Final Activity Report Summary - NOTCH TARGETS (Notch target genes in Drosophila)
Signalling between cells and tissues is critical for their development and for their continued maintenance throughout life. Several key signalling pathways, including the Notch pathway, have been implicated in mediating this communication during development. Misfunction of these pathways contributes to many diseases. For example, altered Notch activity is associated with many cancers. Understanding how Notch controls changes in cell behaviours is therefore of major importance.
The goal of my project was to discover how Notch can alter cell behaviours, by identifying genes that are regulated by Notch. The strategy built on a genome-wide analysis (expression microarray) of all the genes whose expression was altered within 30 minutes of activating the Notch receptor. The main objectives were: 1) To analyse gene-expression profiles from Notch activated cells; 2) To mine data from other studies to identify targets with the highest probability of contributing to the Notch response; 3) To validate these as targets by showing that their expression is regulated by Notch; 4) To determine the biological and developmental functions of the novel targets and how they contribute to the Notch response. By necessity, only a small number of genes can be fully analysed functionally and one major outcome of the first 3 packages was the prioritisation of genes for stage 4 analysis.
The first 2 milestones have been achieved, giving a list of potential Notch target genes in several cell lines. The overlap reveals a core response to Notch in Drosophila and the data mining led me to further focus on 3 genes : Polychaetoid (pyd, the orthologue of ZO-1 in mammals), dTraf-1 (the orthologue of Traf-4 in mammals) and CG12290 encoding a 7-pass trans-membrane receptor. All three genes are associated with binding sites for Su(H) (the transcription factor mediating Notch signal) that are conserved in all Drosophila species for which the genome is available. This is indicative of an evolutionary conservation over more than 60 million years, suggesting that these sites are functionally relevant. The importance of these sites was further tested by combining the fragments with a fluorescent indicator (firefly luciferase) and measuring the response to activated Notch. Based on such studies we have shown that dTraf-1 and CG12290 are indeed targets of Notch, while Pyd may not be. Nevertheless, the functional investigations involved all 3 genes, since previous studies have linked Pyd to the Notch pathway.
My further studies have shown important developmental functions for the 3 proteins coded by pyd, dTraf1 and CG12290 and have helped to discover more about the way that these proteins work. To do this I had to make mutations that interfere with the function of the genes, to generate transgenic lines producing different fragments of the proteins and to make antibodies that could reveal the distribution of the proteins within cells in vivo. All of these tools will be of value to the community and are already being shared with other groups in the EU.
As a consequence of these studies, I have demonstrated contributions of Pyd and Traf to the regulation of sensory organ development. This has revealed (1) that Pyd helps to regulate the amount of Notch available to signal at the membrane by linking it to an enzyme that regulates Notch trafficking within the cell; 2) that there is cross-talk between Notch and the Toll / NF-kB pathway (that mediates immunity and cell stress responses) via Traf1, which is important in regulating cell fates. This latter may therefore shed light on the interplay between the Notch and NF-kB pathways reported in several human cancers and in particular in T-cell cancers.
The goal of my project was to discover how Notch can alter cell behaviours, by identifying genes that are regulated by Notch. The strategy built on a genome-wide analysis (expression microarray) of all the genes whose expression was altered within 30 minutes of activating the Notch receptor. The main objectives were: 1) To analyse gene-expression profiles from Notch activated cells; 2) To mine data from other studies to identify targets with the highest probability of contributing to the Notch response; 3) To validate these as targets by showing that their expression is regulated by Notch; 4) To determine the biological and developmental functions of the novel targets and how they contribute to the Notch response. By necessity, only a small number of genes can be fully analysed functionally and one major outcome of the first 3 packages was the prioritisation of genes for stage 4 analysis.
The first 2 milestones have been achieved, giving a list of potential Notch target genes in several cell lines. The overlap reveals a core response to Notch in Drosophila and the data mining led me to further focus on 3 genes : Polychaetoid (pyd, the orthologue of ZO-1 in mammals), dTraf-1 (the orthologue of Traf-4 in mammals) and CG12290 encoding a 7-pass trans-membrane receptor. All three genes are associated with binding sites for Su(H) (the transcription factor mediating Notch signal) that are conserved in all Drosophila species for which the genome is available. This is indicative of an evolutionary conservation over more than 60 million years, suggesting that these sites are functionally relevant. The importance of these sites was further tested by combining the fragments with a fluorescent indicator (firefly luciferase) and measuring the response to activated Notch. Based on such studies we have shown that dTraf-1 and CG12290 are indeed targets of Notch, while Pyd may not be. Nevertheless, the functional investigations involved all 3 genes, since previous studies have linked Pyd to the Notch pathway.
My further studies have shown important developmental functions for the 3 proteins coded by pyd, dTraf1 and CG12290 and have helped to discover more about the way that these proteins work. To do this I had to make mutations that interfere with the function of the genes, to generate transgenic lines producing different fragments of the proteins and to make antibodies that could reveal the distribution of the proteins within cells in vivo. All of these tools will be of value to the community and are already being shared with other groups in the EU.
As a consequence of these studies, I have demonstrated contributions of Pyd and Traf to the regulation of sensory organ development. This has revealed (1) that Pyd helps to regulate the amount of Notch available to signal at the membrane by linking it to an enzyme that regulates Notch trafficking within the cell; 2) that there is cross-talk between Notch and the Toll / NF-kB pathway (that mediates immunity and cell stress responses) via Traf1, which is important in regulating cell fates. This latter may therefore shed light on the interplay between the Notch and NF-kB pathways reported in several human cancers and in particular in T-cell cancers.