Mediator subunit MED25 links the jasmonate receptor to transcriptionally active chromatin

Abstract

Jasmonoyl-isoleucine (JA-Ile), the active form of the plant hormone jasmonate (JA), is sensed by the F-box protein CORONATINE INSENSITIVE 1 (COI1), a component of a functional Skp-Cullin-F-box E3 ubiquitin ligase complex. Sensing of JA-Ile by COI1 rapidly triggers genome-wide transcriptional changes that are largely regulated by the basic helix-loop-helix transcription factor MYC2. However, it remains unclear how the JA-Ile receptor protein COI1 relays hormone-specific regulatory signals to the RNA polymerase II general transcriptional machinery. Here, we report that the plant transcriptional coactivator complex Mediator directly links COI1 to the promoters of MYC2 target genes. MED25, a subunit of the Mediator complex, brings COI1 to MYC2 target promoters and facilitates COI1-dependent degradation of jasmonate-ZIM domain (JAZ) transcriptional repressors. MED25 and COI1 influence each other's enrichment on MYC2 target promoters. Furthermore, MED25 physically and functionally interacts with HISTONE ACETYLTRANSFERASE1 (HAC1), which plays an important role in JA signaling by selectively regulating histone (H) 3 lysine (K) 9 (H3K9) acetylation of MYC2 target promoters. Moreover, the enrichment and function of HAC1 on MYC2 target promoters depend on COI1 and MED25. Therefore, the MED25 interface of Mediator links COI1 with HAC1-dependent H3K9 acetylation to activate MYC2-regulated transcription of JA-responsive genes. This study exemplifies how a single Mediator subunit integrates the actions of both genetic and epigenetic regulators into a concerted transcriptional program.

Keywords: COI1; MED25; MYC2; jasmonate; nuclear hormone receptor.

Fig. 1.

Enrichment of COI1 and MED25 on the promoters of JAZ8 and ERF1. (A) Schematic diagrams of JAZ8, ERF1, and PCR amplicons indicated as letters A–D used for ChIP-qPCR. (B) ChIP-qPCR showing the enrichment of COI1 on the chromatin of JAZ8 and ERF1. Chromatin of WT plants was immunoprecipitated using anti-COI1 antibody. (C) ChIP-qPCR showing enrichment of MED25 on the chromatin of JAZ8 and ERF1. Chromatin of MED25-myc plants was immunoprecipitated using anti-myc antibody. (D) ChIP-qPCR showing enrichment of COI1 on the TSS regions of JAZ8 and ERF1 upon JA-Ile stimulation. WT plants were treated with 30 μM JA-Ile for the indicated times before cross-linking, and chromatin from each sample was immunoprecipitated using anti-COI1 antibody. (E) ChIP-qPCR showing enrichment of MED25 on the TSS regions of JAZ8 and ERF1 upon JA-Ile stimulation. MED25-myc plants were treated with 30 μM JA-Ile for the indicated times before cross-linking. Chromatin of each sample was immunoprecipitated using anti-myc antibody. (F) ChIP-qPCR assays showing that myc2-2 impairs the enrichment of COI1 on the TSSs of JAZ8 and ERF1 before and after JA-Ile stimulation. WT and myc2-2 plants were treated with or without 30 μM JA-Ile for 15 min before cross-linking, and chromatin of each sample was immunoprecipitated using anti-COI1 antibody. (G) ChIP-qPCR assays showing that myc2-2 impairs the enrichment of MED25 on the TSSs of JAZ8 and ERF1 before and after JA-Ile stimulation. MED25-myc and MED25-myc/myc2-2 plants were treated with or without 30 μM JA-Ile for 15 min before cross-linking, and chromatin of each sample was immunoprecipitated using anti-myc antibody. For B–G, precipitated DNA was quantified by qPCR, and the DNA enrichment is shown as a percentage of input DNA. ACTIN7 (ACT7) was used as a nonspecific binding site. Error bars indicate SD of three independent experiments (n = 3). ANOVA was performed for statistical analysis; bars with different letters are significantly different from each other (P < 0.01).

Fig. S1.

