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Genomic occupancy of Runx2 with global expression profiling identifies a novel dimension to control of osteoblastogenesis

Hai Wu1, Troy W Whitfield2, Jonathan A R Gordon1, Jason R Dobson34, Phillip W L Tai1, Andre J van Wijnen5, Janet L Stein1, Gary S Stein1 and Jane B Lian1*

Author Affiliations

1 Department of Biochemistry, University of Vermont College of Medicine and Vermont Cancer Center, 89 Beaumont Avenue, Burlington, VT 05405, USA

2 Department of Cell & Developmental Biology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01655, USA

3 Current address: Center for Computational Molecular Biology, Department of Molecular Biology, Cell Biology & Biochemistry, Brown University, 115 Waterman Street, Providence, RI 02912, USA

4 Current address: Department of Computer Science, Brown University, 115 Waterman Street, Providence, RI 02912, USA

5 Current address: Departments of Orthopedic Surgery and Biochemistry & Molecular Biology, Mayo Clinic, Medical Sciences Building 3-69, 200 First Street SW, Rochester, MN 55905, USA

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Genome Biology 2014, 15:R52  doi:10.1186/gb-2014-15-3-r52

Published: 21 March 2014

Abstract

Background

Osteogenesis is a highly regulated developmental process and continues during the turnover and repair of mature bone. Runx2, the master regulator of osteoblastogenesis, directs a transcriptional program essential for bone formation through genetic and epigenetic mechanisms. While individual Runx2 gene targets have been identified, further insights into the broad spectrum of Runx2 functions required for osteogenesis are needed.

Results

By performing genome-wide characterization of Runx2 binding at the three major stages of osteoblast differentiation - proliferation, matrix deposition and mineralization - we identify Runx2-dependent regulatory networks driving bone formation. Using chromatin immunoprecipitation followed by high-throughput sequencing over the course of these stages, we identify approximately 80,000 significantly enriched regions of Runx2 binding throughout the mouse genome. These binding events exhibit distinct patterns during osteogenesis, and are associated with proximal promoters and also non-promoter regions: upstream, introns, exons, transcription termination site regions, and intergenic regions. These peaks were partitioned into clusters that are associated with genes in complex biological processes that support bone formation. Using Affymetrix expression profiling of differentiating osteoblasts depleted of Runx2, we identify novel Runx2 targets including Ezh2, a critical epigenetic regulator; Crabp2, a retinoic acid signaling component; Adamts4 and Tnfrsf19, two remodelers of the extracellular matrix. We demonstrate by luciferase assays that these novel biological targets are regulated by Runx2 occupancy at non-promoter regions.

Conclusions

Our data establish that Runx2 interactions with chromatin across the genome reveal novel genes, pathways and transcriptional mechanisms that contribute to the regulation of osteoblastogenesis.