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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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BZR1 interacts with TOPLESS through the EAR motif(a) A yeast two-hybrid assay shows the interaction between BZR1 and TPL. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 50 mM 3AT medium.(b) Mutation in the EAR motif abolishes the BZR1-TPL interaction. Core Leu residues of the EAR motif were substituted to Ala. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 1 mM 3AT medium.(c) BZR1 interacts with TPL and other TPRs. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 5 mM 3AT medium. TPL-N: N-terminal domain of TPL (1–344), TPR1-N : 1–343, TPR2-N : 1–335, TPR3-N : 1–335 and TPR4-N : 1–344.(d) BZR1 interacts with TPL through EAR motif in vivo. Arabidopsis mesophyll protoplasts were transfected with TPL-Myc together with bzr1-1D-GFP or bzr1-1DΔEAR-GFP, and the extracted proteins were immunoprecipitated by anti-GFP antibody. Gel blots were probed with anti-Myc or anti-GFP antibody.(e) TPL binds to DWF4 promoter through BZR1. GST-TPL-N (amino acids 1–344) was incubated with a biotin-labelled DWF4 promoter DNA (400 bps) together with MBP or MBP-BZR1 and pulled down with streptavidin agarose beads. Gel blots were probed with anti-GST or anti-MBP antibody. The full scans of the gel blots (d,e) are shown in Supplementary Fig. 4.(f) The TPL DNA-binding on the promoters of CPD and DWF4 are enhanced by BR treatment. TPL DNA-binding was determined by ChIP assay using TPLp::TPL-HA transgenic plants. One-week seedlings grown on the medium containing 2 µM PPZ were treated with mock (M) or 100 nM BL for 4 hr before crosslinking. Error bars indicate the s.d. (n=3) and **: P<0.01 by Student’s t-test.
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Figure 4: BZR1 interacts with TOPLESS through the EAR motif(a) A yeast two-hybrid assay shows the interaction between BZR1 and TPL. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 50 mM 3AT medium.(b) Mutation in the EAR motif abolishes the BZR1-TPL interaction. Core Leu residues of the EAR motif were substituted to Ala. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 1 mM 3AT medium.(c) BZR1 interacts with TPL and other TPRs. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 5 mM 3AT medium. TPL-N: N-terminal domain of TPL (1–344), TPR1-N : 1–343, TPR2-N : 1–335, TPR3-N : 1–335 and TPR4-N : 1–344.(d) BZR1 interacts with TPL through EAR motif in vivo. Arabidopsis mesophyll protoplasts were transfected with TPL-Myc together with bzr1-1D-GFP or bzr1-1DΔEAR-GFP, and the extracted proteins were immunoprecipitated by anti-GFP antibody. Gel blots were probed with anti-Myc or anti-GFP antibody.(e) TPL binds to DWF4 promoter through BZR1. GST-TPL-N (amino acids 1–344) was incubated with a biotin-labelled DWF4 promoter DNA (400 bps) together with MBP or MBP-BZR1 and pulled down with streptavidin agarose beads. Gel blots were probed with anti-GST or anti-MBP antibody. The full scans of the gel blots (d,e) are shown in Supplementary Fig. 4.(f) The TPL DNA-binding on the promoters of CPD and DWF4 are enhanced by BR treatment. TPL DNA-binding was determined by ChIP assay using TPLp::TPL-HA transgenic plants. One-week seedlings grown on the medium containing 2 µM PPZ were treated with mock (M) or 100 nM BL for 4 hr before crosslinking. Error bars indicate the s.d. (n=3) and **: P<0.01 by Student’s t-test.

Mentions: In several transcription factors, the EAR motif has been known to mediate transcriptional repression through interaction with the Groucho/Tup1-like transcriptional corepressor TOPLESS (TPL) and the homologous TOPLESS RELATED (TPR) proteins25–28. Our recent tandem affinity purification of BZR1 complex followed by mass spectrometry analysis suggested that TPL associates with BZR1 in vivo29. We therefore tested whether BZR1 directly interacts with TPL and TPRs. Indeed, in the yeast two-hybrid assay, BZR1 interacted with TPL and also other TPRs (Fig. 4a,c). Deletion of the EAR motif or mutation of the conserved Leu residues in the EAR motif abolished the BZR1-TPL interaction (Fig. 4a,b), indicating that BZR1 interacts with TPL through the EAR motif. To confirm the BZR1-TPL interaction in vivo, we performed co-immunoprecipitation (co-IP) assays using Arabidopsis mesophyll protoplasts expressing bzr1-1D-GFP or bzr1-1DΔEAR-GFP and TPL-Myc. The TPL-Myc protein was co-immunoprecipitated by anti-YFP antibody specifically when bzr1-1D-GFP was co-expressed (Fig. 4d), confirming the in vivo BZR1-TPL interaction. The deletion of EAR motif greatly reduced the TPL-BZR1 interaction in vivo (Fig. 4d), which is consistent with the result of the yeast two-hybrid assay (Fig. 4a). Thus, both yeast two-hybrid and co-IP assays demonstrate that BZR1 interacts with TPL through the EAR motif.


