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

• 批准号: 10761821
• 负责人: Minji Kim
• 金额: $ 24.59万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-14 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10761821
• 关键词: 3-Dimensional; Algorithms; Alleles; Architecture; Bar Codes; Behavior; Binding; Binding Proteins; Brain; CCCTC-binding factor; Cell Culture Techniques; Cell Differentiation process; Cell Nucleus; Cells; 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; Encapsulated; 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; Maps; Measures; Mediating; Mentors; Mesoderm; Methods; Methylation; Microfluidics; Molecular; Mouse Strains; Mus; Mutate; Nature; Organism; Partner in relationship; Pattern; Phase; Population; 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; Visualization; Walking; Work; autism spectrum disorder; autosome; 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

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。