Exploiting Natural Genetic and Organismic Variation to Identify the DNA Motifs Regulating Transcription

了解非编码DNA是如何调节基因表达的，对于解决人类基因组中的无数问题至关重要 生物技术与人类健康。然而，事实证明，这一努力是一个重大挑战，部分原因是 基因组似乎编码了许多高度错综复杂的调控网络。特别是在脊椎动物中，调节 启动子或增强子等元件是高度多样化的，包含数十个DNA基序，间隔 插入的序列。传统的注重保护的序列分析策略对 随着调控DNA的快速进化，甚至经常重新定位，挑选出功能基序。 转录起始点(TSS)是基因调控的里程碑。精确的TSS使定义DNA基序成为可能 在功能上与转录有关。TSS还允许锚定和比较监管区域 或与直接序列保守无关的进化过程中的同源基因。虽然相距遥远 相关生物通常缺乏同源的调控DNA，具体到什么程度还有待探索 序列基序被选择性地保守以驱动基因的表达。因此，我研制出了带帽的小RNA- SEQ(csRNA-seq)，它准确地映射了稳定的(蛋白质编码和非编码的RNA)和 不稳定的转录本(增强子RNA，不同的转录本)揭示基因组范围内活跃的调控元件。 Csrna-seq只需要总rna作为起始材料，因此可以在几乎任何真核生物中进行tsss分析。 可以从中提取核糖核酸的有机体。真核生物，从单细胞原生动物到人类，在有机体上有所不同， 遗传和监管的复杂性。我假设这种多样性的频谱与TSS图谱相结合 (csRNA-seq)，可以用来发现关键的DNA基序，并随后发现调节 横跨欧亚大陆的基因表达。类似于考古学家在史前遗址的工作，测绘 沿着生命之树的TSS‘挖掘’祖先的、不那么复杂的基因调控状态。这些见解 也应该有助于更好地解释人类基因组。为了探索这一中心假设，我试图 1)实施csRNA-seq分析和可视化工具，并促进比较分析 跨物种的注释调控特征，2)确定介导转录启动的转铁蛋白结合位点 在Eukarya，3)追踪Tf结合位点的进化和使用以及它们在调控中的空间组织 同源基因或基因组的元件。在准备过程中，我已经生成了42个尤卡里亚人的数据 跨越了20亿年的转录组进化，并加入了一个特殊的生物信息学小组，该小组 也提供了一个独特的培训机会，对我成功过渡到独立至关重要。 这一提议如果成功，将揭示介导转录的主要DNA基序，并显著扩大我们的 真核基因调控的机制理解。此外，它还将提供一种新的方法来 捕获新生的tsss(csRNA-seq)，这是一个便于分析的免费软件套件，是一个用于轻松数据的数据门户 访问和浏览，向更大的科学界提供独特的数据集。

Understanding how non-coding DNA regulates gene expression is critical to addressing myriad problems in biotechnology and human health. This endeavor, however, has proven a major challenge, in part, as the genome appears to encode many highly convoluted regulatory networks. Particularly in vertebrates, regulatory elements such as promoters or enhancers are highly diverse and contain of dozens of DNA motifs spaced by intervening sequences. Traditional sequence analysis strategies that focus on conservation are ineffective to single out the functional motifs as regulatory DNA evolves rapidly and often even relocates. The transcription start site (TSS) is a landmark of gene regulation. Accurate TSS enables to define DNA motifs functionally associated with transcription. TSSs further allow anchoring and comparing the regulatory regions or orthologous genes across evolution, independent of direct sequence conservation. Although distantly related organisms typically lack homologous regulatory DNA, it remains to be explored to what extent specific sequence motifs are selectively conserved to drive expression gene. I therefore developed capped small RNA- seq (csRNA-seq), which accurately maps the TSS of both stable (protein coding and non-coding RNAs) and unstable transcripts (enhancer RNAs, divergent transcripts) to reveal active regulatory elements genome-wide. csRNA-seq only requires total RNA as starting material, thus enabling TSSs profiling in virtually any eukaryotic organism from which RNA can be extracted. Eukarya, from unicellular protists to humans, vary in organismic, genetic and regulatory complexity. I hypothesize that this spectrum in diversity, combined with TSS mapping (csRNA-seq), can be exploited to uncover the key DNA motifs and subsequently TF networks that regulate gene expression across the Eukarya. Analogous to the work of an archeologist at prehistoric sites, mapping TSSs along the tree of life ‘excavates’ ancestral, less convoluted states of gene regulation. These insights should also be instrumental to better interpret the human genome. To explore this central hypothesis, I seek to 1) implement tools for the analysis and visualization of csRNA-seq and facilitate the comparative analysis of annotated regulatory features across species, 2) identify the TF binding sites mediating transcription initiation across Eukarya, 3) trace the evolution and usage of TF binding sites and their spatial organization in regulatory elements of orthologous genes or sets of genes. In preparation, I have generated data for 42 Eukarya spanning over 2 billion years of transcriptome evolution and joined an exceptional bioinformatics group, which also provides a unique an opportunity for training critical for my successful transition to independence. This proposal, if successful, will reveal the major DNA motifs mediating transcription and markedly expand our mechanistic understanding of eukaryotic gene regulation. Furthermore, it will provide a novel method to capture nascent TSSs (csRNA-seq), a free software suite to facilitate analysis, a data portal for easy data access and browsing, and unique dataset to the greater scientific community.