title: Analysis of steric effects in DamID profiling of transcription factor target genes.
abstract: DNA adenine methyltransferase identification (DamID) is an enzymatic technology for detecting DNA regions targeted by chromatin-associated proteins. Proteins are fused to bacterial DNA adenine methyltransferase (Dam) and expressed in cultured cells or whole organisms. Here, we used DamID to detect DNA regions bound by the cardiac-restricted transcription factors (TFs) NKX2-5 and SRF, and ubiquitously-expressed co-factors ELK1 and ELK4. We compared targets bound by these TFs as N- and C-terminal fusions with Dam, for both wild type (WT) NKX2-5 and mutant proteins mimicking those found in congenital heart disease. Overall, DamID is highly robust: while the orientation of WT Dam fusions can affect the size of the target sets, their signatures remained largely reproducible. Furthermore, a severe NKX2-5 mutant lacking the homeodomain showed strong steric effects negatively impacting target discovery. The extent of steric effect is likely to be dependent on the protein in question and the orientation of Dam fusion.
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia; Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia; Systems Biology Institute, Australia.
authors.forename: Mirana
authors.initials: M
authors.lastname: Ramialison
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia; Children's Medical Research Institute, University of Sydney, 214 Hawkesbury Road, Westmead, NSW 2145, Australia.
authors.forename: Ashley J
authors.initials: AJ
authors.lastname: Waardenberg
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia; St. Vincent's Clinical School, University of New South Wales, Kensington 2052, Australia.
authors.forename: Nicole
authors.initials: N
authors.lastname: Schonrock
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia.
authors.forename: Tram
authors.initials: T
authors.lastname: Doan
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia.
authors.forename: Danielle
authors.initials: D
authors.lastname: de Jong
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia; St. Vincent's Clinical School, University of New South Wales, Kensington 2052, Australia. Electronic address: r.bouveret@victorchang.edu.au.
authors.forename: Romaric
authors.initials: R
authors.lastname: Bouveret
authors.affiliation: Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia; St. Vincent's Clinical School, University of New South Wales, Kensington 2052, Australia; School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington 2052, Australia. Electronic address: r.harvey@victorchang.edu.au.
authors.forename: Richard P
authors.initials: RP
authors.lastname: Harvey
mesh_terms: D000818:Animals; D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D006330:Heart Defects, Congenital / Q000235:genetics / Q000378:metabolism*; D000072576:Homeobox Protein Nkx-2.5 / Q000378:metabolism; D006801:Humans; D051379:Mice; D026362:Serum Response Factor / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D050807:ets-Domain Protein Elk-1 / Q000378:metabolism; D050808:ets-Domain Protein Elk-4 / Q000378:metabolism
chemical_list: D002843:Chromatin; C494113:Elk1 protein, mouse; C494194:Elk4 protein, mouse; D000072576:Homeobox Protein Nkx-2.5; C484469:Nkx2-5 protein, mouse; D026362:Serum Response Factor; D050807:ets-Domain Protein Elk-1; D050808:ets-Domain Protein Elk-4; D004247:DNA; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)
keywords: Congenital heart disease; DNA adenine methyltransferase identification (DamID); NKX2-5; Quality control; Steric effect