Biology subjects

Marshall, O. J.

Publications and source records attributed to Marshall, O. J..

3 recordsLinked to original sources

bioRxiv2020

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

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 colour. 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.

molecular biology↗

bioRxiv2020

Psi promotes Drosophila wing growth through transcriptional repression of key developmental networks

Psi, the sole FUSE Binding Protein (FUBP) family single stranded DNA/RNA binding protein in Drosophila, is essential for proper cell and tissue growth, however its mechanism of function remains unclear. Here we use Targeted DamID combined with RNA-sequencing to generate the first genome-wide binding and expression profiles for Psi. Surprisingly, we demonstrate Psi drives growth in the Drosophila wing through transcriptional repression of key developmental pathways (e.g. Wnt, Notch and TGF{beta}). Thus, Psi patterns tissue growth by directly repressing transcription of developmental growth suppressors. Analysis of direct Psi targets identified novel growth inhibitors, including Tolkin (Zinc metallopeptidase implicated in TGF{beta} signalling), Ephexin (Rho-GEF) and emp (CD36 scavenger receptor-related protein). Their depletion not only suppressed impaired growth associated with Psi knockdown, but alone was sufficient to drive wing overgrowth. Thus, Psi drives wing growth twofold, through direct activation of Myc and through transcriptional repression of growth inhibitors comprising core developmental pathways.

developmental biology↗

bioRxiv2020

Membrane-bound GFP-labelled vectors for Targeted DamID allow simultaneous profiling of expression domains and DNA binding

Targeted DamID (TaDa) allows highly efficient cell-type-specific profiling of protein-DNA interactions. Cell-type-specificity, however, is governed by the GAL4/UAS system, which can exhibit differences in expression patterns depending upon the genomic insertion site and the UAS promoter strength. The TaDa system uses a bicistronic transcript to reduce the translation rates of Dam-fusion proteins, presenting the possibility of using the primary ORF within in the transcript to label expression domains and precisely identified the profiled cell populations in experimental samples. Here, we describe new TaDa vectors, pTaDaG, pTaDaG2 and pTaDaM, that use myristoylated-GFP or myristoylated-mCherry as the primary ORF. Differing lengths of the myristoylation sequence between the two GFP plasmids allows additional translational control. The mCherry plasmid allows profiled cells to be visualised when using the NanoDam system, in which an anti-GFP nanobody is fused to Dam to profile the localisation of GFP-fusion proteins. Fly lines created with this system allow easy visualisation of expression domains under both fluorescent dissecting and confocal microscopes without the use of antibody staining, whilst faithfully profiling protein-DNA interactions via Targeted DamID.

genomics↗