A highly sensitive in vivo footprinting technique for condition-dependent identification of cis elements

Knowing which regions of a gene are targeted by transcription factors during induction or repression is essential for understanding the mechanisms responsible for regulation. Therefore, we re-designed the traditional in vivo footprinting method to obtain a highly sensitive technique, which allows id...

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Main Authors:	Gorsche, Rita, Jovanovic, Birgit, Gudynaite-Savitch, Loreta, Mach, Robert L., Mach-Aigner, Astrid R.
Format:	Online
Language:	English
Published:	Oxford University Press 2014
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874196/

id	pubmed-3874196
recordtype	oai_dc
spelling	pubmed-38741962013-12-28 A highly sensitive in vivo footprinting technique for condition-dependent identification of cis elements Gorsche, Rita Jovanovic, Birgit Gudynaite-Savitch, Loreta Mach, Robert L. Mach-Aigner, Astrid R. Methods Online Knowing which regions of a gene are targeted by transcription factors during induction or repression is essential for understanding the mechanisms responsible for regulation. Therefore, we re-designed the traditional in vivo footprinting method to obtain a highly sensitive technique, which allows identification of the cis elements involved in condition-dependent gene regulation. Data obtained through DMS methylation, HCl DNA cleavage and optimized ligation-mediated PCR using fluorescent labelling followed by capillary gel electrophoresis are analysed by ivFAST. In this work we have developed this command line-based program, which is designed to ensure automated and fast data processing and visualization. The new method facilitates a quantitative, high-throughput approach because it enables the comparison of any number of in vivo footprinting results from different conditions (e.g. inducing, repressing, de-repressing) to one another by employing an internal standard. For validation of the method the well-studied upstream regulatory region of the Trichoderma reesei xyn1 (endoxylanase 1) gene was used. Applying the new method we could identify the motives involved in condition-dependent regulation of the cbh2 (cellobiohydrolase 2) and xyn2 (endoxylanase 2) genes. Oxford University Press 2014-01-01 2013-10-03 /pmc/articles/PMC3874196/ /pubmed/24097437 http://dx.doi.org/10.1093/nar/gkt883 Text en © The Author(s) 2013. Published by Oxford University Press. http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Gorsche, Rita Jovanovic, Birgit Gudynaite-Savitch, Loreta Mach, Robert L. Mach-Aigner, Astrid R.
spellingShingle	Gorsche, Rita Jovanovic, Birgit Gudynaite-Savitch, Loreta Mach, Robert L. Mach-Aigner, Astrid R. A highly sensitive in vivo footprinting technique for condition-dependent identification of cis elements
author_facet	Gorsche, Rita Jovanovic, Birgit Gudynaite-Savitch, Loreta Mach, Robert L. Mach-Aigner, Astrid R.
author_sort	Gorsche, Rita
title	A highly sensitive in vivo footprinting technique for condition-dependent identification of cis elements
title_short	A highly sensitive in vivo footprinting technique for condition-dependent identification of cis elements
title_full	A highly sensitive in vivo footprinting technique for condition-dependent identification of cis elements
title_fullStr	A highly sensitive in vivo footprinting technique for condition-dependent identification of cis elements
title_full_unstemmed	A highly sensitive in vivo footprinting technique for condition-dependent identification of cis elements
title_sort	highly sensitive in vivo footprinting technique for condition-dependent identification of cis elements
description	Knowing which regions of a gene are targeted by transcription factors during induction or repression is essential for understanding the mechanisms responsible for regulation. Therefore, we re-designed the traditional in vivo footprinting method to obtain a highly sensitive technique, which allows identification of the cis elements involved in condition-dependent gene regulation. Data obtained through DMS methylation, HCl DNA cleavage and optimized ligation-mediated PCR using fluorescent labelling followed by capillary gel electrophoresis are analysed by ivFAST. In this work we have developed this command line-based program, which is designed to ensure automated and fast data processing and visualization. The new method facilitates a quantitative, high-throughput approach because it enables the comparison of any number of in vivo footprinting results from different conditions (e.g. inducing, repressing, de-repressing) to one another by employing an internal standard. For validation of the method the well-studied upstream regulatory region of the Trichoderma reesei xyn1 (endoxylanase 1) gene was used. Applying the new method we could identify the motives involved in condition-dependent regulation of the cbh2 (cellobiohydrolase 2) and xyn2 (endoxylanase 2) genes.
publisher	Oxford University Press
publishDate	2014
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874196/
_version_	1612041915798126592

A highly sensitive in vivo footprinting technique for condition-dependent identification of cis elements

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