Synthetic HoxA to dissect transcriptional regulatory logic

项目摘要 这项提议的“大图景”是，利用我们新发现的能力来构建大型合成DNA(1Mb规模) 我们将以极高的效率和精确度，提出一种新的基因系统遗传学范式 在一个独特的主要发育调节器--HoxA集群的背景下进行的监管。我们用酵母来制作 数百个哺乳动物基因座快速变异，具有高精度和低成本的特点。利用这一点 技术，我们已经组装并提供了134kb和170kb长的包含整个大鼠 Hoxa集群，以及与之对应的鼠标。我们可以提供精密设计的大型HoxA结构 到Hprt1基因座上的异位位置，并努力传递到等位基因位置。vbl.使用 这些强大的新工具，我们描述了如何将大鼠和小鼠HoxA簇传递给小鼠的Hprt1 ES细胞。使用异源大鼠HoxA簇使我们能够在内部比较两者的HOX基因座 同一细胞中的物种使用一系列染色质“基于组学”的读数。HoxA星系团非常 在DNA水平上高度保守；我们将利用老鼠的HoxA，因为它是一种啮齿动物基因，它很可能 小鼠的补体功能，但密集地覆盖着基因组变体(平均每10个碱基中有一个 横跨HoxA)。这些实验将在数据丰富的系统中使用MES细胞的体外分化进行 到运动神经元。这些实验可以在已经生成的HoxA+/+、+/-或-/-ES的环境中进行 细胞。其目的包括提出这样一个问题：HOX集群是否需要它们的基因组环境包含长的- 染色质边界和初始基因表达的距离调控元件？(目标 1)，试图在HoA/-基因敲除背景(AIM)中与我们的异位合成基因座进行功能互补 2)并询问是否有可能在HOX集群内建立新的监管结构域？(目标3)。

Project Summary The “big picture” of this proposal is that using our newfound ability to build big synthetic DNAs (1 Mb scale) at will and with great efficiency and precision, we propose a new paradigm for “systems genetics” of gene regulation in the context of a unique master developmental regulator, the HoxA cluster. We use yeast to make hundreds of mammalian gene loci variants rapidly, with high precision and cos-effective. Leveraging this technology, we have already assembled and deliver 134kb and 170 kb long constructs containing the entire rat HoxA cluster, as well as the mouse counterparts. We can deliver precision-engineered large HoxA constructs to either an ectopic location at the Hprt1 locus, and working on delivering to the allelic location as well. Using these powerful new tools, we describe how we can deliver the rat and mouse HoxA clusters to Hprt1 in mouse ES cells. Using the heterologous rat HoxA clusters allows us to internally compare the Hox loci from both species in the same cell using a range of chromatin “omics-based” readouts. The HoxA cluster is extremely highly conserved at the DNA level; we will take advantage of the rat HoxA, since it is a rodent gene it is likely to complement function in the mouse, but is densely carpeted with genomic variants (one per 10 bp on average across HoxA). These experiments will be done in a data-rich system using in vitro differentiation of mES cells to motor neurons. These experiments can be done in the context of already generated HoxA+/+, +/–, or –/– ES cells. The aims include asking the question Do Hox clusters require their genomic context containing long- distance regulatory elements for the establishment of chromatin boundaries and initial gene expression? (Aim 1), attempting functional complementation with our ectopic synthetic loci in HoA-/- knockout background (Aim 2) and asking Is it possible to establish novel regulatory domains within Hox clusters? (Aim 3).