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，以100万美元的速度构建大型合成DNA（1 Mb规模）， 我们将以极大的效率和精确度，提出一个新的基因“系统遗传学”范式， 在一个独特的主发育调节剂的背景下，HoxA集群的调控。我们用酵母 数百种哺乳动物基因位点的变异迅速，具有高精度和coseffective。利用这一 技术，我们已经组装和交付134 kb和170 kb长的结构，含有整个大鼠 HoxA簇，以及小鼠对应物。我们可以提供精确设计的大型HoxA结构 转移到Hprt 1基因座的异位位置，并致力于转移到等位基因位置。使用 这些强大的新工具，我们描述了我们如何可以提供大鼠和小鼠HoxA集群Hprt 1在小鼠 ES细胞。使用异源大鼠HoxA簇使我们能够在内部比较来自两个组织的Hox基因座。 使用一系列染色质“基于组学”的读数，在同一细胞中检测不同种类的染色质。HoxA星系团 在DNA水平上高度保守;我们将利用大鼠HoxA基因，因为它是啮齿动物基因， 补体功能，但密集地覆盖着基因组变体（平均每10 bp一个 跨越HoxA）。这些实验将在一个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产生的HoxA+/+、+/-或-/- ES的背景下进行 细胞目的包括提出这样一个问题：Hox簇是否需要它们的基因组背景包含长- 距离调控元件的建立染色质边界和初始基因表达？（目标 1），尝试在HoA-/-敲除背景下与我们的异位合成基因座进行功能互补（Aim 2)并问是否有可能在Hox簇内建立新的调控域？(Aim 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).