Epigenetic landscapes and regulatory divergence of human craniofacial traits

描述(由申请人提供)：在发育过程中，脑神经脊细胞(CNCC)在建立颅面形态和确定其物种特异性变异方面发挥着重要作用。为了了解人类的独特特征，除了研究模式生物中的cNCCs外，还必须研究人的cNCCs及其衍生物。由于人类NC的形成发生在妊娠3-6周，遗传学研究基本上无法获得，因此我们在培养皿中建立了与颅面发育高度相关的基于人类多能干细胞的CNCC分化模型。此外，我们将我们的模型扩展到黑猩猩cNCC，使我们能够识别区分人类cNCC和我们最近的进化近亲的cNCC的分子特征。首先，我们建议表征人类和黑猩猩cNCC的表观遗传景观和转录本，并识别这种独特的细胞类型所利用的保守的和物种特有的顺式调控元件。由于除我们的报告外，任何生物体的NCC的染色质修改图谱尚未公开获得，我们将从一组人类和黑猩猩迁徙后的cNCC中生成染色质标记图谱，并辅之以转录组分析。因此，我们将创建“参考表观基因组”，它将被注释为活跃和稳定的增强子和启动子。我们已经确定了2000多个调控元件，这些元件在人类和黑猩猩的染色质标记中显示出强烈的物种特异性偏见，认为人类特有的CNCC分子特征确实存在，并可能是人类特有的头面部差异的基础。这些数据集将为未来研究人类颅面进化、发育和疾病的转录和表观遗传学基础提供丰富的资源。其次，我们将在体内分析候选的人类特异性头面部增强剂的活性。为此，将使用小鼠胚胎中的转基因报告程序分析在人类cNCC中获得或失去活性增强子签名的50个人类调控元件的活性，并与50个同源黑猩猩区域进行比较。因此，我们将产生一套经过验证的人类特异性颅面增强剂，可以在机制研究中进一步探索。对于10个人类特有的增强子，表现出活性的获得或丧失，有趣的活动模式，或与人类颅面发育或疾病相关的增强基因，我们将利用BAC重组工程进一步开发方正转基因系，并将其分发到颅面社区。

DESCRIPTION (provided by applicant): During development, cranial Neural Crest Cells (cNCCs) play major roles in establishing craniofacial morphology and determining its species-specific variation. To understand human distinctive features it is imperative to study human cNCCs and their derivatives in addition to cNCCs from model organisms. Since human NC formation occurs at 3 to 6 weeks of gestation and is largely inaccessible to genetic studies, we have established a human pluripotent stem cell-based cNCC differentiation model in the dish with high relevance to craniofacial development. Moreover, we have extended our model to chimpanzee cNCCs, allowing us to identify molecular features that distinguish human cNCCs from those of our closest evolutionary cousins. First, we propose to characterize epigenetic landscapes and transcriptomes of human and chimpanzee cNCCs and to identify conserved and species-specific cis-regulatory elements utilized by this unique cell type. Since chromatin modification maps from NCCs of any organism are not yet publicly available beyond our report, we will generate chromatin marking profiles from a cohort of human and chimp post- migratory cNCCs, complemented with transcriptome analyses. Thus, we will create "reference epigenomes" that will be annotated for active and poised enhancers and promoters. We have already identified over 2000 regulatory elements that show strong species-specific bias in their chromatin marks in human versus chimpanzee, arguing that human-specific cNCC molecular features do exist and may underlie human-specific craniofacial divergence. These datasets will provide a rich resource for future investigations of the transcriptional and epigenetic basis of human craniofacial evolution, development, and disease. Second, we will analyze candidate human-specific craniofacial enhancer activity in vivo. To this end, transgenic reporter assays in mouse embryos will be used to analyze the activity of 50 human regulatory elements that either gained or lost active enhancer signature in human cNCCs, as compared to the 50 orthologous chimpanzee regions. Thus, we will generate a validated set of human-specific craniofacial enhancers that can be further explored in mechanistic studies. For 10 selected human-specific enhancers exhibiting gain or loss of activity, interesting activity patterns, or relevant associatin with craniofacial development or disease in humans, we will utilize BAC recombineering to further develop founder transgenic lines that will be distributed to the craniofacial community.