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

等位基因特异性染色质相互作用在发育过程转录调控中的作用

基本信息

批准号：
10343821
负责人：
Minji Kim
金额：
$ 12.96万
依托单位：
JACKSON LABORATORY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-05 至 2022-12-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10343821
关键词：
3-Dimensional ATAC-seq Algorithms Alleles Architecture Bar Codes Behavior Binding Binding Proteins Brain CCCTC-binding factor Cell Culture Techniques Cell Differentiation process Cell Nucleus Cells ChIP-seq Chromatin Chromatin Interaction Analysis by Paired-End Tag Sequencing Chromatin Loop Chromosomes Communication Complex Computational algorithm Crossbreeding Data Data Analyses Data Set Defect Development Developmental Biology Dimensions Diploidy Disease Dominant Genes Downstream Enhancer Drops Ectoderm Embryo Endoderm Enhancers Epigenetic Process Event Experimental Designs Future Gene Expression Gene Expression Regulation Genes Genetic Genetic Transcription Genome Genome Mappings Genomic Imprinting Graph Harvest Health Heterogeneity High-Throughput Nucleotide Sequencing Human Hybrids In Vitro Inbred Strain Individual Knowledge Laboratory mice Link Liver Mammals Measures Mediating Mentors Mesoderm Methods Methylation Microfluidics Molecular Mouse Strains Mus Mutate Nature Organism Partner in relationship Pattern Phase Population Problem Solving Process Proteins Public Health RNA Polymerase II Regulatory Element Reproducibility Research Personnel Resolution Role Schizophrenia Skin System Techniques Technology Testing Therapeutic Intervention Time Tissues Training Transcriptional Regulation Variant Walking Work autism spectrum disorder autosome base cell type daughter cell developmental disease embryonic stem cell experimental study gastrulation genome-wide genomic data graph theory high dimensionality human genome sequencing imprint improved insertion/deletion mutation insight mouse model multipotent cell prevent programs promoter signal processing single molecule stem cells three-dimensional modeling tissue repair transcriptome sequencing two-dimensional

项目摘要

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.

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如何区分特定的细胞类型。大多数研究仅限于测量转录水平的一维基因组数据（RNA- 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适应基因调节和分化。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。通过丰富特定蛋白质因子CCCTC结合因子（CTCF）和RNA聚合酶II （RNAPII），可以询问聚集的大量细胞中的染色质结构和基因调节（CHIA-PET）和单个分子（chia-drop）。我们将利用F1混合小鼠菌株中高度不同的基因组源自交配实验室鼠标和野生鼠标以分配高通量测序的读数父母来源，从而揭示了等位基因特异性基因表达和染色质相互作用。我们建议至：（i）确定调节元件和甲基化状态之间的等位基因特异性相互作用是否在小鼠胚胎干细胞（MESC）驱动等位基因特异性基因表达，（ii）量化细胞到细胞的异质性多重染色质相互作用。随后，我们将MESC分为三个谱系特异性前体外胚层，中胚层和内胚层体外。通过执行chia-pet，我们可以确定哪个（如果有）在此过程之后，增强子，启动子和CTCF之间的预先建立的相互作用持续或消失。 CHIA-DROP数据可能会捕获其中的动力学。在整个K99和R00阶段，我们将继续开发可以：（i）定量评估复制的可重复性的计算算法实验，（ii）确定具有统计学上显着的差异相互作用，（iii）跟踪并量化单次差异相互作用等位基因特异性多重相互作用的分子动力学和异质性。为了取得成功，研究人员将将她的知识领域扩展到发育生物学，并获得额外的动手 3D基因组映射技术和小鼠胚胎干细胞培养的实验训练，收获和分化技术。共同，这些整个基因组的沟通联系在监管元素和建筑蛋白将提供有关基因调节和基因组印迹机制的见解胃。