Molecular Plant Advance Access originally published online on October 23, 2007
Molecular Plant 2008 1(1):27-41; doi:10.1093/mp/ssm009
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© The Author 2007. Published by Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS.
The Development of Protein Microarrays and Their Applications in DNA–Protein and Protein–Protein Interaction Analyses of Arabidopsis Transcription Factors
a Peking-Yale Joint Center for Plant Molecular Genetics and Agrobiotechnology, College of Life Sciences, and the National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871, China
b Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA
c Section of Plant Biology, College of Biological Sciences, University of California, Davis 95616, USA
1 To whom correspondence should be addressed. E-mail xingwang.deng{at}yale.edu.
We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein–DNA and protein–protein interaction analyses. For protein–DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein–DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale.
* These authors contributed equally to this work.
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