ABSTRACT: Cellular responses to signalling pathways are often highly dynamic, however most analyses of developmental signalling pathways focus on a single endpoint. Here we have analyzed the temporal changes in transcription following a short Notch activation treatment and have related these to the recruitment of the Notch pathway transcription factor, CSL [Suppressor of Hairless, Su(H), in Drosophila], and to the state of RNA Polymerase II (Pol II) binding. A total of 154 genes showed significant differential expression over time and their expression profiles stratified into 14 clusters based on temporal and quantitative differences in their responses. These differences were partially reflected in the profiles of Pol II and Su(H) binding. However neither could fully account for the different response profiles. Furthermore, the timing of the different responses was unaffected by more prolonged Notch activation. Instead our data suggest that regulatory relationships between genes that segregate into different response clusters can partially account for the stratification. Thus feed-forward repression, where products of early responding Enhancer of split bHLH genes (E(spl)bHLH) inhibit expression of endogenous repressors, is one mechanism that explains the profile of genes that exhibit delayed up-regulation. E(spl)bHLH genes may therefore be responsible for co-ordinating the Notch response of a wide spectrum of other targets, explaining their critical functions in many developmental and disease contexts. DmD8 cells were cultured in Schneiders medium (Invitrogen) supplemented with 10% FBS (Sigma), 5ug/ml insulin (Sigma) and 5% penicillin / streptomycin (Sigma) according to standard protocols. Notch signalling was initiated by replacing cell media with 2mM EDTA in PBS. The cells were stimulated for 5 minutes before washing out EDTA using normal culture media. The cells were then collected at 18 time points at 0M-bM-^@M-^Y, 5M-bM-^@M-^Y, 10M-bM-^@M-^Y, 15M-bM-^@M-^Y, 20M-bM-^@M-^Y, 25M-bM-^@M-^Y, 30M-bM-^@M-^Y, 35M-bM-^@M-^Y, 40M-bM-^@M-^Y, 50M-bM-^@M-^Y, 60M-bM-^@M-^Y, 70M-bM-^@M-^Y, 80M-bM-^@M-^Y, 90M-bM-^@M-^Y, 100M-bM-^@M-^Y, 110M-bM-^@M-^Y, 120M-bM-^@M-^Y and 150M-bM-^@M-^Y after stimulation in 4 independent time trials. For control samples, for each time trial, media was replaced with fresh media to mimic the addition and removal of EDTA in corresponding experimental samples and then collected at equivalent times. For each trial 50 M-BM-5g of the untreated control RNA sample of each time point was pooled to create a trial specific reference sample. Samples from trials #1 and #3 were labelled with Cy5 and trails #2 and #4 as dye-swap with Cy3.