MYC2-dependent orchestration of different branches of JA responses. In response to JA-Ile, MYC2 positively regulates a group of intermediate transcription factors (TFs) (ANAC019, ANAC055, etc.), which, in turn, regulate the expression of downstream wound-responsive genes (VSP1 as a marker). MYC2 also negatively regulates a group of intermediate TFs (ERF1, ORA59, etc.), which, in turn, regulate the expression of downstream pathogen-responsive genes (PDF1.2 as a marker). Note that the positive regulation of wound response by MYC2 occurs relatively early (peaked expression occurs within 6 h after hormone elicitation), whereas the negative regulation of pathogen response by MYC2 occurs relatively late (peaked expression occurs after 48 h after JA application). MYC2 also directly regulates the expression of a group of immediate JA-responsive genes (peaked expression occurs within 1 h after hormone elicitation), including JAZs and JA biosynthetic genes.

Fig. 2.

MED25 interacts with COI1 and facilitates COI1-dependent degradation of JAZ1. (A) Y2H assays showing that MED25 interacts with COI1. Transformed yeast strain was plated on SD medium lacking His, Leu, and Trp (SD/-3). (B) In vitro pull-down assays between MED25^551-836-MBP and COI1-His. COI1-His was pulled down by MED25^551-836-MBP immobilized on amylose resin. Protein bound to amylose resin was eluted and analyzed by immunoblotting using anti-COI1 antibody. (C) MED25 associates with COI1 in LCI assays. (Top) LUC images of N. benthamiana leaves coinfiltrated with various constructs are shown in the lower quadrant of the circle. The pseudocolor bar shows the range of luminescence intensity. (Scale bar, 1 cm.) (D) Co-IP assay between MED25 and COI1. Proteins extracted from WT and COI1-myc plants were immunoprecipitated using anti-myc antibody and immunoblotted using anti-MED25 antibody. (E) JA-Ile–triggered degradation of the JAZ1-GUS reporter in WT and med25-4 backgrounds. Seven-day-old seedlings were treated with 30 μM JA-Ile for the indicated durations before quantification of GUS activity. Error bars indicate SD of three independent experiments (n = 3). (F) Pull-down assays between JAZ1-His and COI1. Protein extracts from COI1-myc and COI1-myc/med25-4 seedlings were incubated with recombinant JAZ1-His protein in the presence or absence of 30 μM JA-Ile. COI1 was pulled down by JAZ1-His immobilized on nickel-nitrilotriacetic acid (Ni-NTA; Novagen) resin and eluted and analyzed by immunoblotting using anti-myc antibody. Bands were quantified using ImageJ.

Fig. S2.

Mapping of the protein domains involved in MED25–COI1 interaction using LCI assays. (A) Based on the schematic protein structure of MED25, full-length MED25 or its derivatives (MED25-nLUC or MED25-nLUC derivatives) were tested for interactions with COI1 (cLUC-COI1). N. benthamiana leaves cotransformed with MED25-nLUC or MED25-nLUC derivatives and cLUC-COI1 were imaged 72 h after Agrobacterium infiltration. (B) Based on the schematic protein structure of COI1, full-length COI1 or its derivatives (cLUC-COI1 or cLUC-COI1 derivatives) were tested for interaction with MED25 (MED25-nLUC). N. benthamiana leaves cotransformed with cLUC-COI1 or cLUC-COI1 derivatives and MED25-nLUC were imaged 72 h after Agrobacterium infiltration. ACID, activator-interacting domain; GD, glutamine-rich domain; LRR, leucine-rich repeat; MD, middle domain; vWF-A, von Willebrand factor A domain. In A and B, the pseudocolor bar shows the range of luminescence intensity.

Fig. 3.

