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

Abstract

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

Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2

“…Our finding that EZH2 attenuates osteoblastogenesis is relevant to skeletal deformities seen in congenital human disorders and consistent with accruing data on epigenetic regulation of osteoblast differentiation (22,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)59). Our initial studies with mice lacking functional Ezh2 expression in mesenchymal progenitor cells show premature closure of the cranial sutures characteristic of craniosynostosis.…”

“…For example, a recent study has demonstrated that suppression of Ezh2 expression is potentially controlled by up-regulation of Runx2 in mature osteoblasts (59). