Testis-specific activation of gene expression

多细胞动物的每个细胞表达（转录）所有可用基因的一个子集。有些基因在所有细胞中转录，有些在细胞类型的子集中转录，而有些只在单一细胞类型中转录。细胞中正确的基因表达，在发育和分化过程中开启和关闭基因，对于正常的细胞功能、正常的生物体发育和终身健康至关重要。每个细胞的基因表达库是由细胞的身份决定的，并在发育和细胞分化过程中建立。精子是非常特殊的细胞，能够执行几种独特的生物功能，包括使卵子受精。在人类和大多数其他动物中，基因组中所有基因的10-20%只在睾丸中转录，在注定要分化成精子的细胞中转录，这种表达对精子的形成和功能至关重要，因此对正常生殖至关重要。令人惊讶的是，人们对睾丸特异性基因表达的调控因素知之甚少，尽管这对生育能力至关重要。在这个项目中，我们将使用果蝇，黑腹果蝇，来研究睾丸特异性表达基因的转录是如何被激活的。我们之前已经确定了一组蛋白质，它们作为一个复合体一起工作，在靶基因的调控区域与DNA结合，并激活它们的表达。这个复合体中的四种蛋白质包含我们预测直接与DNA结合的区域。我们知道，睾丸特异性基因的起始位点只需要一个非常短的DNA序列区域就能激活它们。然而，当比较受该复合体调控的1000多个基因时，这些短区域的实际DNA序列彼此之间几乎没有相似性。这个复合物是如何识别明显不同的DNA片段，与它们结合，并激活相邻的基因，而忽略了其他DNA位点的？我们将使用一组独立的实验方法，结合相互关联和综合的计算分析来回答这个问题。1)我们将确定我们复合物中每个DNA结合蛋白的DNA序列结合偏好。这将使我们能够看到这些序列在整个基因组中的位置——我们希望它们在睾丸表达基因的位置富集。2)我们将确定基因组中睾丸细胞中每种复杂蛋白的所有位点——我们希望它们在睾丸表达基因的位置富集。3)我们将制造突变版本的蛋白质，这些蛋白质仍然可以对复合体做出贡献，但不能再结合DNA，然后识别在睾丸细胞中发现这些突变蛋白质的基因组中的所有位点，以及这对基因表达的影响-我们预计某些位点的结合会丢失，与这些位点相关的基因将不再表达。4)这些实验的结果将使我们更清楚地了解决定睾丸特异性基因表达以响应我们的蛋白质复合物的生物学规则。为了确定我们是否真的理解了这些规则，我们将测试一组基因控制区域的突变版本的活动，我们预测应该或不应该激活基因表达。5)最后，新进化的基因通常只在睾丸中表达，它们的表达需要我们的调节蛋白复合物。这些基因是如何获得这种表达模式的？我们将通过将我们的复合物引导到基因组的不同区域并评估将其定位到特定区域是否足以激活附近DNA序列的表达来开始测试这一点。

Each cell in a multicellular animal expresses (transcribes) a subset of all the available genes. Some genes are transcribed in all cells, some in a subset of cell types, while some are transcribed only in a single cell type. Correct gene expression in cells, both turning on and turning off genes in development and differentiation, is essential for normal cellular function, for normal organism development, and for lifelong health. The gene expression repertoire of each individual cell is determined by the cell's identity, and is established during development and cellular differentiation. Sperm are extremely specialised cells, capable of performing several unique biological functions including fertilisation of the egg. In humans, and most other animals, 10-20% of all the genes in the genome are transcribed exclusively in testes, in cells destined to differentiate into sperm, and this expression is critical for the formation and function of sperm, and thus for normal reproduction. Surprisingly, very little is known about the regulatory elements that drive testis-specific gene expression, despite how critically important this is for fertility.In this project we will use the fruit fly, Drosophila melanogaster, to investigate how transcription of testis-specifically expressed genes is activated. We have previously identified a set of proteins that work together as a complex to bind to DNA at the regulatory regions of target genes and activate their expression. Four of the proteins within this complex contain regions that we predict bind directly to DNA. We know that only a very short region of DNA sequence at the start site of testis-specific genes is needed for them to be active. However, the actual DNA sequences of these short regions bear little similarity with one another, when comparing between the over 1000 genes regulated by this complex. How is it that this complex recognises apparently very different stretches of DNA, binds to them, and causes activation of the adjacent gene, while ignoring other DNA sites?We will answer this question using a set of independent experimental approaches coupled with interlinked and integrative computational analyses.1) We will determine the DNA sequence binding preference for each of the DNA-binding proteins in our complex. This will allow us to look at where these sequences are found in the whole genome - we expect them to be enriched at the positions of testis-expressed genes. 2) We will identify all the sites in the genome at which each of complex proteins are found in cells in the testis - we expect them to be enriched at the positions of testis-expressed genes. 3) We will make mutant versions of the proteins that can still contribute to the complex but that can no longer bind DNA, and then identify all the sites in the genome at which these mutant proteins are found in cells in the testis, and what effect this has on gene expression - we expect binding at some sites to be lost, and that the genes associated with these sites will no longer be expressed. 4) The results of these experiments will generate a clearer understanding of the biological rules that determine testis-specific gene expression in response to our protein complex. To determine whether we actually understand these rules we will test the activity of a set of mutant versions of gene control regions that we predict should, or should not, activate gene expression.5) Finally, newly evolving genes are often expressed exclusively in testes, and their expression requires our regulatory protein complex. How do these genes gain this expression pattern? We will begin to test this by directing our complex to different regions of the genome and evaluating whether targeting it to a particular region is sufficient to activate expression of nearby DNA sequences.