COI1 and MED25 affect each other’s enrichment on the promoters of JAZ8 and ERF1. (A) Schematic diagrams of JAZ8, ERF1, and the PCR amplicons indicated as letters A–D used for ChIP-qPCR. (B) Sequential ChIP analysis showing that COI1 and MED25 co-occupy the promoters of JAZ8 and ERF1. Chromatin of MED25-myc plants was immunoprecipitated with anti-COI1 antibody, and then with anti-myc antibody. (C) ChIP-qPCR assays showing that med25-4 impairs the enrichment of COI1 on the TSSs of JAZ8 and ERF1 upon JA-Ile stimulation. WT and med25-4 plants were treated with or without 30 μM JA-Ile for 15 min before cross-linking, and chromatin of each sample was immunoprecipitated using anti-COI1 antibody. (D) ChIP-qPCR assays showing that coi1-2 impairs the enrichment of MED25 on the TSSs of JAZ8 and ERF1 upon JA-Ile stimulation. MED25-myc and MED25-myc/coi1-2 plants were treated with or without 30 μM JA-Ile for 15 min before cross-linking, and chromatin of each sample was immunoprecipitated using anti-myc antibody. For B–D, the precipitated DNA was quantified by qPCR, and DNA enrichment is displayed as a percentage of input DNA. ACT7 was used as a nonspecific binding site. Error bars indicate SD of three independent experiments (n = 3). ANOVA was performed for statistical analysis; bars with different letters are significantly different from each other (P < 0.01).

Fig. S3.

MED25 and COI1 do not affect the protein levels of each other. (A) Effect of the med25-4 mutation on the protein levels of COI1. WT and med25-4 plants were treated with or without 30 μM JA-Ile for indicated durations, and proteins were extracted for immunoblotting using anti-COI1 antibody. ACTIN11 (ACT11) was used as a loading control. (B) Effect of the coi1-2 mutation on the protein levels of MED25. WT and coi1-2 plants were treated with or without 30 μM JA-Ile for indicated durations, and proteins were extracted for immunoblotting using anti-MED25 antibody. ACT11 was used as a loading control.

Fig. 4.

MED25 interacts with HAC1, which is recruited to the promoters of JAZ8 and ERF1. (A) Y2H assays showing that MED25 interacts with HAC1 but not HAC5 and HAC12. Transformed yeast strains were plated on SD medium lacking His, Ade, Leu, and Trp (SD/-4). (B) Co-IP assay of MED25 with HAC1. Proteins extracted from WT and MED25-myc plants were immunoprecipitated using anti-myc antibody and immunoblotted using anti-HAC1 antibody. The arrow indicates the position of HAC1. (C) LCI assays showing that MED25 interacts with HAC1. (Top) LUC images of N. benthamiana leaves coinfiltrated with the different construct combinations are shown in the lower quadrant of the circle. The pseudocolor bar shows the range of luminescence intensity. (Scale bar, 1 cm.) (D) Schematic diagrams of JAZ8, ERF1, and PCR amplicons indicated as letters A–D used for ChIP-qPCR. (E) ChIP-qPCR showing enrichment of HAC1 on the chromatin of JAZ8 and ERF1. Chromatin of HAC1-GFP plants was immunoprecipitated using anti-GFP antibody. (F) ChIP-qPCR showing enrichment of HAC1 on the TSSs of JAZ8 and ERF1 upon JA-Ile stimulation. HAC1-GFP plants were treated with 30 μM JA-Ile for the indicated durations before cross-linking, and the chromatin of each sample was immunoprecipitated using anti-GFP antibody. For E and F, the precipitated DNA was quantified by qPCR, and DNA enrichment is displayed as a percentage of input DNA. ACT7 was used as a nonspecific binding site. Error bars indicate the SD of three independent experiments (n = 3). ANOVA was performed for statistical analysis; bars with different letters are significantly different from each other (P < 0.01).

Fig. S4.

