Dissecting the logic of mammalian gene regulation using synthetic biology and single-cell sequencing

项目总结 在发育过程中，一系列细胞外信号通过诱导不同类型的细胞形成 许多基因的差异表达。尽管所有基因都经历相同的信号，但一些基因的表达 基因增加了，一些减少了，而另一些保持不变。因此，基因表达谱就是 任何细胞类型的特征都必须源于每个基因组位点以不同的方式解释信号。 在包括癌症和癌症在内的许多情况下，基因组座位失去忠实的信号解释都是致病的。 神经精神疾病。因此，对单个基因组座位如何解释细胞外信号的理解 是一个根本性但尚未解决的问题。理解顺式监管以应对 细胞外信号是双重的。首先，对信号(注意力、持续时间、身份)的任何操纵都会导致 在令人困惑的解释中存在着无数的多效性效应。第二，多个顺式监管要素 (Cres)跨越大的基因组窗口共同工作，以指定其目标基因的表达。即使是在 在后CRISPR时代，同时操作多个CRE仍然具有挑战性 大的基因组区域去卷积它们对靶基因调控的相对贡献。这项提议旨在 利用合成生物学和单细胞测序相结合的方法来解决这些挑战。 在HoxA簇，细胞外信号如维甲酸(RA)和Wnt诱导建立 不同的转录、表观遗传和拓扑域，通过细胞分裂稳定遗传。在……里面 Aim1，我们将重新连接HoxA簇以响应与细胞外信号完全正交的 基因组的其余部分。这将使转录因子结合的独立操作在顺式和 对跨监管环境的变化，以确定它们在建立和 维持HoxA对分化信号的反应。 我们不太可能从对单个基因座的研究中收集到普遍适用的基因调控原则。在……里面 AIM2我们将开发一种使用单细胞作为单独实验的技术，以大幅增加 Cre之间的相互作用可以在任何位置被发现的规模。这项技术将应用于 剖析神经细胞类型规范中涉及的基因的调控格局。然后，我们将使用 建立其他基因座顺式调控的预测模型的大数据集。

PROJECT SUMMARY During development, a series of extracellular signals lead to the formation of different cell-types by inducing the differential expression of many genes. Although all genes experience the same signal, the expression of some genes is increased, some reduced, and others stay the same. Therefore, the gene expression profile that is characteristic of any cell-type must result from each genomic locus interpreting the signal in a distinct manner. The loss of faithful signal interpretation by genomic loci is pathogenic in many contexts including cancer and neuropsychiatric disease. Thus, an understanding of how individual genomic loci interpret extracellular signals is a fundamental yet unresolved problem. The challenge in understanding cis-regulation in response to extracellular signals is two-fold. First, any manipulation of the signal (concentration, duration, identity) leads to myriad pleiotropic effects in trans that confound interpretation. Second, multiple cis-regulatory elements (CREs) work together across large genomic windows to specify the expression of their target gene. Even in this post-CRISPR era, it has remained challenging to simultaneously manipulate multiple CREs across these large genomic regions to deconvolve their relative contributions to target gene regulation. This proposal seeks to solve these challenges using a combination of synthetic biology and single-cell sequencing. At the HoxA cluster, extracellular signals such as retinoic acid (RA) and Wnt induce the establishment of distinct transcriptional, epigenetic and topological domains that are stably inherited through cell divisions. In Aim1, we will rewire the HoxA cluster to respond to an extracellular signal that is completely orthogonal to the rest of the genome. This will enable the independent manipulation of transcription factor binding in cis and changes to the trans-regulatory environment to determine their relative contributions in establishing and maintaining the HoxA response to differentiating signals. We are unlikely to glean generally applicable principles of gene regulation from studies of a single locus. In Aim2 we will develop a technology that uses single-cells as individual experiments to massively increase the scale at which interactions between CREs can be uncovered at any locus. This technology will be applied to dissect the regulatory landscapes of genes involved in neuronal cell-type specification. We will then use the large dataset to develop a predictive model of cis-regulation at other loci.