Nuclear Architecture-driven Cellular Heterogeneity in Stem Cell and Hematopoietic Differentiation

干细胞和造血分化中核结构驱动的细胞异质性

• 批准号: 10242691
• 负责人: Puja Agrawal
• 金额: $ 5.1万
• 依托单位: VERSITI WISCONSIN, INC.
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-05 至 2022-09-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242691
• 关键词: 3-Dimensional; Adult; Architecture; Automobile Driving; Binding; Binding Sites; Biological Models; Bone Marrow; CCCTC-binding factor; CRISPR/Cas technology; Cell Differentiation process; Cell Lineage; Cells; Cellular biology; Chromatin; Chromatin Loop; Clinical; DNA; Data; Development; Disease; Distal; Elements; Enhancers; Environment; Fellowship; Foundations; Future; Gene Expression; Gene Expression Regulation; Genetic Transcription; Genome; Half-Life; Hematopoiesis; Hematopoietic; Heterogeneity; Inner Cell Mass; Maintenance; Measures; Mediating; Messenger RNA; Mus; Myeloid Cells; Neighborhoods; Nuclear; Physicians; Play; Positioning Attribute; Process; Proteins; RNA Splicing; Regulation; Regulatory Element; Research; Residencies; Role; Scientific Advances and Accomplishments; Scientist; Site; Stem cell pluripotency; Sum; Tissues; Training; Transcript; Transcriptional Activation; Variant; base; blastocyst; cell growth regulation; chromosome conformation capture; embryonic stem cell; experimental study; genome sequencing; hematopoietic differentiation; hematopoietic stem cell differentiation; mRNA Expression; personalized medicine; pluripotency; prevent; programs; promoter; self-renewal; stem cell differentiation; stem cell model; stem cells; symposium; transcription factor; whole genome

Differentiation is the consequence of two steps, an exit from pluripotency and an entrance into a specific lineage. Cells are known to be primed for differentiation based on cell-to-cell variation in gene expression, or cellular heterogeneity, although the mechanism remains unknown. Fundamentally, gene expression is regulated by nuclear architecture, defined as the three dimensional, temporally-regulated architecture of chromatin. This study focuses on the role of nuclear architecture in setting the foundation for cellular heterogeneity to permit the exit of a stem cell from pluripotency and entrance into hematopoiesis. Two key contributors to nuclear architecture are enhancers and CCCTC-binding factor (CTCF) binding sites. Enhancers are cis-regulatory elements that play a critical role in regulating lineage-specific transcription. CTCF binds sites throughout the genome and regulates gene expression by permitting or preventing enhancer- promoter interactions. Neither regulatory element has been studied as a modulator of cellular heterogeneity. This study will show that the nuclear architecture of a cell is vital to directing stem cell differentiation into hematopoietic lineages through regulation of cellular heterogeneity. This proposal focuses on how stem cells exit pluripotency through changes in Nanog expression (Aim 1), and successively enter hematopoietic lineages by observing changes in the hematopoietic-essential HoxA cluster (Aim 2). For Aim 1, the role of nuclear architecture in heterogeneity will be examined by single-cell RT- qPCR through the CRISPR/Cas9 mediated deletion in murine embryonic stem cells (ESCs) of 1) the three Nanog-associated enhancers and 2) the CTCF sites bounding Nanog. For Aim 2, CTCF sites within the HoxA cluster will be deleted in ESCs. These cells will then be differentiated to hematopoietic lineages and changes in heterogeneity evaluated using single-cell RT-qPCR. In both aims, changes in enhancer-promoter interactions will be investigated using Chromosome Conformation Capture (3C). In sum, although cellular heterogeneity is critical to stem cell differentiation and lineage-specification, its mechanism remains unknown. This study will establish the paradigm that nuclear architecture modulations are responsible for cellular heterogeneity and driving differentiation. Better understanding of the regulation of stem cell differentiation and hematopoiesis will further our ability to understand how these processes are perturbed in disease and development. Importantly, as the role of personalized medicine and stem cells as therapies grows in the clinical arena, the understanding of the nuances of stem cell differentiation and biology this fellowship will provide the applicant is a rigorous training ground for her future as an independent physician- scientist. The guidance in the vibrant and intellectual environment, as well as the clinical shadowing, conference attendance and coursework described in this fellowship, will position the applicant to be a competitive candidate for research-based residency programs.

分化是两个步骤的结果，退出多能性和进入多能性。 特殊的血统。已知细胞基于基因的细胞间变异而被引发分化。 表达或细胞异质性，尽管机制仍然未知。基本上，基因 表达受核结构的调节，核结构被定义为三维的、时间调节的 染色质的结构这项研究的重点是核结构的作用，在奠定基础， 细胞异质性以允许干细胞从多能性退出并进入造血。两 核结构的关键贡献者是增强子和CCCTC结合因子（CTCF）结合位点。 增强子是在调节谱系特异性转录中起关键作用的顺式调节元件。CTCF 结合整个基因组的位点，并通过允许或阻止增强子- 启动子相互作用这两种调控元件都没有被研究为细胞异质性的调节剂。 这项研究将表明，细胞的核结构对于指导干细胞分化为 造血谱系通过调节细胞异质性。 这项提案的重点是干细胞如何通过Nanog表达的变化退出多能性（Aim 1），并通过观察造血必需的HoxA的变化， 集群（目标2）。对于目标1，核结构在异质性中的作用将通过单细胞RT-PCR检测。 通过CRISPR/Cas9介导的鼠胚胎干细胞（ESC）中的缺失的qPCR：1）三种 Nanog相关的增强子和2）结合Nanog的CTCF位点。对于目标2，HoxA内的CTCF位点 将在ESC中删除群集。然后这些细胞将分化为造血谱系并发生变化。 使用单细胞RT-qPCR评价异质性。在这两个目标中，增强子-启动子相互作用的变化 将使用染色体构象捕获（3C）进行研究。 总之，尽管细胞异质性对干细胞分化和谱系特化至关重要，但其 机制仍然未知。本研究将建立核结构调控的范式， 负责细胞异质性和驱动分化。更好地理解茎的调节 细胞分化和造血将进一步提高我们理解这些过程是如何受到干扰的能力 疾病和发展。重要的是，个性化医疗和干细胞作为治疗方法的作用 在临床竞技场上，对干细胞分化和生物学的细微差别的理解， 奖学金将为申请人提供一个严格的培训基地，为她的未来作为一个独立的医生- 科学家在充满活力和智力环境的指导，以及临床阴影， 会议出席和课程描述在这个奖学金，将定位申请人是一个 有竞争力的候选人为研究为基础的住院医师计划。