Mapping of the protein domains involved MED25–HAC1 interaction using Y2H assays. (A) Based on the schematic protein structure of MED25, full-length MED25 or its derivatives (pGBKT7-MED25 or pGBKT7-MED25 derivatives) were tested for interaction with HAC1 (pGADT7-HAC1). Yeast cells cotransformed with pGBKT7-MED25 or pGBKT7-MED25 derivatives (baits) and pGADT7-HAC1 (prey) were grown on selective media lacking Ade, His, Leu, and Trp (SD/-4) to test protein interaction. (B) Based on the schematic protein structure of HAC1, full-length HAC1 or its derivatives (pGADT7-HAC1 or pGADT7-HAC1 derivatives) were tested for interaction with MED25 (pGBKT7-MED25). Yeast cells cotransformed with pGADT7-HAC1 or pGADT7-HAC1 derivatives (prey) and pGBKT7-MED25 (bait) were grown on selective media lacking Ade, His, Leu, and Trp (SD/-4) to test protein interaction. ACID, activator-interacting domain; GD, glutamine-rich domain; LRR, leucine-rich repeat; MD, middle domain; PHD, plant homeodomain; vWF-A, von Willebrand factor A domain; Znf-TAZ, transcription adaptor putative Zinc finger (TAZ)-type Zinc finger; Znf-ZZ, two Zinc binding domain (ZZ)-type Zinc finger.

Fig. S5.

Phenotypic and molecular characterization of pHAC1:HAC1-GFP/hac1-4 (HAC1-GFP) transgenic plants. (A, Left) The hac1-4 mutants exhibited a late-flowering phenotype, whereas the flowering times of HAC1-GFP #1 and #3 were comparable to those of WT plants, indicating that HAC1-GFP protein retains the biological function as endogenous HAC1. (A, Right) Relative RNA levels of HAC1 are also shown. Plants were grown under long-day (LD, 16-h white light/8-h dark) conditions. (B) qRT-PCR shows that HAC1-GFP #1 and #3 rescue the phenotype of hac1-4 in terms of JA-Ile–induced gene expression. Ten-day-old seedlings were treated with or without 30 μM JA-Ile for the indicated times. Error bars indicate SD of three independent experiments (n = 3).

Fig. 5.

Depletion of HAC1 impairs JA-responsive gene expression and reduces H3K9ac accumulation on the promoters of JAZ8 and ERF1. (A) qRT-PCR showing JA-Ile–induced expression of indicated genes in WT and hac1-4. WT and hac1-4 plants were treated with or without 30 μM JA-Ile for the indicated durations. Error bars indicate SD of three independent experiments (n = 3). ANOVA was performed for statistical analysis; bars with different letters are significantly different from each other (P < 0.01). (B) Hierarchical clustering of the selected JA-Ile–responsive genes showing reduced expression in hac1-4 plants at the indicated time points. (C) Protein gel analyses showing global H3K9ac levels in the WT and hac1-4 in response to JA-Ile. WT and hac1-4 plants were treated with or without 30 μM JA-Ile for 30 min before extraction of nuclear proteins for immunoblotting using the indicated antibodies. Bands were quantified using ImageJ. (D) Schematic diagrams of JAZ8, ERF1, and PCR amplicons indicated as letters A–D used for ChIP-qPCR. (E) ChIP-qPCR assays showing that hac1-4 impairs the enrichment of H3K9ac on the TSS regions of JAZ8 and ERF1 in response to JA-Ile. WT and hac1-4 plants were treated with or without 30 μM JA-Ile for 30 min before cross-linking, and chromatin of each sample was immunoprecipitated using anti-H3 and anti-H3K9ac antibodies. Precipitated DNA was quantified by qPCR, and H3K9ac levels are normalized to H3. (F) ChIP-qPCR assays showing that hac1-4 impairs the enrichment of Pol II C-terminal domain (CTD) on the TSS regions of JAZ8 and ERF1 in response to JA-Ile stimulation. WT and hac1-4 plants were treated with or without 30 μM JA-Ile for 30 min before cross-linking, and chromatin of each sample was then immunoprecipitated using anti-Pol II CTD antibody. Precipitated DNA was quantified by qPCR, and DNA enrichment is displayed as a percentage of input DNA. For E and F, ACT7 was used as a nonspecific binding site. Error bars indicate SD of three independent experiments (n = 3). ANOVA was performed for statistical analysis; bars with different letters are significantly different from each other (P < 0.01).

Fig. S6.

