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对人类疾病和人类独特表型的进化都有贡献。然而，我们缺乏方法来预测基因组中的哪些变异会影响调控功能，或者确定它们的影响程度。目前的模型是基于基因调控元件（如进化保护）功能的描述性数据，因此由于缺乏直接的实验证据，解释力较低，因此潜在的高假阳性率。在这里，我提出了一项研究来量化增强子序列中突变的影响，并开发这种突变的预测模型。使用大规模平行报告基因试验（MPRA），我将量化人类特异性替代增强子的影响，我们之前确定的增强子显示了人类新皮层发育中活性的进化增益。在平行的方法中，我将从人类和黑猩猩的诱导多能干细胞中提取神经干细胞，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替换联系起来。然后，我将使用这两条经验结果线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、转录因子结合位点预测、进化守恒和生化标记来预测增强子替换的功能效应。这种双管齐下的方法使用来自调控分析和生化比较的数据，将使我能够通过序列变化和差异染色质活性来识别改变调控活性的替代。最后，我将通过交叉比较MPRA和生化比较以及分析物种之间的基因表达差异，通过研究模型在人类和黑猩猩神经干细胞系中预测的具有重大影响的变异来验证模型。该项目将为人类大脑发育的进化提供具体的见解，并通过增强子序列的变化为调节功能的进化提供一般的见解。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基因变异。一种预测基因调控元件替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和生物学的方式。

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.