Abstract
Targeted DamID (TaDa) is an increasingly popular method of generating cell-type-specific DNA-binding profiles in vivo. Although sensitive and versatile, TaDa requires the generation of new transgenic fly lines for every protein that is profiled, which is both time-consuming and costly. Here, we describe the FlyORF-TaDa system for converting an existing FlyORF library of inducible open reading frames (ORFs) to TaDa lines via a genetic cross, with recombinant progeny easily identifiable by eye color. Profiling the binding of the H3K36me3-associated chromatin protein MRG15 in larval neural stem cells using both FlyORF-TaDa and conventional TaDa demonstrates that new lines generated using this system provide accurate and highly reproducible DamID-binding profiles. Our data further show that MRG15 binds to a subset of active chromatin domains in vivo. Courtesy of the large coverage of the FlyORF library, the FlyORF-TaDa system enables the easy creation of TaDa lines for 74% of all transcription factors and chromatin-modifying proteins within the Drosophila genome.
Highlights
Characterizing the specific protein–DNA interactions that underlie gene expression is essential for understanding the biology of any given tissue
A stop codon positioned directly after the FRT5 site allows the FlyORF-Targeted DamID (TaDa) line to be used as a DNA adenine methylase (Dam)-only control for DamID signal normalization (Supplementary Figure S1B)
The FlyORF-TaDa system places fast and straightforward celltype-specific profiling of transcription factors (TFs) binding within the reach of any fly lab, allowing the profiling of over 74% of all TFs and chromatinassociated proteins via a simple genetic cross
Summary
Characterizing the specific protein–DNA interactions that underlie gene expression is essential for understanding the biology of any given tissue. A key recent finding has been that the majority of TFs can act as either an activator or as a repressor, with their function determined by the surrounding TF community (Stampfel et al 2015). For ChIPseq, a lack of appropriate antibodies is a considerable impediment, combined with the difficulty of profiling TFs that are not directly bound to DNA. These issues are solved by DamID, a technique in which the TF of interest is expressed as a fusion protein with Escherichia coli DNA adenine methylase (Dam) and the resulting enriched adenine methylation surrounding the TF-binding sites profiled. DamID effectively profiles all TFs coming into proximity with DNA and requires no antibodies for profiling (for review, see Aughey et al 2019)
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