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

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

BZR1 interacts with TOPLESS through the EAR motif(a) A yeast two-hybrid assay shows the interaction between BZR1 and TPL. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 50 mM 3AT medium.(b) Mutation in the EAR motif abolishes the BZR1-TPL interaction. Core Leu residues of the EAR motif were substituted to Ala. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 1 mM 3AT medium.(c) BZR1 interacts with TPL and other TPRs. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 5 mM 3AT medium. TPL-N: N-terminal domain of TPL (1–344), TPR1-N : 1–343, TPR2-N : 1–335, TPR3-N : 1–335 and TPR4-N : 1–344.(d) BZR1 interacts with TPL through EAR motif in vivo. Arabidopsis mesophyll protoplasts were transfected with TPL-Myc together with bzr1-1D-GFP or bzr1-1DΔEAR-GFP, and the extracted proteins were immunoprecipitated by anti-GFP antibody. Gel blots were probed with anti-Myc or anti-GFP antibody.(e) TPL binds to DWF4 promoter through BZR1. GST-TPL-N (amino acids 1–344) was incubated with a biotin-labelled DWF4 promoter DNA (400 bps) together with MBP or MBP-BZR1 and pulled down with streptavidin agarose beads. Gel blots were probed with anti-GST or anti-MBP antibody. The full scans of the gel blots (d,e) are shown in Supplementary Fig. 4.(f) The TPL DNA-binding on the promoters of CPD and DWF4 are enhanced by BR treatment. TPL DNA-binding was determined by ChIP assay using TPLp::TPL-HA transgenic plants. One-week seedlings grown on the medium containing 2 µM PPZ were treated with mock (M) or 100 nM BL for 4 hr before crosslinking. Error bars indicate the s.d. (n=3) and **: P<0.01 by Student’s t-test.
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Figure 4: BZR1 interacts with TOPLESS through the EAR motif(a) A yeast two-hybrid assay shows the interaction between BZR1 and TPL. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 50 mM 3AT medium.(b) Mutation in the EAR motif abolishes the BZR1-TPL interaction. Core Leu residues of the EAR motif were substituted to Ala. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 1 mM 3AT medium.(c) BZR1 interacts with TPL and other TPRs. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 5 mM 3AT medium. TPL-N: N-terminal domain of TPL (1–344), TPR1-N : 1–343, TPR2-N : 1–335, TPR3-N : 1–335 and TPR4-N : 1–344.(d) BZR1 interacts with TPL through EAR motif in vivo. Arabidopsis mesophyll protoplasts were transfected with TPL-Myc together with bzr1-1D-GFP or bzr1-1DΔEAR-GFP, and the extracted proteins were immunoprecipitated by anti-GFP antibody. Gel blots were probed with anti-Myc or anti-GFP antibody.(e) TPL binds to DWF4 promoter through BZR1. GST-TPL-N (amino acids 1–344) was incubated with a biotin-labelled DWF4 promoter DNA (400 bps) together with MBP or MBP-BZR1 and pulled down with streptavidin agarose beads. Gel blots were probed with anti-GST or anti-MBP antibody. The full scans of the gel blots (d,e) are shown in Supplementary Fig. 4.(f) The TPL DNA-binding on the promoters of CPD and DWF4 are enhanced by BR treatment. TPL DNA-binding was determined by ChIP assay using TPLp::TPL-HA transgenic plants. One-week seedlings grown on the medium containing 2 µM PPZ were treated with mock (M) or 100 nM BL for 4 hr before crosslinking. Error bars indicate the s.d. (n=3) and **: P<0.01 by Student’s t-test.
Mentions: In several transcription factors, the EAR motif has been known to mediate transcriptional repression through interaction with the Groucho/Tup1-like transcriptional corepressor TOPLESS (TPL) and the homologous TOPLESS RELATED (TPR) proteins25–28. Our recent tandem affinity purification of BZR1 complex followed by mass spectrometry analysis suggested that TPL associates with BZR1 in vivo29. We therefore tested whether BZR1 directly interacts with TPL and TPRs. Indeed, in the yeast two-hybrid assay, BZR1 interacted with TPL and also other TPRs (Fig. 4a,c). Deletion of the EAR motif or mutation of the conserved Leu residues in the EAR motif abolished the BZR1-TPL interaction (Fig. 4a,b), indicating that BZR1 interacts with TPL through the EAR motif. To confirm the BZR1-TPL interaction in vivo, we performed co-immunoprecipitation (co-IP) assays using Arabidopsis mesophyll protoplasts expressing bzr1-1D-GFP or bzr1-1DΔEAR-GFP and TPL-Myc. The TPL-Myc protein was co-immunoprecipitated by anti-YFP antibody specifically when bzr1-1D-GFP was co-expressed (Fig. 4d), confirming the in vivo BZR1-TPL interaction. The deletion of EAR motif greatly reduced the TPL-BZR1 interaction in vivo (Fig. 4d), which is consistent with the result of the yeast two-hybrid assay (Fig. 4a). Thus, both yeast two-hybrid and co-IP assays demonstrate that BZR1 interacts with TPL through the EAR motif.

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