REVERSE GENOMICS OF REGULATORY ELEMENTS GOVERNING SPLICING

描述（由申请人提供）：我们个体基因组的差异产生了大部分人类多样性。人类基因组计划已经过去了十年，基因组如何指导表型仍然是一个谜。像ENCODE这样的联盟试图通过使用全基因组数据集将功能输出与序列相关联来识别人类和其他模式生物中的功能DNA元件。然而，这些研究并不一定能提高我们解释DNA元件在新环境下或突变时如何发挥作用的能力。这种理解对于预测序列改变对表型的影响以及为未来的医学或技术目的设计生物学将是至关重要的。DNA元件的组合充当控制特定功能如转录、剪接、定位和沉默的密码。破译这些代码是困难的，因为有限的自然变体集合通常不足以控制诸如序列组成或元素组合之类的变量。要证明特定的序列对基因表达有因果作用，需要仔细控制反向遗传学研究。在全基因组范围内进行这样的实验是困难的，因为我们无法（1）快速改变单个遗传元件的序列和背景，（2）量化数千个这样的变化的后果。我的中心愿景是破译控制基因表达的顺式调控代码，通过将反向遗传学实验扩展到基因组规模，使用定义的合成DNA文库的多重测量。我将建立在我的工作开发下一代基因合成技术和多路复用的报告分析，系统地确定如何通过做成千上万的控制实验测试同时进行哺乳动物基因表达序列的一致行动。在这里，我们将应用这些技术发展来研究遗传调控元件如何控制前mRNA剪接的过程。控制剪接的主要序列元件，即剪接供体、受体和分支位点，不能传递足够的信息来单独指定外显子包含或排除。已知其他调控元件，如外显子或内含子剪接增强子和抑制子，影响复杂密码子中的剪接，所述复杂密码子可基于组织或细胞类型而变化。我们将利用这里提出的新技术发展来系统地询问和改进剪接代码。研究剪接将有助于集中开发一套完整的工具和技术，这将使我们能够攻击其他形式的控制基因调控的顺式元件。

DESCRIPTION (provided by applicant): Differences in our individual genomes give rise to most of human diversity. A decade removed from the Human Genome Project, much of how the genome directs phenotype still remains a mystery. Consortiums like ENCODE seek to identify functional DNA elements in humans and other model organisms by correlating functional outputs with sequence using genome-wide data sets. However, these studies do not necessarily improve our ability to interpret how DNA elements act in new contexts or when mutated. Such an understanding will be critical to predict the effects of sequence alterations on phenotype and to engineer biology for future medicinal or technological purposes. Combinations of DNA elements act as codes controlling particular functions like transcription, splicing, localization, and silencing. Deciphering these codes is difficult, as the limited set of natural variants is typically insufficient to control for variables such as sequence composition or element combinations. Proving that particular sequences have causative effects on gene expression requires carefully controlled reverse genetic studies. Conducting such experiments on genome-wide scales is difficult because of our inability to (1) rapidly alter the sequence and context of individual genetic elements and (2) quantify the consequences of thousands of such changes. My central vision is to decipher cis-regulatory codes controlling gene expression by scaling reverse genetics experiments to genomic scales using multiplexed measurements of defined synthetic DNA libraries. I will build upon my work developing next-generation gene synthesis technologies and multiplexed reporter assays to systematically determine how sequences governing mammalian gene expression act in concert by doing thousands of controlled experimental tests simultaneously. Here, we will apply these technological developments to study how genetic regulatory elements control the process of pre-mRNA splicing. The major sequence elements controlling splicing, namely the splice donor, acceptors, and branch sites, do not convey enough information to specify exon inclusion or exclusion alone. Other regulatory elements, such as exonic or intronic splicing enhancers and suppressors, are known to affect splicing in a complex code that can vary based on tissue or cell type. We will systematically interrogate and refine the splicing code by leveraging the new technological developments proposed here. Studying splicing will help focus development of a complete suite of tools and technologies, which will later let us attack other forms of cis-elements controlling gene regulation.