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.
| Original language | English |
|---|---|
| Pages (from-to) | 75-82 |
| Number of pages | 8 |
| Journal | Genomics |
| Volume | 109 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 1 Mar 2017 |
Keywords
- Congenital heart disease
- DNA adenine methyltransferase identification (DamID)
- NKX2-5
- Quality control
- Steric effect
Cite this
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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; Harvey, Richard P.
In: Genomics, Vol. 109, No. 2, 01.03.2017, p. 75-82.Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - Analysis of steric effects in DamID profiling of transcription factor target genes
AU - Ramialison, Mirana
AU - Waardenberg, Ashley J.
AU - Schonrock, Nicole
AU - Doan, Tram
AU - de Jong, Danielle
AU - Bouveret, Romaric
AU - Harvey, Richard P.
PY - 2017/3/1
Y1 - 2017/3/1
N2 - 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.
AB - 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.
KW - Congenital heart disease
KW - DNA adenine methyltransferase identification (DamID)
KW - NKX2-5
KW - Quality control
KW - Steric effect
UR - http://www.scopus.com/inward/record.url?scp=85011966248&partnerID=8YFLogxK
U2 - 10.1016/j.ygeno.2017.01.006
DO - 10.1016/j.ygeno.2017.01.006
M3 - Article
VL - 109
SP - 75
EP - 82
JO - Genomics
JF - Genomics
SN - 0888-7543
IS - 2
ER -