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

项目摘要/摘要 发育生物学的一个基本挑战是剖析一个多能细胞是如何分化成 特定单元格类型。大多数研究仅限于测量转录水平的一维基因组数据。 SEQ)、蛋白质结合强度(CHIP-SEQ)和染色质可及性(ATAC-SEQ)。这些数据集缺乏直接的 各种调节元件之间沟通的证据，这些元件适应基因调节和 差异化。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 家滴。通过丰富特定的蛋白质因子CCCTC结合因子(CTCF)和RNA聚合酶II (RNAPII)，人们可以询问聚集的块状细胞中的染色质结构和基因调控(CHIA-PET)。 而且是在一个分子中(chia-drop)。我们将利用F1杂交小鼠品系中高度不同的基因组 来自实验室小鼠和野生小鼠的交配，为其分配高通量测序读数 亲本来源，从而解开等位基因特异的基因表达和染色质相互作用。我们建议 以：(I)确定调控元件和甲基化状态之间的等位基因特异性相互作用是否 小鼠胚胎干细胞(MESCs)驱动等位基因特异的表达，(Ii)量化细胞间的异质性 多重染色质相互作用。我们将随后将mESCs区分为三个特定的谱系 外胚层、中胚层和内胚层的前体在体外。通过进行CHIA-PET，我们可以识别出，如果有的话， 在这一过程之后，促进剂、启动子和CTCF之间预先建立的相互作用中的许多仍然存在或消失。 Chia-Drop数据可能会捕捉到其中的动态。在整个K99和R00阶段，我们将 继续开发能够：(I)定量评估复制的可重复性的计算算法 实验，(Ii)确定具有统计学意义的差异交互作用，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， 研究人员将把她的知识领域扩展到发育生物学，并获得更多的实践经验 3D基因组图谱技术和小鼠胚胎干细胞培养、收获和 差异化技术。总之，这些调控元件和基因组之间的全基因组通讯联系 建筑蛋白将提供对基因调控和基因组印迹机制的见解 原肠形成。

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.