Roles of allele-specific chromatin interactions in transcription regulation during development

项目总结/摘要 发育生物学的一个基本挑战是剖析一个多能细胞如何分化成一个 特定细胞类型。大多数研究仅限于测量转录水平（RNA-1）的一维基因组数据。 seq）、蛋白结合强度（ChIP-seq）和染色质可及性（ATAC-seq）。这些数据集缺乏直接的 调节基因调节的各种调节元件之间的通信的证据， 分化为了解决这个问题，我们将利用尖端的3D基因组技术，ChIA-PET和 ChIA-Drop。通过富集特异性蛋白因子CCCTC结合因子（CTCF）和RNA聚合酶II （RNAPII），人们可以询问聚集体细胞中的染色质结构和基因调控（ChIA-PET）。 和在单个分子中（ChIA-Drop）。我们将利用F1杂交小鼠品系中高度不相似的基因组 通过将实验室小鼠和野生小鼠交配以将高通量测序读数分配给 亲本起源，从而解开等位基因特异性基因表达和染色质相互作用。我们提出 目的：（i）确定是否存在调节元件和甲基化状态之间的等位基因特异性相互作用， 小鼠胚胎干细胞（mESC）驱动等位基因特异性基因表达，（ii）量化细胞间异质性 多重染色质相互作用。随后，我们将mESCs分为三个谱系特异性 前体外胚层、中胚层和内胚层。通过进行ChIA-PET，我们可以识别哪些，如果有的话， 增强子、启动子和CTCF之间预先建立的相互作用在此过程后持续或消失。 ChIA-Drop数据将潜在地捕获其中的动态。在K99和R 00阶段，我们将 继续开发计算算法，可以：（i）定量评估重复的再现性 实验，（ii）识别统计上显著的差异相互作用，以及（iii）跟踪和量化单个 等位基因特异性多重相互作用的分子动力学和异质性。为了实现这些目标， 研究员将扩展她知识领域到发育生物学，并接受额外的实践 3D基因组图谱技术和小鼠胚胎干细胞培养，收获， 分化技术。总之，这些调控元件之间的基因组范围的通信联系， 建筑蛋白质将提供深入了解基因调控和基因组印记机制， 原肠胚形成

PROJECT SUMMARY / ABSTRACT A fundamental challenge in developmental biology is to dissect how one multipotent cell differentiates into a specific cell type. Most studies are limited to 1-dimensional genomic data that measure transcription level (RNA- seq), protein binding intensity (ChIP-seq), and chromatin accessibility (ATAC-seq). These datasets lack direct evidence of communication between various regulatory elements that accommodate gene regulation and differentiation. To solve this problem, we will leverage cutting-edge 3D genome technologies, ChIA-PET and ChIA-Drop. By enriching for specific protein factors CCCTC binding factor (CTCF) and RNA Polymerase II (RNAPII), one can interrogate chromatin architecture and gene regulation in aggregated bulk cells (ChIA-PET) and in a single molecule (ChIA-Drop). We will exploit the highly dissimilar genomes in F1 hybrid mouse strains derived from mating a laboratory mouse and a wild mouse to assign high-throughput sequencing reads to parental origin, thereby unraveling the allele-specific gene expression and chromatin interactions. We propose to: (i) determine whether allele-specific interactions between regulatory elements and methylation status in mouse embryonic stem cells (mESCs) drive allele-specific gene expression, (ii) quantify cell-to-cell heterogeneity of multiplex chromatin interactions. We will subsequently differentiate mESCs into three lineage-specific precursors ectoderm, mesoderm, and endoderm in vitro. By performing ChIA-PET, we can identify which, if any, of the pre-established interactions among enhancers, promoters, and CTCF persist or vanish after this process. ChIA-Drop data will potentially capture the dynamics therein. Throughout the K99 and R00 phases, we will continue to develop computational algorithms that can: (i) quantitatively assess reproducibility of replicate experiments, (ii) identify statistically significant differential interactions, and (iii) trace and quantify single- molecule dynamics and heterogeneity of allele-specific multiplex interactions. To succeed in these aims, the investigator will expand her knowledge domain to developmental biology and receive additional hands-on experimental training in 3D genome mapping technologies and mouse embryonic stem cell culture, harvest, and differentiation techniques. Together, these genome-wide communication links between regulatory elements and architectural protein will provide insights into gene regulation and genomic imprinting mechanisms during gastrulation.