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TOPLESS mediates brassinosteroid-induced transcriptional repression through interaction with BZR1.

Oh E, Zhu JY, Ryu H, Hwang I, Wang ZY - Nat Commun (2014)

Bottom Line: Specific deletion or mutation of an evolutionarily conserved ERF-associated amphiphilic repression (EAR) motif at the carboxy terminus abolishes BZR1's abilities to regulate gene expression and cell elongation, but these defects are rescued by TPL fusion to the EAR motif-mutated BZR1.A triple tpl mutant (tpl;tpr1;tpr4) shows reduced BR sensitivity and suppresses the gain-of-function bzr1-1D mutant phenotype.Our study demonstrates key roles of the EAR motif and TPL in BR regulation of gene expression and plant growth.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Plant Biology, Carnegie Institution for Science, 260 Panama Street, Stanford, California 94305, USA [2].

ABSTRACT
Brassinosteroid (BR) regulates plant development by activating the transcription factor brassinazole resistant 1 (BZR1), which activates and represses different target genes to switch cellular programmes. The mechanisms that determine BZR1's transcriptional activities remain largely unknown. Here we show that BZR1 represses target genes by recruiting the Groucho/TUP1-like transcriptional corepressor TOPLESS (TPL). Specific deletion or mutation of an evolutionarily conserved ERF-associated amphiphilic repression (EAR) motif at the carboxy terminus abolishes BZR1's abilities to regulate gene expression and cell elongation, but these defects are rescued by TPL fusion to the EAR motif-mutated BZR1. The EAR motif in BZR1 mediates recruitment of TPL to BZR1-repressed promoters. A triple tpl mutant (tpl;tpr1;tpr4) shows reduced BR sensitivity and suppresses the gain-of-function bzr1-1D mutant phenotype. BR repression of gene expression also requires histone deacetylases that interact with TPL. Our study demonstrates key roles of the EAR motif and TPL in BR regulation of gene expression and plant growth.

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EAR motif mediates BZR1 regulation of gene expression(a,b) The deletion of EAR motif abolishes BZR1 regulation of gene expression. Seedlings were grown on the medium containing 2 µM PPZ in the dark for 5 days before harvesting for RNA extraction. Gene expression levels are normalized to that of PP2A and are shown relative to the expression levels in wild type (Col-0).(c,d) The EAR motif-deleted bzr1-1D neither represses CPD expression (c) nor activates SAUR15 expression (d). Four-week-old leaves of transgenic plants expressing bzr1-1D-GR or bzr1-1DΔEAR-GR were treated with mock or 40 µM of dexamethasone (DEX) for 4 hr. Ratios of expression levels in DEX-treated to mock-treated are shown. Each bar represents independent transgenic plant.(e,f) The EAR motif-deleted bzr1-1D cannot activate PRE5 promoter activity (e) and cannot repress CPD promoter activity (f) in a transient gene expression assay. The 2 kb of PRE5 promoter or 1 kb of CPD promoter fused to fire fly luciferase reporter gene was co-transfected with 35S::bzr1-1D or 35S::bzr1-1DΔEAR into Arabidopsis mesophyll protoplast. The fire fly luciferase activities were normalized by renilla luciferase as an internal control and are shown relative to that of protoplasts transfected with 35S::GFP (GFP). ** : P<0.01 and * : P<0.05 by Student’s t-test.(g) The deletion of EAR motif does not affect BZR1 DNA-binding. Transgenic plants overexpressing BZR1 and BZR1ΔEAR were treated with 100 nM BL and crosslinked for the ChIP assays. In the ChIP assays, the enrichment of DNA was calculated as the ratio between transgenic plants and wild type control (Col-0), normalized to that of the PP2A coding region as an internal control.Error bars in (a,b,e,f,g) indicate the s.d. (n=3).
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Figure 2: EAR motif mediates BZR1 regulation of gene expression(a,b) The deletion of EAR motif abolishes BZR1 regulation of gene expression. Seedlings were grown on the medium containing 2 µM PPZ in the dark for 5 days before harvesting for RNA extraction. Gene expression levels are normalized to that of PP2A and are shown relative to the expression levels in wild type (Col-0).(c,d) The EAR motif-deleted bzr1-1D neither represses CPD expression (c) nor activates SAUR15 expression (d). Four-week-old leaves of transgenic plants expressing bzr1-1D-GR or bzr1-1DΔEAR-GR were treated with mock or 40 µM of dexamethasone (DEX) for 4 hr. Ratios of expression levels in DEX-treated to mock-treated are shown. Each bar represents independent transgenic plant.(e,f) The EAR motif-deleted bzr1-1D cannot activate PRE5 promoter activity (e) and cannot repress CPD promoter activity (f) in a transient gene expression assay. The 2 kb of PRE5 promoter or 1 kb of CPD promoter fused to fire fly luciferase reporter gene was co-transfected with 35S::bzr1-1D or 35S::bzr1-1DΔEAR into Arabidopsis mesophyll protoplast. The fire fly luciferase activities were normalized by renilla luciferase as an internal control and are shown relative to that of protoplasts transfected with 35S::GFP (GFP). ** : P<0.01 and * : P<0.05 by Student’s t-test.(g) The deletion of EAR motif does not affect BZR1 DNA-binding. Transgenic plants overexpressing BZR1 and BZR1ΔEAR were treated with 100 nM BL and crosslinked for the ChIP assays. In the ChIP assays, the enrichment of DNA was calculated as the ratio between transgenic plants and wild type control (Col-0), normalized to that of the PP2A coding region as an internal control.Error bars in (a,b,e,f,g) indicate the s.d. (n=3).

