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Progressive recruitment of Runx2 to genomic targets despite decreasing expression during osteoblast differentiation

Authors

  • Steven Pregizer,

    1. Department of Biochemistry & Molecular Biology, University of Southern California, Los Angeles, California
    2. Institute for Genetic Medicine, University of Southern California, Los Angeles, California
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  • Sanjeev K. Baniwal,

    1. Department of Biochemistry & Molecular Biology, University of Southern California, Los Angeles, California
    2. Institute for Genetic Medicine, University of Southern California, Los Angeles, California
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  • Xiting Yan,

    1. Department of Biological Sciences, Molecular and Computational Biology Program, University of Southern California, Los Angeles, California
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  • Zea Borok,

    1. Department of Biochemistry & Molecular Biology, University of Southern California, Los Angeles, California
    2. Department of Medicine, University of Southern California, Los Angeles, California
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  • Baruch Frenkel

    Corresponding author
    1. Department of Biochemistry & Molecular Biology, University of Southern California, Los Angeles, California
    2. Institute for Genetic Medicine, University of Southern California, Los Angeles, California
    3. Department of Orthopaedic Surgery, University of Southern California, Los Angeles, California
    • USC IGM CSC 240, 2250 Alcazar Street, Los Angeles, CA 90033.
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Abstract

The mRNAs encoding Runx2, a master osteoblast transcription factor, and its target gene Osteocalcin (OC), are commonly used as markers of osteoblast differentiation. We found that while OC mRNA levels do indeed increase during development of the osteoblast phenotype in MC3T3-E1 cultures, Runx2 mRNA levels surprisingly decrease. Neither translational control of Runx2 (based on Western analysis) nor regulation of its DNA-binding ability (assessed by electrophoretic mobility shift assay) could explain the unexpected opposite patterns of Runx2 and OC expression. Instead, a series of chromatin immunoprecipitation (ChIP) assays during osteoblast differentiation revealed that early on, when Runx2 protein amount and DNA-binding activity are maximal, it is practically absent from the OC promoter. At later stages, Runx2 is recruited to the OC promoter while Runx2 mRNA, protein, and in vitro DNA binding progressively decrease. We also followed Runx2 occupancy at a novel genomic target discovered by ChIP-Chip analysis of cells in which the OC promoter is maximally occupied. The results revealed that Runx2 is recruited to this locus and to the OC promoter with a remarkably similar temporal pattern. These observations highlight a mechanism that restrains Runx2-mediated transcriptional control by confining its access to genomic targets to a narrow window of time. The need for such stringent control is consistent with the severe consequences of Runx2 over-expression in vivo. J. Cell. Biochem. 105: 965–970, 2008. © 2008 Wiley-Liss, Inc.

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