Bian, Ming-Di

Li, Hong-Yu

Lin, ChenTao

Lin, Jian-Zhong

Liu, Bin

Liu, Jun

Peng, Yu-Chong

Zhang, Chun-Yu

Zhang, Yong-Xing

Zhao, Tao

English

Tao Zhao

Jun Liu

Hong-Yu Li

Jian-Zhong Lin

Ming-Di Bian

Chun-Yu Zhang

Yong-Xing Zhang

Yu-Chong Peng

Bin Liu

ChenTao Lin

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Using hybrid transcription factors to study gene function in rice

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UCLAUCLA Previously Published WorksTitleUsing hybrid transcription factors to study gene function in ricePermalinkhttps://escholarship.org/uc/item/94r8x2ctJournalScience China Life Sciences, 58(11)ISSN1674-7305AuthorsZhao, TaoLiu, JunLi, Hong-Yuet al.Publication Date2015-11-01DOI10.1007/s11427-015-4937-x Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaSCIENCE CHINA Life Sciences © The Author(s) 2015. This article is published with open access at link.springer.com life.scichina.com   link.springer.com                   †Contributed equally to this work *Corresponding author (email: liubin05@caas.cn) • LETTER TO THE EDITOR • November 2015  Vol.58  No.11: 1160–1162  doi: 10.1007/s11427-015-4937-x Using hybrid transcription factors to study gene function in rice ZHAO Tao1†, LIU Jun1†, LI Hong-Yu1†, LIN Jian-Zhong2†, BIAN Ming-Di3†, ZHANG Chun-Yu1, ZHANG Yong-Xing1, PENG Yu-Chong2, LIU Bin1* & LIN ChenTao4 1Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, Changsha 410082, China; 3Laboratory of Soil and Plant Molecular Genetics, College of Plant Science, Jilin University, Changchun 130062, China; 4Department of Molecular, Cell & Developmental Biology, University of California, Los Angeles, CA90095, USA Received September 14, 2015; accepted September 18, 2015; published online September 24, 2015  Citation:  Zhao T, Liu J, Li HY, Lin JZ, Bian MD, Zhang CY, Zhang YX, Peng YC, Liu B, Lin CT. Using hybrid transcription factors to study gene function in rice. Sci China Life Sci, 2015, 58: 1160–1162, doi: 10.1007/s11427-015-4937-x    Dear Editors,  Transcription factors (TF) play important roles in plant growth and development, which determine crop productivi-ty, quality, and stress tolerance. However, the basic mecha-nism governing transcription regulation by TF remains largely unclear in plants. It is well known that TF proteins contain at least two functional domains, the DNA-binding domain and the transcription regulatory domain. The VP16 transcription activation domain of human Herpes simplex virus and the EAR transcription suppression domain of many plant TF proteins are two of the best-known transcrip-tion regulatory domains [1,2]. It has been reported that both VP16 and EAR domains exert their respective transcription activation/repression functions in heterologous protein and/or cellular environment [24], supporting a hypothesis that the transcription regulatory domains function inde-pendent of other functional domains of TF proteins. How-ever, this hypothesis has not been examined systematically. We reasoned that a phenotypic analysis of transgenic ex-pression of all or most TF genes from a plant species in the form of fusion proteins, which are composed of the coding sequences of TFs fused to the VP16 or EAR domain, would allow a systematic test of this hypothesis, and we reported here the overall result of such an experiment.  We cloned cDNAs for 1,685 out of the 3,148 predicted transcription factor genes from rice (Oryza sativa subsp. Japonica) (Figure 1A, Table S1 in Supporting Information), and prepared 2,980 HTF Ti plasmid constructs that express the EAR4 (tetrameric repeats of EAR)-TF fusion proteins or the VP64 (tetrameric repeats of VP16)-TF fusion proteins under the control of the maize ubiquitin promoter [5] (Fig-ure 1B, Table S2 in Supporting Information). We obtained 57,751 independent transgenic lines in the Japonica rice accession Kitaake that express a total of 2,470 HTFs con-structs. On average, 23 independent transgenic lines were generated per construct (Figure 1C, Table S3 in Supporting Information). Approximately 13% (7,719/57,751 events) of transgenic lines showed phenotypic alterations in compari-son to the wild-type parents, including changes in mor-phology, heading date, or grain yield (Figure S1 in Sup-porting Information). Expression of 957 HTF fusion pro-teins, including 343 EAR4 HTF and 614 VP64 HTF pro-teins, caused the phenotypic alteration in at least three in-dependent transgenic lines, and they were investigated in more details (Figure 1D). 