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领域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，但并不是NIH支持的主要对象。R15 该奖项特别希望让本科生接触到理解所需的基本概念 生物医学研究和器官系统的分子和细胞基础研究，包括 其中包括胎儿的骨骼。我们相信，我们在这里提出的项目和地点是一个完美的 与R15区域奖的目标相匹配。 哺乳动物矮小相关转录因子基因(RUNX1、Runx2和Runx3)长期以来一直被认为与 作为骨发育的调节因子，Runx2和Runx3在骨形成中的作用更为突出。运行2 被认为是最早的成骨细胞分化的主要调节因子，在膜内和 骨质疏松症软骨内骨化的遗传学分析及遗传学研究 修改后的小鼠模型。哺乳动物矮小相关转录因子基因Runx2和Runx3是 公认在骨骼形成中具有协同作用。我们将研究其分子机制。 在软骨细胞成熟和成骨细胞发育的协同调控中， Runx2和Runx3内源调控下表达绿色荧光蛋白的小鼠系 利用专为Runx2或Runx3-富含的细胞进行基因表达(转录组)研究的序列 通过荧光激活细胞分选(FACS)获得表达细胞。此外，我们还将解决基因问题 基因表达在分子水平敲除Runx2和Runx3的剂量效应 单倍体充足的Runx2+/EGFP和Runx3+/EGFP杂交小鼠的荧光细胞分析。 为了避免使用不纯的细胞群体进行RNA-Seq研究的问题，我们正在建立 在Runx2或Runx3启动子控制下表达绿色荧光蛋白的小鼠株系。我们的目标是 利用RNA-Seq转录组数据阐明Runx2和Runx3的基因调控机制 流式细胞术早期特异性富含Runx2或Runx3表达的细胞 当这些基因第一次活跃时，胚胎发生。我们全面的全基因组转录图谱 Runx2和Runx3将作为一个宝贵的基因组资源来促进我们对它们的理解 相互关联的胚胎骨骼发育管理，以及提供急需的基因 向研究界提供资源。