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Genes (Basel). 2014 Dec 19;5(4):1115-31. doi: 10.3390/genes5041115.

Next-generation sequencing of genomic DNA fragments bound to a transcription factor in vitro reveals its regulatory potential.

Author information

1
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. yukio.kurihara@riken.jp.
2
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. yuko.makita@riken.jp.
3
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. mkawa@riken.jp.
4
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. hamasaki@psc.riken.jp.
5
The United Graduate School of Agricultural Science, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. yyy@gifu-u.ac.jp.
6
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. minami@riken.jp.

Abstract

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.

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