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: 9681934
• 负责人: Puja Agrawal
• 金额: $ 4.95万
• 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/9681934
• 关键词: 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; Dimensions; Disease; Distal; Elements; Enhancers; Environment; Fellowship; Foundations; Future; Gene Expression; Genes; Genetic Transcription; Genome; Half-Life; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Heterogeneity; Inner Cell Mass; Maintenance; Measures; Mediating; Messenger RNA; Modeling; 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 cells; Sum; Tissues; Training; Transcript; Transcriptional Activation; Variant; base; blastocyst; cell growth regulation; chromosome conformation capture; embryonic stem cell; experimental study; genome sequencing; mRNA Expression; personalized medicine; pluripotency; prevent; programs; promoter; self-renewal; stem cell differentiation; 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，多能性的退出和进入多能性