A valid strategy for precise identifications of transcription factor binding sites in combinatorial regulation using bioinformatic and experimental approaches

doi:10.1186/1746-4811-9-34

	A valid strategy for precise identifications of transcription factor binding sites in combinatorial regulation using bioinformatic and experimental approaches
	Wang, Hailong 1; Guan, Shan ; Zhu, Zhixin 1; Wang, Yan ; Lu, Yingqing
	2013
发表期刊	PLANT METHODS
ISSN	1746-4811
卷号	9
摘要	Background: Transcription factor (TF) binding sites (cis element) play a central role in gene regulation, and eukaryotic organisms frequently adapt a combinatorial regulation to render sophisticated local gene expression patterns. Knowing the precise cis element on a distal promoter is a prerequisite for studying a typical transcription process; however, identifications of cis elements have lagged behind those of their associated trans acting TFs due to technical difficulties. Consequently, gene regulations via combinatorial TFs, as widely observed across biological processes, have remained vague in many cases. Results: We present here a valid strategy for identifying cis elements in combinatorial TF regulations. It consists of bioinformatic searches of available databases to generate candidate cis elements and tests of the candidates using improved experimental assays. Taking the MYB and the bHLH that collaboratively regulate the anthocyanin pathway genes as examples, we demonstrate how candidate cis motifs for the TFs are found on multi-specific promoters of chalcone synthase (CHS) genes,and how to experimentally test the candidate sites by designing DNA fragments hosting the candidate motifs based on a known promoter (us1 allele of Ipomoea purpurea CHS-D in our case) and applying site-mutagenesis at the motifs. It was shown that TF-DNA interactions could be unambiguously analyzed by assays of electrophoretic mobility shift (EMSA) and dual-luciferase transient expressions, and the resulting evidence precisely delineated a cis element. The cis element for R2R3 MYBs including Ipomoea MYB1 and Magnolia MYB1, for instance, was found to be ANCNACC, and that for bHLHs (exemplified by Ipomoea bHLH2 and petunia AN1) was CACNNG. A re-analysis was conducted on previously reported promoter segments recognized by maize C1 and apple MYB10, which indicated that cis elements similar to ANCNACC were indeed present on these segments, and tested positive for their bindings to Ipomoea MYB1. Conclusion: Identification of cis elements in combinatorial regulation is now feasible with the strategy outlined. The working pipeline integrates the existing databases with experimental techniques, providing an open framework for precisely identifying cis elements. This strategy is widely applicable to various biological systems, and may enhance future analyses on gene regulation.
关键词	cis element MYB bHLH EMSA Dual-luciferase transient expression assay
学科领域	Biochemical Research Methods ; Plant Sciences
DOI	10.1186/1746-4811-9-34
收录类别	SCI
语种	英语
WOS关键词	DNA-BINDING ; ARABIDOPSIS-THALIANA ; ANTHOCYANIN BIOSYNTHESIS ; PROTEIN-PROTEIN ; MORNING GLORY ; MYB PROTEIN ; LOOP-HELIX ; GENE ; EXPRESSION ; PROMOTER
WOS研究方向	Science Citation Index Expanded (SCI-EXPANDED)
WOS记录号	WOS:000324550300001
出版者	BIOMED CENTRAL LTD
文献子类	Article
出版地	LONDON
资助机构	National Science Foundation of China(National Natural Science Foundation of China (NSFC)) ; Chinese Academy of Sciences(Chinese Academy of Sciences) ; Ministry of Science and Technology(Ministry of Science, ICT & Future Planning, Republic of Korea)
作品OA属性	Green Published, gold
引用统计	被引频次：12[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	http://ir.ibcas.ac.cn/handle/2S10CLM1/27858
专题	系统与进化植物学国家重点实验室
作者单位	1.Chinese Acad Sci, Inst Bot, State Key Lab Systemat & Evolutionary Bot, Beijing 100093, Peoples R China 2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
推荐引用方式 GB/T 7714	Wang, Hailong,Guan, Shan,Zhu, Zhixin,et al. A valid strategy for precise identifications of transcription factor binding sites in combinatorial regulation using bioinformatic and experimental approaches[J]. PLANT METHODS,2013,9.
APA	Wang, Hailong,Guan, Shan,Zhu, Zhixin,Wang, Yan,&Lu, Yingqing.(2013).A valid strategy for precise identifications of transcription factor binding sites in combinatorial regulation using bioinformatic and experimental approaches.PLANT METHODS,9.
MLA	Wang, Hailong,et al."A valid strategy for precise identifications of transcription factor binding sites in combinatorial regulation using bioinformatic and experimental approaches".PLANT METHODS 9(2013).

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