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

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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.