Identifying enhancers with human-specific developmental functions

描述(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。这些特征源于身体适应，例如大脑尺寸的增加和肢体形态的变化，这需要在发育过程中发生变化。尽管长期以来，人们一直认为基因调控的变化推动了人类独特特征的进化，但人类特有的发育调控功能的体内证据仍然难以捉摸。此外，尽管对基因组中的功能元件进行了密集的注释，如ENCODE项目，但没有类似的努力来注释独特的人类顺式调节功能。这项研究的目的是确定和表征体内具有人类特有功能的发育增强剂，并研究它们在人类进化中的作用。我们提案的目的是基于大量的初步数据。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实质性的变化。利用小鼠转基因增强子试验，我们已经鉴定了8个在发育过程中作为增强子的“人类加速的”保守非编码序列(HACNs)。与该提案的目的特别相关的是，我们还表明，我们数据集中进化最快的元件HACNS1是一个发育增强子，相对于黑猩猩和恒河猴的同源元件，它获得了一个强大的肢体表达结构域。这一领域包括假定的前腕和近侧拇指。在这里，我们将在这些先前研究的基础上，生成更复杂的非编码元件中人类特异性序列加速图，包括转录因子结合位点数据和基因组范围内注释调节功能所产生的芯片序列数据。我们将使用这些数据结合计算和实验过滤器，例如极端进化限制和p300的招募，以预测增强子和小鼠转基因技术，以识别和表征具有人类特有活动的增强子。我们将使用合成增强剂和生化方法来确定人类特有的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HACNS1中人类特有的功能变化的进化相关性，方法是使用基因打靶将HACNS1的小鼠同源基因替换为人类增强子，并评估这种基因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的准确的分子进化事件。 公共卫生相关性：了解人类形态和发育的遗传基础是美国国立卫生研究院公共卫生使命的核心。除了人类起源的基本问题，研究人类进化史与人类健康直接相关，因为许多常见的人类疾病可能部分是由于序列变化的影响，这些变化在我们进化的早期是有利的，但现在是有害的。这一假说的证据来自人类和黑猩猩的比较：许多带有基因成分的普遍人类疾病，包括皮肤癌、心脏病、疟疾感染、阿尔茨海默病、多发性硬化症和重大精神病，在黑猩猩中似乎不常见、不同或不存在。因此，我们的研究可能会揭示人类特有疾病的遗传和进化基础。

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. PUBLIC HEALTH RELEVANCE: Understanding the genetic basis of human morphology and development is central to the public health mission of the NIH. Beyond the fundamental question of human origins, studying human evolutionary history is directly relevant to human health, as many common human diseases may in part be due to the effects of sequence changes that were advantageous early in our evolution but are now harmful. Evidence for this hypothesis stems from human and chimpanzee comparisons: many prevalent human diseases with a genetic component, including skin cancers, heart disease, malaria infection, Alzheimer disease, multiple sclerosis and major psychoses, appear to be uncommon, different or absent in chimpanzees. Our research may thus shed light on the genetic and evolutionary basis of diseases that are unique to our species.