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Genes 2014, 5(4), 1115-1131; doi:10.3390/genes5041115

Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential

Synthetic Genomics Research Team, Biomass Engineering Program Cooperation Division, RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan
The United Graduate School of Agricultural Science, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan
Author to whom correspondence should be addressed.
Received: 17 November 2014 / Revised: 13 December 2014 / Accepted: 16 December 2014 / Published: 19 December 2014
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Several transcription factors (TFs) coordinate to regulate expression of specific genes at the transcriptional level. In Arabidopsis thaliana it is estimated that approximately 10% of all genes encode TFs or TF-like proteins. It is important to identify target genes that are directly regulated by TFs in order to understand the complete picture of a plant’s transcriptome profile. Here, we investigate the role of the LONG HYPOCOTYL5 (HY5) transcription factor that acts as a regulator of photomorphogenesis. We used an in vitro genomic DNA binding assay coupled with immunoprecipitation and next-generation sequencing (gDB-seq) instead of the in vivo chromatin immunoprecipitation (ChIP)-based methods. The results demonstrate that the HY5-binding motif predicted here was similar to the motif reported previously and that in vitro HY5-binding loci largely overlapped with the HY5-targeted candidate genes identified in previous ChIP-chip analysis. By combining these results with microarray analysis, we identified hundreds of HY5-binding genes that were differentially expressed in hy5. We also observed delayed induction of some transcripts of HY5-binding genes in hy5 mutants in response to blue-light exposure after dark treatment. Thus, an in vitro gDNA-binding assay coupled with sequencing is a convenient and powerful method to bridge the gap between identifying TF binding potential and establishing function. View Full-Text
Keywords: plant; transcription factor; HY5; in vitro binding; next-generation sequencing plant; transcription factor; HY5; in vitro binding; next-generation sequencing

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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Kurihara, Y.; Makita, Y.; Kawashima, M.; Hamasaki, H.; Yamamoto, Y.Y.; Matsui, M. Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential. Genes 2014, 5, 1115-1131.

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