Developing a multi-species platform for understanding mammalian gene control

转录因子（TF）是与DNA结合并调节基因表达的蛋白质。与蛋白质编码基因突变相比，相对较少的功能性调控突变已被表征。 该提案的总体目标是识别和确定进化上保守的和物种特异性的DNA区域的功能，这些区域与TF相互作用并调节特定细胞类型中的基因。 最近的技术进步允许TF结合DNA序列的全基因组实验分离已经揭示，在任何给定的细胞类型中，存在比基因更多的TF结合DNA片段。因此，这就产生了对新方法的巨大需求，以过滤数十万TF-DNA相互作用，以影响基因功能的最重要的相互作用。 比较物种之间的DNA是一种长期以来确定基因功能的方法。我们最近的发现，比较TF-DNA相互作用的肝脏组织从5种脊椎动物，揭示了保守的TF-DNA相互作用显着丰富的肝脏TF网络和重要的保守的肝脏途径，如凝血因子的生产和脂质代谢。然而，仍然非常需要开发一个真正的多物种平台，它可以捕获正常的生物过程，但仍然适合实验操作。在这项计划中，我们将首先建立一个新的多物种TF-DNA相互作用图谱，从几个哺乳动物物种，包括人，狗，小鼠，大鼠和牛的内皮细胞分离。然后，我们将测试我们的预测，保守的调控区域是重要的功能，通过执行有针对性的调控DNA缺失。总的来说，这项工作将使我们能够在哺乳动物基因控制的进化方面取得新的发现。

Transcription factors (TF) are proteins that bind to DNA and regulate the expression of genes. Compared to protein-coding gene mutations, relatively few functional regulatory mutations have been characterized. The overarching goal of this proposal is to identify and determine the function of evolutionarily conserved and species-specific DNA regions that interact with TFs and regulate genes in specific cell types. Recent technological advances allowing genome-wide experimental isolation of TF-bound DNA sequences have revealed that in any given cell type, there are more TF bound DNA segments than there are genes. Consequentially, this has created a great need for new ways to filter hundreds of thousands of TF-DNA interactions down to the most essential ones that impact gene function. Comparing DNA between species is a long-established way to identify gene function. Our recent discoveries, comparing TF-DNA interactions in liver tissue from 5 vertebrate species, revealed that conserved TF-DNA interactions significantly enriched for liver TF networks and important conserved liver pathways such as blood clotting factor production and lipid metabolism. There remains, however, a great need to develop a truly multi-species platform that captures normal biological processes, yet is still amenable to experimental manipulation. In this proposal, we will first create a new multi-species TF-DNA interaction atlas in endothelial cells isolated from several mammalian species, including human, dog, mouse, rat and cow. We will then test our prediction that conserved regulatory regions are the important functional ones by performing targeted regulatory DNA deletions. Overall, this work will allow us to make new discoveries regarding the evolution of mammalian gene control.