Final Report Summary - FRUITLESS TARGETS (Transcriptional regulation of male courtship behaviour: understanding Fruitless molecular networks)
In this proposal we aimed to define the direct regulatory targets of fruitless (Fru) by using Chromatin Immunoprecipitation coupled to ultra high-throughput sequencing (ChIP-Seq) to map regions throughout the Drosophila genome bound by Fru proteins. We proposed undertaking ChIP using a system in which the transcription factor of interest is tagged with a short (~23 amino acid) sequence (herein known as the biotin ligase recognition peptide, or BLRP), which is capable of being recognised by the E.coli biotin ligase enzyme, BirA (de Boer et al., 2003; Mito et al., 2005). Expression of BirA under the control of a cell type specific promoter then allows the specific biotinylation of this tag in vivo. Biotinylated proteins complexed with bound DNA can then be readily isolated from the tissues of these animals by exploiting the extremely strong affinity (Kd=10-15) that streptavidin has for biotin (de Boer et al., 2003), followed by DNA isolation and direct deep sequencing of the DNA.
The first step of this project required the generation of BLRP tagged versions of fruitless. We successfully generated constructs that carry the FruA, FruB or FruC isoform of fruitless (the three protein isoforms of Fru shown to be important for courtship behaviour; Sabrina Jörchel, unpublished data, and (Billeter et al., 2006) tagged with a C-terminal BLRP epitope. Each of these tagged isoforms can be immunoprecipitated from cell lysates using Streptavidin coupled magnetic beads, when co-expressed with the BirA enzyme.
These constructs have been used to drive expression in drosophila S2 cell lines for initial pilot ChIP-Seq experiments from which we have identified lists of potential Fru target regions for each isoform.
Furthermore, we have used these constructs to generate transgenic fly lines that express these tagged isoforms under the control of the GAL4-UAS system. These transgenic lines have initially been crossed to UAS-BirA; fru-GAL4 lines to generate flies that express both BirA and Fru-BLRP in all fruitless neurons in the fly. ChIP-Seq experiments utilising these flies are currently in progress.
A second major aim of the proposal was to identify fruitless dependent gene expression changes in subsets of fruitless expressing neurons. We originally proposed the use of a method known as TU-tagging, to isolate mRNA from specific subpopulations of fru neurons (Cleary et al., 2005), followed by direct next-generation RNA sequencing (Solexa) to obtain an unbiased high-throughput profile of Fru directed expression changes (Wilhelm and Landry, 2009).
Initial experiments suggested that we were not able to enrich the specific RNA populations from the cells we were interested in to a level that we found satisfactory using the TU-tagging method. As such, I am currently employing another technique to isolate RNA from specific cell populations.
This new technique involves expressing mCD8-GFP in subsets of neurons and using this marker to collect these labelled cells either by hand (based on GFP expression) or via a CD8 antibody coupled to magnetic beads. Thus far, this technique looks to be more promising for the enrichment of cell type specific RNA transcripts and optimisation of the experimental conditions is currently underway.
The first step of this project required the generation of BLRP tagged versions of fruitless. We successfully generated constructs that carry the FruA, FruB or FruC isoform of fruitless (the three protein isoforms of Fru shown to be important for courtship behaviour; Sabrina Jörchel, unpublished data, and (Billeter et al., 2006) tagged with a C-terminal BLRP epitope. Each of these tagged isoforms can be immunoprecipitated from cell lysates using Streptavidin coupled magnetic beads, when co-expressed with the BirA enzyme.
These constructs have been used to drive expression in drosophila S2 cell lines for initial pilot ChIP-Seq experiments from which we have identified lists of potential Fru target regions for each isoform.
Furthermore, we have used these constructs to generate transgenic fly lines that express these tagged isoforms under the control of the GAL4-UAS system. These transgenic lines have initially been crossed to UAS-BirA; fru-GAL4 lines to generate flies that express both BirA and Fru-BLRP in all fruitless neurons in the fly. ChIP-Seq experiments utilising these flies are currently in progress.
A second major aim of the proposal was to identify fruitless dependent gene expression changes in subsets of fruitless expressing neurons. We originally proposed the use of a method known as TU-tagging, to isolate mRNA from specific subpopulations of fru neurons (Cleary et al., 2005), followed by direct next-generation RNA sequencing (Solexa) to obtain an unbiased high-throughput profile of Fru directed expression changes (Wilhelm and Landry, 2009).
Initial experiments suggested that we were not able to enrich the specific RNA populations from the cells we were interested in to a level that we found satisfactory using the TU-tagging method. As such, I am currently employing another technique to isolate RNA from specific cell populations.
This new technique involves expressing mCD8-GFP in subsets of neurons and using this marker to collect these labelled cells either by hand (based on GFP expression) or via a CD8 antibody coupled to magnetic beads. Thus far, this technique looks to be more promising for the enrichment of cell type specific RNA transcripts and optimisation of the experimental conditions is currently underway.