Mentions: We next analyzed the effects of the EAR motif deletion on BZR1 regulation of target gene expression. BZR1 is known to directly repress BR biosynthesis genes, including DWF4 and CPD, to provide the feed-back regulation of BR level5. The expression levels of DWF4 and CPD were dramatically reduced in bzr1-1D compared to wild type, but was only marginally reduced in the bzr1-1DΔEAR-OX plants (Fig. 2a), indicating that the transcriptional repression activity is impaired in bzr1-1DΔEAR. BZR1 also activates expression of many of its target genes15. While the expression levels of SAURs, IAA19 and PRE5 were highly increased by bzr1-1D, expression of SAUR19, IAA19 and PRE5 was not activated, and SAUR15 expression was only slightly activated by bzr1-1DΔEAR (Fig. 2b), suggesting that the EAR motif is also required for the BZR1-mediated transcriptional activation of target genes.


TOPLESS mediates brassinosteroid-induced transcriptional repression through interaction with BZR1.

Oh E, Zhu JY, Ryu H, Hwang I, Wang ZY - Nat Commun (2014)

EAR motif mediates BZR1 regulation of gene expression(a,b) The deletion of EAR motif abolishes BZR1 regulation of gene expression. Seedlings were grown on the medium containing 2 µM PPZ in the dark for 5 days before harvesting for RNA extraction. Gene expression levels are normalized to that of PP2A and are shown relative to the expression levels in wild type (Col-0).(c,d) The EAR motif-deleted bzr1-1D neither represses CPD expression (c) nor activates SAUR15 expression (d). Four-week-old leaves of transgenic plants expressing bzr1-1D-GR or bzr1-1DΔEAR-GR were treated with mock or 40 µM of dexamethasone (DEX) for 4 hr. Ratios of expression levels in DEX-treated to mock-treated are shown. Each bar represents independent transgenic plant.(e,f) The EAR motif-deleted bzr1-1D cannot activate PRE5 promoter activity (e) and cannot repress CPD promoter activity (f) in a transient gene expression assay. The 2 kb of PRE5 promoter or 1 kb of CPD promoter fused to fire fly luciferase reporter gene was co-transfected with 35S::bzr1-1D or 35S::bzr1-1DΔEAR into Arabidopsis mesophyll protoplast. The fire fly luciferase activities were normalized by renilla luciferase as an internal control and are shown relative to that of protoplasts transfected with 35S::GFP (GFP). ** : P<0.01 and * : P<0.05 by Student’s t-test.(g) The deletion of EAR motif does not affect BZR1 DNA-binding. Transgenic plants overexpressing BZR1 and BZR1ΔEAR were treated with 100 nM BL and crosslinked for the ChIP assays. In the ChIP assays, the enrichment of DNA was calculated as the ratio between transgenic plants and wild type control (Col-0), normalized to that of the PP2A coding region as an internal control.Error bars in (a,b,e,f,g) indicate the s.d. (n=3).
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Figure 2: EAR motif mediates BZR1 regulation of gene expression(a,b) The deletion of EAR motif abolishes BZR1 regulation of gene expression. Seedlings were grown on the medium containing 2 µM PPZ in the dark for 5 days before harvesting for RNA extraction. Gene expression levels are normalized to that of PP2A and are shown relative to the expression levels in wild type (Col-0).(c,d) The EAR motif-deleted bzr1-1D neither represses CPD expression (c) nor activates SAUR15 expression (d). Four-week-old leaves of transgenic plants expressing bzr1-1D-GR or bzr1-1DΔEAR-GR were treated with mock or 40 µM of dexamethasone (DEX) for 4 hr. Ratios of expression levels in DEX-treated to mock-treated are shown. Each bar represents independent transgenic plant.