Abstract
DNA adenine methyltransferase identification (DamID) has emerged as an alternative method to profile protein-DNA interactions; however, critical issues limit its widespread applicability. Here, we present iDamIDseq, a protocol that improves specificity and sensitivity by inverting the steps DpnI-DpnII and adding steps that involve a phosphatase and exonuclease. To determine genome-wide protein-DNA interactions efficiently, we present the analysis tool iDEAR (iDamIDseq Enrichment Analysis with R). The combination of DamID and iDEAR permits the establishment of consistent profiles for transcription factors, even in transient assays, as we exemplify using the small teleost medaka (Oryzias latipes). We report that the bacterial Dam-coding sequence induces aberrant splicing when it is used with different promoters to drive tissue-specific expression. Here, we present an optimization of the sequence to avoid this problem. This and our other improvements will allow researchers to use DamID effectively in any organism, in a general or targeted manner.
Highlights
Animal development is the result of an exquisite orchestration of changes in gene expression in time and space
To overcome these drawbacks and allow a wider, immediate application of the technique, we have made a series of improvements to the original iDamIDseq protocol, resulting in a method that is applicable and provides consistent results. This approach permits transcription factor profiling even in transient applications. We complement these experimental improvements with iDEAR, an analysis pipeline associated with iDam, as a rapid new method for establishing highly reliable profiles of transcription factor-binding sites
When mRNA coding for the fusion E. coli Dam (eDam)-GFP was injected into zygotes, we observed a high number of abnormal embryos at stage 25, 52%, compared with 1% in the control case (Iwamatsu, 2004) (Fig. 1A)
Summary
Animal development is the result of an exquisite orchestration of changes in gene expression in time and space. Two methods are currently used to profile transcription factor-binding regions in the genome: chromatin immunoprecipitation (ChIP) and DNA adenine methyltransferase identification (DamID) (reviewed by Aughey and Southall, 2016; Furey, 2012).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
