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区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，但不是NIH支持的主要接受者。的R15 该奖项特别希望让本科生了解理解所需的基本概念 生物医学研究，并对器官系统的分子和细胞基础进行研究，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选择。 比赛的目标是R15区域奖。 哺乳动物的runt相关转录因子基因（Runx 1，Runx 2和Runx 3）长期以来一直被认为是 作为骨发育的调节因子，Runx 2和Runx 3在骨骼发生中更为突出。Runx2 被鉴定为成骨细胞分化的最早的主调节因子， 通过人类骨骼发育不良的遗传分析和遗传学研究， 改良的小鼠模型。哺乳动物侏儒相关转录因子基因Runx 2和Runx 3， 被认为在骨骼发生中具有协同作用。我们将研究 在软骨细胞成熟和成骨细胞发育的协同调节下， 在Runx 2或Runx 3内源性调节基因的控制下表达EGFP的小鼠系 利用特异性富集Runx 2-或Runx 3-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）获得的表达细胞。此外，我们将解决基因 通过进行基因表达在分子水平敲除Runx 2和Runx 3的剂量效应 分析由单倍足够的Runx 2 +/EGFP和Runx 3 +/EGFP杂交产生的小鼠荧光细胞。 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Runx 2或Runx 3启动子控制下表达EGFP的小鼠系。我们的目标是 利用RNA-Seq转录组数据阐明Runx 2和Runx 3的基因调控机制， 通过流式细胞术在早期阶段获得的特异性富集Runx 2-或Runx 3-表达的细胞， 当这些基因第一次被激活时，胚胎发生。我们全面的全基因组转录谱分析， Runx 2和Runx 3将作为一个有价值的基因组资源，以促进我们对它们的理解。 胚胎骨骼发育的相互关联的管理，以及提供急需的遗传 研究社区的资源。