(e,f) The EAR motif-deleted bzr1-1D cannot activate PRE5 promoter activity (e) and cannot repress CPD promoter activity (f) in a transient gene expression assay. The 2 kb of PRE5 promoter or 1 kb of CPD promoter fused to fire fly luciferase reporter gene was co-transfected with 35S::bzr1-1D or 35S::bzr1-1DΔEAR into Arabidopsis mesophyll protoplast. The fire fly luciferase activities were normalized by renilla luciferase as an internal control and are shown relative to that of protoplasts transfected with 35S::GFP (GFP). ** : P<0.01 and * : P<0.05 by Student’s t-test.(g) The deletion of EAR motif does not affect BZR1 DNA-binding. Transgenic plants overexpressing BZR1 and BZR1ΔEAR were treated with 100 nM BL and crosslinked for the ChIP assays. In the ChIP assays, the enrichment of DNA was calculated as the ratio between transgenic plants and wild type control (Col-0), normalized to that of the PP2A coding region as an internal control.Error bars in (a,b,e,f,g) indicate the s.d. (n=3).
Mentions: We next analyzed the effects of the EAR motif deletion on BZR1 regulation of target gene expression. BZR1 is known to directly repress BR biosynthesis genes, including DWF4 and CPD, to provide the feed-back regulation of BR level5. The expression levels of DWF4 and CPD were dramatically reduced in bzr1-1D compared to wild type, but was only marginally reduced in the bzr1-1DΔEAR-OX plants (Fig. 2a), indicating that the transcriptional repression activity is impaired in bzr1-1DΔEAR. BZR1 also activates expression of many of its target genes15. While the expression levels of SAURs, IAA19 and PRE5 were highly increased by bzr1-1D, expression of SAUR19, IAA19 and PRE5 was not activated, and SAUR15 expression was only slightly activated by bzr1-1DΔEAR (Fig. 2b), suggesting that the EAR motif is also required for the BZR1-mediated transcriptional activation of target genes.

Bottom Line: Specific deletion or mutation of an evolutionarily conserved ERF-associated amphiphilic repression (EAR) motif at the carboxy terminus abolishes BZR1's abilities to regulate gene expression and cell elongation, but these defects are rescued by TPL fusion to the EAR motif-mutated BZR1.A triple tpl mutant (tpl;tpr1;tpr4) shows reduced BR sensitivity and suppresses the gain-of-function bzr1-1D mutant phenotype.Our study demonstrates key roles of the EAR motif and TPL in BR regulation of gene expression and plant growth.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Plant Biology, Carnegie Institution for Science, 260 Panama Street, Stanford, California 94305, USA [2].

ABSTRACT
Brassinosteroid (BR) regulates plant development by activating the transcription factor brassinazole resistant 1 (BZR1), which activates and represses different target genes to switch cellular programmes. The mechanisms that determine BZR1's transcriptional activities remain largely unknown. Here we show that BZR1 represses target genes by recruiting the Groucho/TUP1-like transcriptional corepressor TOPLESS (TPL). Specific deletion or mutation of an evolutionarily conserved ERF-associated amphiphilic repression (EAR) motif at the carboxy terminus abolishes BZR1's abilities to regulate gene expression and cell elongation, but these defects are rescued by TPL fusion to the EAR motif-mutated BZR1. The EAR motif in BZR1 mediates recruitment of TPL to BZR1-repressed promoters. A triple tpl mutant (tpl;tpr1;tpr4) shows reduced BR sensitivity and suppresses the gain-of-function bzr1-1D mutant phenotype. BR repression of gene expression also requires histone deacetylases that interact with TPL. Our study demonstrates key roles of the EAR motif and TPL in BR regulation of gene expression and plant growth.

Show MeSH