The histone deubiquitinase OTLD1 targets euchromatin to regulate plant growth

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.

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

(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.

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

(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.

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

(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.

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

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.

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

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.

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

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.

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

(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.

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

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.