Analysis of steric effects in DamID profiling of transcription factor target genes.
Genomics
; 109(2): 75-82, 2017 03.
Article
in En
| MEDLINE
| ID: mdl-28189763
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.
Key words
Full text:
1
Database:
MEDLINE
Main subject:
Chromatin
/
Site-Specific DNA-Methyltransferase (Adenine-Specific)
/
Gene Expression Regulation
/
Genetic Techniques
/
Heart Defects, Congenital
Type of study:
Prognostic_studies
Limits:
Animals
/
Humans
Language:
En
Journal:
Genomics
Journal subject:
GENETICA
Year:
2017
Type:
Article
Affiliation country:
Australia