Identifying functional enhancer mutations in the evolution of primate brain development

基因调控序列中的遗传变异被认为有助于人类疾病和独特的人类表型的进化。然而，我们缺乏预测基因组中哪些变异会影响调控功能或确定其影响程度的方法。目前的模型是基于描述性数据的功能基因调控元件，如进化保守，因此遭受低解释力，从而潜在的高假阳性率，由于缺乏直接的实验evidence.Here我提出了一项研究，以量化增强子序列内的突变的影响，并开发一个预测模型，这样的突变。使用大规模平行报告分析（MPRA），我将量化人类特异性取代增强子的影响，我们以前确定为显示进化增益的活动在发展中的人类新皮层。在一个平行的方法中，我将从人类和黑猩猩的诱导多能干细胞中获得神经干细胞，作为神经发育的模型。我将通过分析这些细胞系中的H3K27ac组蛋白标记和染色质可及性来进行基因调控生化活性的全基因组比较，并将观察到的差异与人类特异性增强子取代联系起来。然后，我将使用这两条经验结果来开发一个预测模型，以全局识别改变增强子活性的变体。我将进一步使用大范围的全基因组参数，如序列内容，转录因子结合位点的预测，进化保守性，和生化标记来预测增强子取代的功能效应。这种双管齐下的方法使用的数据，从监管化验和生化比较将使我能够确定取代，改变监管活动都通过序列变化和通过差异染色质活性。最后，我将验证该模型通过调查变异与大的影响预测的模型在人类和黑猩猩的神经干细胞系交叉比较MPRA和生化比较，并通过分析基因表达的差异物种之间。该项目将提供具体的见解到人类大脑发育的进化和一般的见解到进化的调节功能，通过增强子序列的变化。我的分析提供的直接实验证据将使我们能够更好地预测影响调节功能的遗传变异。一种预测基因调控元件中取代效应的有效方法可能很容易改变我们理解遗传变异如何影响调控功能的方式，从而改变生物学。

Genetic variants in gene regulatory sequences are thought to contribute both to human disease and the evolution of uniquely human phenotypes. However, we lack the means to predict which variants in the genome will affect regulatory function, or to determine the extent of their effects. Current models are based on descriptive data of the function of gene regulatory elements such as evolutionary conservation, and thus suffer from low explanatory power and thus potentially high false positive rates due to a lack of direct experimental evidence.Here I propose a study to quantify the effects of mutations within enhancer sequences and to develop a predictive model of such mutations. Using a massively parallel reporter assay (MPRA), I will quantify the effects of human-specific substitutions in enhancers we previously identified as showing evolutionary gains in activity in the developing human neocortex. In a parallel approach, I will derive neural stem cells, which serve as a model for neurodevelopment, from induced pluripotent stem cells of humans and chimpanzees. I will conduct genome-wide comparisons of gene regulatory biochemical activity by analysing H3K27ac histone marks and chromatin accessibility in these cell lines and connect observed differences with human-specific enhancer substitutions. I will then use these two lines of empirical results to develop a predictive model to globally identify variants that alter enhancer activity. I will further use a large range of genome-wide parameters such as sequence content, transcription factor binding site predictions, evolutionary conservation, and biochemical markers to predict functional effects of enhancer substitutions. This two-pronged approach using data from a regulatory assay and from biochemical comparisons will enable me to identify substitutions that alter regulatory activity both through sequence change and through differential chromatin activity. Finally, I will validate the model by investigating variants with large effects as predicted by the model in human and chimpanzee neural stem cell lines by cross-comparing MPRA and biochemical comparisons and by analysing gene expression differences between species.This project will provide both specific insights into the evolution of human brain development and general insights into the evolution of regulatory function through enhancer sequence change. The direct experimental evidence my analyses will provide will enable us to better predict genetic variants that influence regulatory function. A powerful way to predict the effects of substitutions in gene regulatory elements may easily transform the way we understand how genetic variation affects regulatory function and thus biology.