DISSECTING NEURAL CELL FATE SPECIFICATION USING TRANSPOSON CALLING CARDS

描述(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。这项提议试图理解在生物体的发育过程中，同一转录因子如何指定多个不同的细胞命运。具体地说，我们对转录因子Orig2感兴趣，它可以促进运动神经元和少突胶质细胞的命运。我们假设Orig2能够通过与其他DNA结合蛋白相互作用来发挥其多种功能，从而使其在不同的细胞环境中结合不同的靶点。使用目前分析转录因子的方法来验证这一假设是困难的，因为它们无法追踪整个细胞谱系的结合，从而无法将祖细胞中的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的方法，转座子“呼叫卡”，来记录神经分化过程中的寡核苷酸结合。该方法需要将转座子的转座酶与转录因子融合，从而使其直接将转座子DNA插入到靠近其结合的基因组中。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。 通过恢复这些呼叫卡以及它们两侧的一些基因组DNA，然后确定它们的DNA序列，就有可能绘制出转录因子的全基因组结合历史。我们建议应用呼叫卡的方法来理解Otl2是如何执行其不同的功能的。由于许多重要的转录因子在发育过程中发挥多种功能，我们从这项工作中获得的见解应该具有广泛的适用性。我们的具体目标是：1)通过神经干细胞的分化来追踪寡核苷酸结合，以了解它如何促进两种不同的细胞命运；2)证实差异结合的靶基因促进运动神经元或少突胶质细胞的命运；3)利用电话卡技术分析活斑马鱼中的寡核苷酸和Ngn2结合。这些目标是可行的：我们已经成功地在酵母和哺乳动物细胞中实现了呼叫卡方法，我们的初步结果证明了转录因子指导的呼叫卡在斑马鱼中的插入。我们相信，进一步开发这项技术并将其应用于理解细胞命运指定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