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

协调细胞增殖和细胞死亡对多细胞生物体的发育具有重要意义。E2F家族蛋白是进化上保守的转录因子，调节对这两个过程都至关重要的基因的表达。在从线虫到哺乳动物的各种生物的发育过程中，改变E2F的活性已经被证明会影响组织中的细胞总数。果蝇基因组编码两个E2F家族基因de2f1和de2f2。其中，de2f1是唯一一个功能与细胞增殖和存活明显相关的E2F家族基因。决定dE2F1在发育过程中功能的一个重要因素是它的表达水平和模式。有趣的是，de2f1可以从四个不同的转录起始点表达，产生四个转录本，只是它们的5‘UTR序列不同。虽然这四个转录本的存在已经知道很多年了，但实际上人们对它们的生物学意义一无所知。最近，我们发现dE2F1蛋白的表达受TSC/Rheb/Tor途径的转录后调控，而de2f1的5‘UTR序列在这一过程中起着重要的作用。此外，我们还发现，在发育过程中，特定的de2f1转录本对dE2F1蛋白的表达有不同的贡献。这些观察结果表明，不同的发育信号可能通过协调四种不同的de2f1转录本的转录和翻译来调节dE2F1的表达。在这个提案中，我将描述我们的实验计划，以系统地确定每个de2f1转录本的表达模式，以及它们在发育过程中对dE2F1蛋白表达的贡献。具体目的：1)研究特定de2f1转录本在发育过程中的表达水平和模式。以往对de2f1表达的研究主要依赖于无法区分特定de2f1转录本的方法。结果，几乎对每个转录本的表达模式一无所知。因此，作为研究特定de2f1转录本的第一步，我们将确定所有四个de2f1转录本在不同发育阶段的表达水平和模式。我们还将定义负责其表达的启动子元件。2)生成转录特异的de2f1突变体，以研究每个转录的生物学作用。我们发现，在启动子不同位置插入的de2f1等位基因以转录特异性的方式影响de2f1的表达。通过利用这些突变的等位基因，我们将调查每个转录本在发育过程中对dE2F1表达的贡献。此外，我们还将研究与这些突变体相关的发育缺陷，以阐明每个de2f1转录本的生物学功能。3)鉴定对TSC失活对dE2F1表达的影响至关重要的de2f1转录本和5‘UTR序列。我们有证据表明，TSC/Rheb/Tor途径通过其5’UTR序列在转录后调控dE2F1的表达。这一点特别有趣，因为四个de2f1转录本之间的唯一差异是它们的5‘UTR序列。我将描述我们的实验计划，以阐明5‘UTR序列对dE2F1表达的功能，以及它们如何介导TSC/Rheb/Tor途径下游的信号。

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.