Identifying enhancers with human-specific developmental functions

描述（由申请人提供）：作为一个物种，我们与其他灵长类动物的区别在于我们的语言能力，我们直立行走的能力，以及我们发明和使用复杂工具的天赋。这些特征起源于身体适应，例如大脑尺寸的增加和四肢形态的变化，这需要发育的变化。虽然长期以来人们一直认为基因调控的变化推动了人类独特性状的进化，但人类特异性发育调控功能的体内证据仍然难以捉摸。此外，尽管在注释基因组中的功能元件方面进行了大量的努力，例如ENCODE项目，但还没有类似的努力来注释独特的人类顺式调节功能。本研究的目的是鉴定和表征具有人类特异性功能的发育增强子，并研究它们在人类进化中的作用。我们建议的目的是基于广泛的初步数据。我们已经开发了一种新的计算方法来识别非编码的，潜在的调节序列，这些序列在脊椎动物物种中高度保守，但在人类进化过程中发生了重大变化。使用小鼠转基因增强子测定，我们已经确定了8个这些“人类加速”保守的非编码序列（HACNS），在开发过程中作为增强子的功能。与本提案的目标特别相关的是，我们还表明，我们的数据集中进化最快的元素，HACNS 1，是一个发育增强子，相对于黑猩猩和恒河猴的orthopathic元素，它获得了一个强大的肢体表达结构域。该区域包括假定的前腕和近端拇指。在这里，我们将建立在这些以前的研究，以产生更复杂的地图，人类特异性序列加速在非编码元件，包括转录因子结合位点数据和ChIP-seq数据产生的全基因组的努力，注释调控功能。我们将使用这些数据结合计算和实验过滤器，如极端进化约束和招聘的p300，预测增强子和小鼠转基因技术，以确定和表征增强子与人类特异性活动。我们将使用合成增强子和生物化学方法来确定人类特异性序列变化如何改变增强子功能。最后，我们将使用反向遗传策略来研究人类特异性功能变化在HACNS 1中的进化相关性，通过使用基因靶向来用人类增强子替换小鼠HACNS 1的直系同源物，并评估这种遗传变化对小鼠发育的影响。这些研究的结果将补充和扩展目前对人类基因组进行功能注释的努力，并将开始揭示产生现代人类的精确分子进化事件。

DESCRIPTION (provided by applicant): As a species, we are distinguished from other primates by our capacity for language, our ability to walk upright, and our talent for inventing and using sophisticated tools. These traits originated in physical adaptations, such as increased brain size and changes in the morphology of the limbs, which required changes in development. Although it has long been thought that changes in gene regulation drove the evolution of uniquely human traits, in vivo evidence for human-specific developmental regulatory functions remains elusive. Moreover, despite intensive efforts to annotate functional elements in the genome, such as the ENCODE project, there is no parallel effort to annotate uniquely human cis-regulatory functions. The goal of this study is to identify and characterize developmental enhancers with human-specific functions in vivo and examine their role in human evolution. The aims of our proposal are based on extensive preliminary data. We have developed a novel computational approach to identify noncoding, potentially regulatory sequences that are highly conserved across vertebrate species, but that changed substantially during human evolution. Using a mouse transgenic enhancer assay, we have identified eight of these "human-accelerated" conserved noncoding sequences (HACNSs) that function as enhancers during development. Of particular relevance for the aims of this proposal, we have also shown that the most rapidly evolving element in our dataset, HACNS1, is a developmental enhancer that has gained a robust limb expression domain relative to the orthologous elements from chimpanzee and rhesus macaque. This domain includes the presumptive anterior wrist and proximal thumb. Here we will build on these previous studies to generate more sophisticated maps of human-specific sequence acceleration in noncoding elements, including transcription factor binding site data and ChIP-seq data generated by genome-wide efforts to annotate regulatory function. We will use these data in conjunction with computational and experimental filters, such as extreme evolutionary constraint and recruitment of p300, to predict enhancers and mouse transgenic technologies to identify and characterize enhancers with human-specific activities. We will use synthetic enhancers and biochemical approaches to determine how human-specific sequence change alters enhancer function. Finally, we will use a reverse genetic strategy to study the evolutionary relevance of the human-specific functional change in HACNS1, by using gene targeting to replace the mouse ortholog of HACNS1 with the human enhancer and evaluating the effect of this genetic change on mouse development. The results from these studies will complement and extend current efforts to functionally annotate the human genome and will begin to reveal the precise molecular evolutionary events that produced modern humans.