Summary of the RNA-seq analysis. (A) Overview of RNA-seq data from JA-Ile–treated WT and hac1-4 plants. Relative percentages of multiple (Multi) mapped reads, unique mapped reads, and unmapped reads are shown. (B) Principal component (PC) analysis showing the relatedness among the gene expression patterns of samples used for RNA-seq analysis. Colors represent different genotypes, and symbols represent treatments. (C) Venn diagrams of JA–up-regulated genes (up-regulated genes in WT after JA-Ile treatment for 1 or 24 h; fold change > 1.5, FDR-adjusted P < 0.05) and HAC1-regulated genes (down-regulated genes in hac1-4 compared with WT after JA-Ile treatment for 1 or 24 h; fold change > 1.5, FDR-adjusted P < 0.05). Genes coregulated by JA-Ile and HAC1 are shown in the overlapping region. The number of differentially expressed genes with significant differential expression (FDR-adjusted P < 0.05) is shown. (D) Venn diagrams of JA–down-regulated genes (down-regulated genes in WT after JA-Ile treatment for 1 or 24 h; fold change > 1.5, FDR-adjusted P < 0.05) and HAC1-regulated genes (up-regulated genes in hac1-4 compared with WT after JA-Ile treatment for 1 or 24 h; fold change > 1.5, FDR-adjusted P < 0.05). Genes coregulated by JA-Ile and HAC1 are shown in the overlapping region. The number of differentially expressed genes with significant differential expression (fold change > 1.5, FDR-adjusted P < 0.05) is shown. (E) GO analysis of HAC1-regulated JA-Ile–induced genes. GO terms (biological processes) enriched in the set of genes identified as JA–up-regulated genes that were down-regulated in hac1-4 compared with WT are shown. The P value was expressed in exponential notation, replacing part of the number with E + n, where E multiplies the preceding number by 10 to the nth power.

Fig. S7.

Enrichment of H3K9ac on the chromatin of JAZ8 and ERF1. (A) Schematic diagrams of JAZ8, ERF1, and the PCR amplicons indicated as letters A–D used for ChIP-qPCR. (B) ChIP-qPCR shows the enrichment of H3K9ac on the chromatin of JAZ8 and ERF1. The chromatin of WT plants was immunoprecipitated using anti-H3 and anti-H3K9ac antibodies, respectively. (C) ChIP-qPCR shows the enrichment of H3K9ac on the TSS regions of JAZ8 and ERF1 in response to JA-Ile elicitation. WT plants were treated with 30 μM JA-Ile for indicated durations before cross-linking, and the chromatin of each sample was immunoprecipitated using anti-H3 and anti-H3K9ac antibodies, respectively. For (B) and (C), the precipitated DNA was quantified by qPCR, and H3K9ac levels are normalized to H3. ACT7 was used as a nonspecific binding site. Error bars indicate the SD of three independent experiments (n = 3). ANOVA was performed for statistical analysis; bars with different letters are significantly different from each other (P < 0.01).

Fig. 6.

Depletion of COI1 or MED25 impairs the function of HAC1 on the promoters of JAZ8 and ERF1. (A) Schematic diagrams of JAZ8, ERF1, and PCR amplicons indicated as letters A–D used for ChIP-qPCR. (B) ChIP-qPCR assays showing that coi1-2 impairs the enrichment of HAC1 on the TSS regions of JAZ8 and ERF1 in response to JA-Ile. HAC1-GFP and HAC1-GFP/coi1-2 plants were treated with or without 30 μM JA-Ile for 30 min before cross-linking, and chromatin of each sample was then immunoprecipitated using anti-GFP antibody. Precipitated DNA was quantified by qPCR, and DNA enrichment is displayed as a percentage of input DNA. (C) ChIP-qPCR assays showing that coi1-2 impairs the enrichment of H3K9ac on the TSS regions of JAZ8 and ERF1 in response to JA-Ile. WT and coi1-2 plants were treated with or without 30 μM JA-Ile for 30 min before cross-linking, and chromatin of each sample was then immunoprecipitated using anti-H3 and anti-H3K9ac antibodies. Precipitated DNA was quantified by qPCR, and H3K9ac levels are normalized to H3. (D) ChIP-qPCR assays showing that med25-4 impairs the enrichment of HAC1 on the TSS regions of JAZ8 and ERF1 in response to JA-Ile. HAC1-GFP and HAC1-GFP/med25-4 plants were treated with or without 30 μM JA-Ile for 30 min before cross-linking, and chromatin of each sample was then immunoprecipitated using anti-GFP antibody. Precipitated DNA was quantified by qPCR, and DNA enrichment is displayed as a percentage of input DNA. (E) ChIP-qPCR assays showing that med25-4 impairs the enrichment of H3K9ac on the TSS regions of JAZ8 and ERF1 in response to JA-Ile. WT and med25-4 plants were treated with or without 30 μM JA-Ile for 30 min before cross-linking, and chromatin of each sample was then immunoprecipitated using anti-H3 and anti-H3K9ac antibodies. Precipitated DNA was quantified by qPCR, and H3K9ac levels are normalized to H3. For B–E, ACT7 was used as a nonspecific binding site. Error bars indicate SD of three independent experiments (n = 3). ANOVA was performed for statistical analysis; bars with different letters are significantly different from each other (P < 0.01).

