DISSECTING NEURAL CELL FATE SPECIFICATION USING TRANSPOSON CALLING CARDS

描述（由申请人提供）：控制生物体发育的转录网络是精确的、高度协调的和复杂的。该提案旨在了解同一个转录因子如何在生物体发育过程中指定多个不同的细胞命运。具体而言，我们感兴趣的转录因子Olig2，它可以促进运动神经元和少突胶质细胞的命运。我们假设Olig2能够通过与其他DNA结合蛋白的相互作用来执行其多种功能，从而使其在不同的细胞环境中结合不同的靶标。使用目前用于分析转录因子的方法来测试这一假设是困难的，因为它们不能在整个细胞谱系中追踪结合，使得不可能将祖细胞中的DNA结合事件与其后代的最终细胞命运相关联。我们建议使用一种新的方法，转座子“名片”，记录Olig2结合在神经分化。该方法需要将转座子的转座酶与转录因子融合，从而使其引导转座子DNA插入其结合的基因组附近。转座子变成了一张“名片”，永久地标记了转录因子对基因组中该位置的访问。 通过回收这些电话卡沿着它们侧翼的一些基因组DNA，然后确定它们的DNA序列，有可能绘制转录因子的全基因组结合历史。我们建议应用Calling Card方法来理解Olig2如何执行其独特的功能。由于许多重要的转录因子在发育过程中执行不止一种功能，因此我们从这项工作中获得的见解应该是广泛适用的。我们的具体目标是：1）通过神经干细胞分化追踪Olig2结合，以了解它如何促进两种不同的细胞命运，2）确认差异结合的靶基因促进运动神经元或少突胶质细胞命运，3）使用Calling Card技术分析活体斑马鱼中Olig2和Ngn2的结合。这些目标是可行的：我们已经在酵母和哺乳动物细胞中成功地实施了Calling Card方法，并且我们的初步结果证明了在斑马鱼中有转录因子指导的Calling Card插入。我们相信，进一步发展这项技术并将其应用于了解细胞命运特化过程的回报将是巨大的。

DESCRIPTION (provided by applicant): Transcriptional networks that control the development of organisms are precise, highly coordinated, and complex. This proposal seeks to understand how the same transcription factor can specify multiple distinct cell fates during the development of an organism. Specifically, we are interested in the transcription factor Olig2, which can promote both a motoneuron and an oligodendrocyte cell fate. We hypothesize that Olig2 is able to perform its multiple functions through interactions with other DNA binding proteins causing it to bind different targets in different cellular contexts. Testing this hypothesis using current methods for the analysis of transcription factors is difficult because they cannot trace binding throughout a cell lineage, making it impossible to correlate DNA-binding events in progenitor cells to the final cell fates of their progeny. We propose to use a novel method, transposon "Calling Cards", to record Olig2 binding during neural differentiation. The method entails fusing the transposase of a transposon to a transcription factor, thereby causing it to direct the insertion of transposon DNA into the genome near where it binds. The transposon becomes a "Calling Card" that permanently marks the transcription factor's visit to that place in the genome. By recovering these Calling Cards along with some of the genomic DNA that flanks them and then determining their DNA sequences, it is possible to map the genome-wide binding history of the transcription factor. We propose to apply the Calling Card method to understand how Olig2 carries out its distinct functions. Since many important transcription factors perform more than one function during development, the insights that we gain from this work should be broadly applicable. Our specific aims are 1) to trace Olig2 binding through neural stem cell differentiation to understand how it promotes two distinct cell fates, 2) to confirm that differentially bound target genes promote motoneuron or oligodendrocyte cell fates, 3) to analyze Olig2 and Ngn2 binding in living zebrafish using Calling Card technology. These aims are feasible: we have successfully implemented the Calling Card method in both yeast and mammalian cells, and our preliminary results demonstrate transcription factor directed insertion of Calling Cards in zebrafish. We are confident that the rewards of further developing this technology and applying it to understand the process of cell fate specification will be substantial