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

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 TF...

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Autores principales: Kurihara, Yukio, Makita, Yuko, Kawashima, Mika, Hamasaki, Hidefumi, Yamamoto, Yoshiharu Y., Matsui, Minami
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4276929/
https://www.ncbi.nlm.nih.gov/pubmed/25534860
http://dx.doi.org/10.3390/genes5041115
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author Kurihara, Yukio
Makita, Yuko
Kawashima, Mika
Hamasaki, Hidefumi
Yamamoto, Yoshiharu Y.
Matsui, Minami
author_facet Kurihara, Yukio
Makita, Yuko
Kawashima, Mika
Hamasaki, Hidefumi
Yamamoto, Yoshiharu Y.
Matsui, Minami
author_sort Kurihara, Yukio
collection PubMed
description 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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spelling pubmed-42769292014-12-30 Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential Kurihara, Yukio Makita, Yuko Kawashima, Mika Hamasaki, Hidefumi Yamamoto, Yoshiharu Y. Matsui, Minami Genes (Basel) Article 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. MDPI 2014-12-19 /pmc/articles/PMC4276929/ /pubmed/25534860 http://dx.doi.org/10.3390/genes5041115 Text en © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Kurihara, Yukio
Makita, Yuko
Kawashima, Mika
Hamasaki, Hidefumi
Yamamoto, Yoshiharu Y.
Matsui, Minami
Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential
title Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential
title_full Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential
title_fullStr Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential
title_full_unstemmed Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential
title_short Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential
title_sort next-generation sequencing of genomic dna fragments bound to a transcription factor in vitro reveals its regulatory potential
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4276929/
https://www.ncbi.nlm.nih.gov/pubmed/25534860
http://dx.doi.org/10.3390/genes5041115
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