agriculture/National Institute of Food and Agriculture, U.S.–Israel Binational Agricultural Research and Development Fund, and U.S.–Israel Binational Science Foundation.

Footnotes

Competing interests: The authors declare that they have no competing interests.

Data and materials availability:

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplementary Fig. 1

Fig. S1. Increased size of leaf epidermal cells in OTLD1 OE-1 and OTLD1 OE-2 plants.

NIHMS1833511-supplement-supplementary_Fig__1.docx (2.1MB, docx)
supplementary Table 1

Table S1. List of tested genes and corresponding PCR primers.

NIHMS1833511-supplement-supplementary_Table_1.docx (39KB, docx)

Data Availability Statement

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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