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

项目摘要 在发育过程中，一系列的细胞外信号通过诱导细胞分化，导致不同细胞类型的形成。 许多基因的差异表达。尽管所有基因都经历相同的信号，但某些基因的表达 有些基因增加了，有些基因减少了，有些基因保持不变。因此，基因表达谱， 任何细胞类型的特征必须由以不同方式解释信号的每个基因组基因座产生。 基因组基因座的忠实信号解释的丧失在许多情况下是致病的，包括癌症和癌症。 神经精神疾病。因此，了解单个基因座如何解释细胞外信号 是一个根本性的尚未解决的问题。理解顺式调节的挑战在于 细胞外信号是双重的。首先，对信号的任何操纵（浓度、持续时间、身份）导致 无数的反式多效性效应混淆了解释。二是多个顺式调控元件 CREs（CREs）在大的基因组窗口中一起工作，以指定其靶基因的表达。即使在 在这个后CRISPR时代，同时操纵多个克雷斯仍然具有挑战性。 大的基因组区域去卷积它们对靶基因调控的相对贡献。该提案寻求 通过结合合成生物学和单细胞测序来解决这些挑战。 在HoxA簇，细胞外信号如视黄酸（RA）和Wnt诱导HoxA簇的建立。 不同的转录、表观遗传和拓扑结构域通过细胞分裂稳定遗传。在 目标1，我们将重新连接HoxA簇，以响应与细胞外信号完全正交的细胞外信号。 其余的基因组。这将使得能够独立地操纵转录因子顺式结合， 跨监管环境的变化，以确定其在建立和 维持HoxA对分化信号的反应。 我们不太可能从单个基因座的研究中收集到普遍适用的基因调控原则。在 目标2：我们将开发一种技术，使用单细胞作为单独的实验，以大规模增加 在任何位点都可以发现克雷斯之间相互作用的规模。这项技术将应用于 剖析参与神经元细胞类型特化的基因的调控景观。然后，我们将使用 大数据集，以开发在其他基因座的顺式调节的预测模型。

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.