Analysis of steric effects in DamID profiling of transcription factor target genes

Ramialison, Mirana, Waardenberg, Ashley J., Schonrock, Nicole, Doan, Tram, de Jong, Danielle, Bouveret, Romaric, and Harvey, Richard P. (2017) Analysis of steric effects in DamID profiling of transcription factor target genes. Genomics, 109 (2). pp. 75-82.

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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.

Item ID: 55655
Item Type: Article (Research - C1)
ISSN: 0888-7543
Keywords: congenital heart disease; DNA adenine methyltransferase identification (DamID); NKX2-5; quality control; steric effect
Copyright Information: Copyright © 2017 Elsevier Inc. All rights reserved.
Funders: National Health and Medical Research Council (NHMRC), Australian Research Council (ARC), Atlantic Philanthropies (AP), Human Frontiers in Science Program (HFSP), Swiss National Science Foundation (SNSF)
Projects and Grants: NHMRC Grant 573703, NHMRC Grant 1061539, NHMRC Grant 1121172, ARC DP0988507, ARC DP140101067, AP 19131, NHMRC Australia Fellowship 573705, NHMRC Senior Principal Research Fellowship 1118576, NHMRC/Heart Foundation Career Development (1049980) Fellowships, SNSF PBEZB-111553, HFSP (LT000245/2010-L), HFSP Fellowship (LT00044/2007-L), EMBO (1133-2009)
Date Deposited: 28 Sep 2018 01:47
FoR Codes: 31 BIOLOGICAL SCIENCES > 3101 Biochemistry and cell biology > 310109 Proteomics and intermolecular interactions (excl. medical proteomics) @ 100%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 100%
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