Genome-Wide Analysis of the Transcriptional Cooperation Between Runx2 And Runx3 During Skeletal Development

骨骼发育过程中 Runx2 和 Runx3 之间转录合作的全基因组分析

• 批准号: 9812042
• 负责人: THOMAS LUFKIN
• 金额: $ 45.9万
• 依托单位: CLARKSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9812042
• 关键词: Address; Award; Biomedical Research; Bone Development; Cells; Chondrocytes; Communities; Data; Development; Dissection; Embryo; Embryonic Development; Environment; Exposure to; Fetal Skeleton; Fluorescence-Activated Cell Sorting; Gene Dosage; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Genes; Genetic Transcription; Genomics; Goals; Grant; Human; In Situ Hybridization; Institution; Knock-in; Knock-out; Knockout Mice; Limb structure; Location; Manuals; Masks; Molecular; Molecular Genetics; Mus; Organ; Osteoblasts; Physiologic Ossification; Population; RNA; RUNX3 gene; Regulation; Reporting; Research; Research Project Grants; Resources; Role; Skeletal Development; Time; Transcript; United States National Institutes of Health; body system; genetic analysis; genetic resource; genome-wide; genome-wide analysis; hands on research; mouse model; mutant; osteoblast differentiation; promoter; skeletal dysplasia; skeletogenesis; transcription factor; transcriptome; transcriptome sequencing; undergraduate student

The goal of the R15 AREA award is to support small-scale research projects at educational institutions that provide baccalaureate or advanced degrees but have not been major recipients of NIH support. The R15 award specifically wants to expose undergraduate students to the basic concepts required to understand biomedical research and conduct research on the molecular and cellular basis of organ systems including among others, the fetal skeleton. We believe that the project and location we are proposing here is a perfect match for the goals of the R15 AREA award. The mammalian runt-related transcription factor genes (Runx1, Runx2 and Runx3) have long been implicated as regulators of bone development with Runx2 and Runx3 being more prominent in skeletogenesis. Runx2 was identified as the earliest master regulator of osteoblast differentiation in both intramembranous and endochondral ossification through genetic analyses of human skeletal dysplasias and studies of genetically modified mouse models. The mammalian runt-related transcription factor genes, Runx2 and Runx3, are recognized to have cooperative roles in skeletogenesis. We will investigate the molecular mechanisms underlying this cooperative regulation of chondrocyte maturation and osteoblast development by generating mouse lines expressing EGFP under the control of either the Runx2 or Runx3 endogenous regulatory sequences for gene expression (transcriptome) studies utilizing cells specifically enriched for Runx2- or Runx3- expressing cells obtained by fluorescence-activated cell sorting (FACS). In addition, we will address the gene dosage effects of knocking out Runx2 and Runx3 at the molecular level by performing gene expression analysis on fluorescing cells of mice resulting from haplo-sufficient Runx2+/EGFP and Runx3+/EGFP crosses. To circumvent the issue of using an impure population of cells for RNA-Seq studies, we are establishing mouse lines expressing EGFP under the control of either the Runx2 or Runx3 promoter. Our goal is to elucidate the gene regulation mechanisms of Runx2 and Runx3 by utilizing RNA-Seq transcriptome data on cells specifically enriched for Runx2- or Runx3-expression obtained by FACS during the early stages of embryogenesis when these genes are first active. Our comprehensive genome-wide transcriptional profiling of Runx2 and Runx3 will serve as a valuable genomic resource to progress our understanding of their interconnected governance of embryonic bone development, as well as providing much needed genetic resources to the research community.

R15地区奖的目标是支持教育机构的小规模研究项目 提供学士学位或高级学位，但不是NIH支持的主要接受者。 R15 奖项特别希望使本科生了解了解的基本概念 生物医学研究和进行有关器官系统分子和细胞基础的研究，包括 除其他外，胎儿骨骼。我们相信我们在这里提议的项目和位置是一个完美的 匹配R15区域奖的目标。 长期以来，哺乳动物RUNT相关的转录因子基因（Runx1，Runx2和Runx3）长期涉及 作为骨骼发育的调节剂，runx2和runx3在骨骼生成中更为突出。 runx2 被确定为膜内和 通过人类骨骼发育不良的遗传分析和基因研究 修改的鼠标模型。哺乳动物RUNT相关的转录因子基因RUNX2和RUNX3是 公认在骨骼生成中具有合作作用。我们将研究分子机制 通过产生这种合作的软骨细胞成熟和成骨细胞开发的协同调节 在Runx2或Runx3内源调节的控制下表达EGFP的小鼠线 基因表达（转录组）研究的序列利用特异性富含Runx2-或Runx3-的细胞的序列 通过荧光激活的细胞分选（FACS）获得的表达细胞。此外，我们将解决该基因 通过执行基因表达在分子水平上敲除Runx2和Runx3的剂量效应 对单倍体充足的Runx2+/EGFP和Runx3+/EGFP交叉产生的小鼠荧光细胞的分析。 为了避免使用不纯细胞群进行RNA-seq研究的问题，我们正在建立 在Runx2或Runx3启动子的控制下表达EGFP的鼠标线。我们的目标是 通过利用RNA-Seq转录组数据来阐明Runx2和Runx3的基因调节机制 特异性的细胞富集了FACS在 当这些基因首先活跃时，胚胎发生。我们全面的全基因组的转录分析 runx2和runx3将是一种有价值的基因组资源，以提高我们对他们的理解 胚胎骨发育的互连治理，并提供急需的遗传 研究社区的资源。