Fig. S8.

Effect of COI1 and MED25 on the protein levels of HAC1. WT, coi1-2, and med25-4 plants were treated with or without 30 μM JA-Ile for indicated durations, and proteins were extracted for immunoblotting using anti-HAC1 antibody. The arrowhead indicates the HAC1-specific band. ACT11 was used as a loading control.

Fig. 7.

MED25 cooperates with both genetic and epigenetic regulators in regulating hormone-induced activation of MYC2. (A) Co-IP assays of MED25, MYC2, and JAZ1 in N. benthamiana. MED25-Flag and MYC2-myc were transiently coexpressed with or without JAZ1-GFP in N. benthamiana leaves. Protein extracts were immunoprecipitated using anti-GFP antibody and analyzed by immunoblotting with anti-Flag, anti-myc, and anti-GFP antibodies. (B) Co-IP assay of MED25, MYC2, and JAZ1 in Arabidopsis. Proteins extracted from WT and JAZ1-GFP plants were immunoprecipitated using anti-GFP antibody and immunoblotted using anti-MED25 and anti-MYC2 antibodies. (C) Co-IP assay between MED25 and COI1. WT and COI1-myc plants were treated with or without 30 μM JA-Ile for the indicated times. Protein from each sample was immunoprecipitated using anti-myc antibody and immunoblotted using anti-MED25 antibody. Bands were quantified using ImageJ. (D) Co-IP assay between MED25 and MYC2. WT and MYC2-myc plants were treated with or without 30 μM JA-Ile for the indicated times. Protein from each sample was immunoprecipitated using anti-myc antibody and immunoblotted using anti-MED25 antibody. Bands were quantified using ImageJ. (E) Proposed working model for the mechanistic roles of MED25 in regulating JA-Ile–induced activation of MYC2. In the resting stage, the MED25–COI1 interaction is relatively strong, whereas the MED25–MYC2 interaction is relatively weak because JAZ repressors compete with MED25 for interaction with MYC transcription factors. Basal levels of MED25 bring COI1 to MYC2 target promoters through physical interaction. In the hormone-mediated transition stage, JA-Ile acts as molecular glue to promote the formation of the COI1–JAZ coreceptor complex, which leads to proteasome-dependent degradation of JAZ repressors. During this stage, the MED25–COI1 interaction is weakened in a hormone-dependent manner, whereas the MED25–MYC2 interaction is enhanced in a hormone-dependent manner. Upon degradation of JAZ repressors, MED25 interacts with MYC2 and recruits HAC1 as well as Pol II to the promoters of MYC2 target genes, and thereby activate their expression. NINJA, Novel Interactor of JAZ; TPL, TOPLESS.

Fig. S9.

Molecular characterization of p35S:JAZ1-GFP (JAZ1-GFP) transgenic plants. WT and JAZ1-GFP plants were treated with or without 30 μM JA-Ile for 15 min, and proteins were extracted for immunoblotting using anti-GFP antibody. ACT11 was used as a loading control.