132 pairs of these 957 HTFs ex-hibited phenotypic alterations in both the EAR4 and VP64 configurations, of which 86, 30 and 16 pairs of EAR4 and VP64 HTFs resulted in similar, different, or opposite phe-notypes, respectively (Figure 1D, Pie diagram). It is sur-prising that only 12% (16/132) of EAR4- and VP64-fusion  Zhao T, et al.   Sci China Life Sci   November (2015) Vol.58 No.11 1161  Figure 1  Using hybrid transcription factors to identify gene functions. A, The distribution of cloned TFs in different gene families. B, Schematic presenta-tions of the HTF constructs. The detailed vector maps and sequences are documented in Supporting Information. C, Transgenic event number distribution of the HTF transgenes. D, An overview of constructs with at least 3 transgenic events showing similar phenotypic alteration. E, Representative image of CV865 and CE865 transgenic plants at adult stage. F, qRT-PCR analysis showing the overexpression of CV865 and CE865 mRNA in each transgenic line. G, Im-munoblot showing the level of CV865 protein in the indicated lines. The CV865 protein was detected with anti-VP16 antibody. The ponceau staining band of Rubisco large subunit was used as loading control. H and I, Statistical analysis of plant height (H) and grain yield per plant (I) of indicated genotypes, means ± SD (**, P<0.001, n>20) are shown. proteins caused opposite phenotypes, suggesting that the activity of a transcription regulatory domain is more de-pendent on other functional domains or the structural con-text than what the previous reports may have suggested. Exactly how a transcription regulatory domain is dependent on other functional domains or the structural context of the HTF proteins remains unclear, but the speculative interpre-tations would include distinct structural impacts of the VP64 or EAR4 modules, different functional effects of fu-sion protein configuration to the activity of individual mod-ule, distinct modes of action of the two modules, and so on. For example, VP16 is known to interact with not only spe-cific host transcription factors, such as Oct-1 and HCF-1[6], but also components of general transcription machinery, such as TBP, TFIIB, and TFIIH [7,8], whereas EAR is only known to interact with specific transcription regulators, such as TPL, to recruit histone deacetylases [9]. It is con-ceivable that VP64-fusion proteins are more likely to inter-act with the general transcription machinery to activate transcription in a heterologous protein and cellular envi-ronment, whereas the EAR4-fusion proteins may be less likely to encounter and interact with the specific transcrip-tion regulators to suppress transcription in the heterologous protein environment. On the other hand, those 16 HTFs that conferred opposite phenotypes in the VP64 or EAR4 configuration provide a relatively simpler explanation for the mode of action of the respective rice TF genes (Table S4 in Supporting Infor-mation). For example, a MYB-like transcription factor (en-coded by LOC_Os06g45890), referred to as TF865 in this study, showed opposite growth phenotype in the EAR4 or VP64 configuration. Transgenic lines expressing the VP64-fusion proteins (CV865) exhibited dwarf phenotype, whereas those expressing the EAR4-fusion proteins (CE865) grew significantly taller than the wild-type control (Figure 1E and 1H). Overexpression of both CV865 and CE865 transgenes were confirmed by the quantitative re-verse transcription PCR (qRT-PCR) or the immunoblot 1162 Zhao T, et al.   