title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins.
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.
authors.affiliation: Centre for Organismal Studies (COS), University of Heidelberg, Im Neuenheimer Feld 230, Heidelberg D-69120, Germany.
authors.forename: Jose Arturo
authors.initials: JA
authors.lastname: Gutierrez-Triana
authors.affiliation: Centre for Organismal Studies (COS), University of Heidelberg, Im Neuenheimer Feld 230, Heidelberg D-69120, Germany.
authors.forename: Juan L
authors.identifier: 0000-0001-9902-6048
authors.initials: JL
authors.lastname: Mateo
authors.affiliation: Deep Sequencing Core Facility, Cell Networks, University of Heidelberg, Im Neuenheimer 267, Heidelberg D-69120, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Ibberson
authors.affiliation: Developmental Genetics of the Nervous System, Max Planck Institute for Medical Research, Jahnstrasse 29, Heidelberg D-69120, Germany.|Focus Program Translational Neuroscience, University Medical Center, Johannes Gutenberg University Mainz, Langenbeckstr. 1, Mainz D-55131, Germany.
authors.forename: Soojin
authors.initials: S
authors.lastname: Ryu
authors.affiliation: Centre for Organismal Studies (COS), University of Heidelberg, Im Neuenheimer Feld 230, Heidelberg D-69120, Germany jochen.wittbrodt@cos.uni-heidelberg.de.
authors.forename: Joachim
authors.identifier: 0000-0001-8550-7377
authors.initials: J
authors.lastname: Wittbrodt
mesh_terms: D000465:Algorithms; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002843:Chromatin / Q000378:metabolism*; D019295:Computational Biology / Q000379:methods*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D030541:Databases, Genetic; D004625:Embryo, Nonmammalian; D005786:Gene Expression Regulation / Q000235:genetics; D009990:Oryzias / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)
keywords: Chromatin profiling; DamID; Epigenetics; Transcription factor; Transcriptional regulation