FlyORF-TaDa allows rapid generation of new lines for in vivo cell-type-specific profiling of protein-DNA interactions in Drosophila melanogaster

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.

Keywords: Chromatin; DamID; Development; Neural stem cells; Transcription; Transcription factor.

Figure 1

The FlyORF-TaDa system generates new lines for cell-type specific profiling. (A) Schematic representation of the FlyORF-TaDa system. When the FlyORF-TaDa donor line is crossed to a FlyORF line and a heat-shock-inducible flippase (hs-Flp), recombination between compatible FRT5 sites generates a new TaDa line for the ORF in the progeny. These lines can be screened for in the subsequent generation by eye color. Dashed lines show GAL4/UAS-induced transcription; *stop codon. (B) Correlation of DNA-binding profiles generated for MRG15 in NSCs using the FlyORF-TaDa system, compared to conventional TaDa (Pearson’s correlation is shown).

Figure 2

Characteristics of MRG15 binding in NSCs. (A) MRG15 binds to regions of Yellow chromatin in NSCs. Profiles obtained via FlyORF-TaDa and conventional TaDa are shown; peaks were called on the average binding profile of the combined MRG15 datasets. NSC chromatin state data and colors are from Marshall and Brand (2017). A 300-kb region of chrX is illustrated. (B) Significance and odds ratio (θ) of enrichment of MRG15 peak occupancy within NSC chromatin states as assessed via Fisher’s exact test.

Figure 3

GO term enrichment for genes covering MRG15-bound peaks identifies terms for both metabolism and developmental neurogenesis (non-redundant GO terms covering <1000 genes illustrated). Metabolism-related terms are colored in orange; developmental terms are colored in red. All terms illustrated were significantly enriched (all adjusted P-values <10⁻²⁸).