Sci China Life Sci   November (2015) Vol.58 No.11 analyses (Figure 1F and 1G). In comparison to that of the wild-type control, the grain yield per plant of transgenic lines expressing the EAR4-fusion protein (CE865) or the VP64-fusion protein (CV865) increased (50%100%) or decreased (10%20%), respectively (Figure 1I). These re-sults are consistent with the hypothesis that this MYB-like TF gene may regulate rice growth and development or me-tabolism to affect grain yield. Further study of other HTF that caused various phenotypic variations will likely reveal the functional roles and modes of action for other rice TF genes. Transcription factors regulate almost all aspects of biological processes by dynamically modulating target gene transcription. A large percentage of genes, ranged from 1%6% in different Eukaryotic species, encode TFs [10]. Only a relatively small fraction of eukaryotic TFs’ DNA sequence preferences are experimentally determined, but their biological functions are much less determined. The functional redundancy of many plant TF genes imposes a major technical difficulty in the study of physiological roles of many TF genes. The HTF transgenic resource reported here provides a valuable tool complement to the loss-of-function resources reported previously for the func-tional study of rice genes. This work is supported by the MOA Transgenic Research Grant (2010ZX08010-002 to ICS-CAAS), Agricultural Science and Technology Innovation Program of CAAS, and National Natural Science Foundation of China (31422041, 31371649, 31301298 and 31170172). 1 Sadowski I, Ma J, Triezenberg S, Ptashne M. GAL4-VP16 is an un-usually potent transcriptional activator. Nature, 1988, 335: 563564 2 Kagale S, Rozwadowski K. EAR motif-mediated transcriptional re-pression in plants: an underlying mechanism for epigenetic regulation of gene expression. Epigenetics, 2011, 6: 141146 3 Ohta M, Matsui K, Hiratsu K, Shinshi H, Ohme-Takagi M. Repres-sion domains of class II ERF transcriptional repressors share an es-sential motif for active repression. Plant Cell, 2001, 13: 19591968 4 Herve C, Dabos P, Bardet C, Jauneau A, Auriac MC, Ramboer A, Lacout F, Tremousaygue D. In vivo interference with AtTCP20 func-tion induces severe plant growth alterations and deregulates the ex-pression of many genes important for development. Plant Physiol, 2009, 149: 14621477 5 Cornejo MJ, Luth D, Blankenship KM, Anderson OD, Blechl AE. Activity of a maize ubiquitin promoter in transgenic rice. Plant Mol Biol, 1993, 23: 567581 6 Wysocka J, Herr W. The herpes simplex virus VP16-induced com-plex: the makings of a regulatory switch. Trends Biochem Sci, 2003, 28: 294304 7 Xiao H, Pearson A, Coulombe B, Truant R, Zhang S, Regier JL, Triezenberg SJ, Reinberg D, Flores O, Ingles CJ, et al. Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53. Mol Cell Biol, 1994, 14: 70137024 8 Hall DB, Struhl K. The VP16 activation domain interacts with multi-ple transcriptional components as determined by protein-protein cross-linking in vivo. J Biol Chem, 2002, 277: 4604346050 9 Wang L, Kim J, Somers DE. Transcriptional corepressor TOPLESS complexes with pseudoresponse regulator proteins and histone deacetylases to regulate circadian transcription. Proc Natl Acad Sci USA, 2013, 110: 761766 10 Jin J, Zhang H, Kong L, Gao G, Luo J. PlantTFDB 3.0: a portal for the functional and evolutionary study of plant transcription factors. Nucleic Acids Res, 2014, 42: D11821187  Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.  Supporting Information Figure S1  The number of HTFs in EAR4 or VP64 configuration showing indicated phenotypic alterations. Table S1  The list of 1685 transcription factor genes cloned into pDONR vector. Table S2  The list of 2980 HTF constructs. Table S3  The numbers of independent transgenic events of each HTF construct. Table S4  The list of 16 pairs of EAR4- and VP64-fusion proteins causing opposite phenotypes. The supporting information is available online at life.scichina.com and link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors. 

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Using hybrid transcription factors to study gene function in rice.

Using hybrid transcription factors to study gene function in rice.

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