A Highly Sensitive in Vivo Footprinting Technique for Condition-Dependent Identification of Cis Elements

Nucleic Acids Res. 2014 Jan;42(1):e1. doi: 10.1093/nar/gkt883. Epub 2013 Oct 3.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Cellulose 1,4-beta-Cellobiosidase / genetics
  • DNA Cleavage
  • DNA Footprinting / methods*
  • DNA Methylation
  • Endo-1,4-beta Xylanases / genetics
  • Polymerase Chain Reaction
  • Regulatory Elements, Transcriptional*
  • Software
  • Trichoderma / genetics

Substances

  • Endo-1,4-beta Xylanases
  • Cellulose 1,4-beta-Cellobiosidase