Investigating Transcriptional and Post-transcriptional Regulation of dE2F1 Expression during Drosophila Development

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Coordinating cellular proliferation and cell death is important for development of multi-cellular organisms. E2F family proteins are evolutionarily conserved transcription factors that regulate expression of genes that are crucial for both processes. Altering E2F activity during development of various organisms, ranging from nematodes to mammals, has been shown to affect the total number of cells in a tissue. The Drosophila genome encodes two E2F family genes, de2f1 and de2f2. Among them, de2f1 is the only E2F family gene whose function is clearly associated with cellular proliferation and survival. One important factor that determines the function of dE2F1 during development is its expression level and pattern. Interestingly, de2f1 can be expressed from four different transcription start sites, producing four transcripts that only differ in their 5’UTR sequences. Although the existence of the four transcripts have been known for many years, virtually nothing is known about their biological significance. Recently, we have discovered that dE2F1 protein expression is post-transcriptionally regulated by the TSC/Rheb/Tor pathway, and that the 5’UTR sequences of de2f1 is important in this process. Furthermore, we have found that specific de2f1 transcripts differentially contribute to dE2F1 protein expression during development. These observations raise the possibility that various developmental signals regulate dE2F1 expression by coordinating transcription and translation of the four different de2f1 transcripts. In this proposal, I will describe our experimental plans to systematically determine the expression pattern of each de2f1 transcript and their contribution to dE2F1 protein expression during development. Specific Aims: 1) Investigation of the expression level and pattern of specific de2f1 transcripts during development. Previous studies on de2f1 expression mainly relied on methods that cannot distinguish specific de2f1 transcripts. As a consequence, virtually nothing is known about expression pattern of each transcript. Therefore, as a first step to investigate specific de2f1 transcripts, we will determine the expression levels and patterns of all four de2f1 transcripts at different stages of development. We will also define promoter elements that are responsible for their expression. 2) Generation of transcript-specific de2f1 mutants to investigate the biological role of each transcript. We have discovered that de2f1 alleles with insertions at different locations of the promoter affect de2f1 expression in a transcript-specific manner. By taking advantage of these mutant alleles, we will investigate the contribution of each transcript to the dE2F1 expression during development. In addition, we will also investigate the developmental defects that are associated with these mutants to elucidate the biological function of each de2f1 transcript. 3) Identification of the de2f1 transcript and 5’UTR sequences that are important for the effect of TSC inactivation on dE2F1 expression. We have evidence to suggest that the TSC/Rheb/Tor pathway post-transcriptionally regulates dE2F1 expression through their 5’UTR sequences. This is particularly interesting since the only difference between the four de2f1 transcripts is in their 5’UTR sequences. I will describe our experimental plans to elucidate the function of 5’UTR sequences on dE2F1 expression and how they mediate the signal downstream of the TSC/Rheb/Tor pathway.