Recruitment of AtWHY1 and AtWHY3 by a distal element upstream of the kinesin gene AtKP1 to mediate transcriptional repression Ji-Yuan Xiong Æ Cheng-Xia Lai Æ Zhe Qu Æ Xue-Yong Yang Æ Xing-Hua Qin Æ Guo-Qin Liu Received: 26 February 2009 / Accepted: 22 July 2009 / Published online: 11 August 2009 Ó Springer Science+Business Media B.V. 2009 Abstract A 43-bp distal element, the AtKP1-related ele- ment (KPRE), was previously shown to repress the promoter activity of the kinesin gene AtKP1 in Arabidopsis thaliana. In order to identify KPRE-binding factor 1 (KBF1), a combination of ion-exchange chromatography, gel-filtration chromatography and DNA-affinity chromatography was used to purify KBF1 from whole cell extracts of Arabidopsis seedlings. Mass spectrometric identification showed that KBF1 contains two members of the whirly family of tran- scription factors, AtWHY1 and AtWHY3. KBF1 is a single and double-stranded DNA-binding factor. A ChIP assay showed that AtWHY1 and AtWHY3 bind to the upstream region of AtKP1 gene in vivo. Over-expression of AtWHY1 and AtWHY3 led to an obvious decrease of AtKP1 tran- scripts, based on quantitative real-time PCR analysis. Interestingly, salicylic acid treatment resulted in an increase of AtWHY1 and AtWHY3 transcripts, and a decrease of AtKP1 transcripts. Thus, AtWHY1 and AtWHY3, as two components of KBF1, can be recruited at the KPRE site to mediate the transcriptional repression of AtKP1. Our results prove that AtKP1 is a new downstream target of the whirly family of transcription factors. Keywords KPRE-binding factor 1 Á Whirly family of transcription factors Á Transcriptional repression Á Kinesin Á AtKP1-related element Introduction Transcription factors play important roles in the activation and repression of gene transcription by binding to the regulatory regions of genes (Jones et al. 1988). The inter- actions between transcription factors and promoters at the DNA level are a decisive step in transcriptional regulation, in which a multitude of DNA-protein and protein-protein interactions activate or repress gene transcription by forming functional and regulatory complexes (Suzuki et al. 1998). Transcription is a cumulative result of multiple reactions and components, and the elucidation of critical reactions such as DNA-protein interactions will increase understanding of the whole process, and facilitate further investigation of gene function. Transcription factors are members of a superfamily that performs different functions in the process of cell devel- opment (Hori et al. 2003; Perez-Rodriguez et al. 2005), differentiation (Reimold et al. 2001; Igarashi et al. 2007), and signal transduction (Uno et al. 2000; Middleton et al. 2007), etc. The whirly family of transcription factors are plant-specific proteins, and are predicted to have the ability to bind to single-stranded DNA (Desveaux et al. 2005). The first identified member of the whirly proteins is PBF-2, which activates potato PR-10a gene expression in an ERE element-dependent manner. The cDNA for PBF-2 was cloned and termed StWHY1, and demonstrated to play roles in defense gene regulation (Desveaux et al. 2000, 2004). Analysis of its crystal structure showed that four StWHY1 molecules associate with cyclic C4 symmetry, and the Electronic supplementary material The online version of this article (doi:10.1007/s11103-009-9533-7) contains supplementary material, which is available to authorized users. J.-Y. Xiong Á C.-X. Lai Á Z. Qu Á X.-Y. Yang Á X.-H. Qin Á G.-Q. Liu (&) State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, 100193 Beijing, China e-mail: Liu@cau.edu.cn 123 Plant Mol Biol (2009) 71:437–449 DOI 10.1007/s11103-009-9533-7