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。这一建议旨在了解在生物体发育过程中，相同的转录因子如何能够指定多种不同的细胞命运。具体来说，我们对转录因子Olig2感兴趣，它可以促进运动神经元和少突胶质细胞的细胞命运。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。使用目前的转录因子分析方法来验证这一假设是困难的，因为它们不能在整个细胞谱系中追踪结合，这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联系起来。我们建议使用一种新颖的方法，转座子“名片”，来记录神经分化过程中的Olig2结合。该方法需要将转座子的转座酶融合到转录因子上，从而使其直接将转座子DNA插入到它结合的基因组附近。转座子变成了一张“名片”，永久地标志着转录因子访问了基因组中的那个位置。

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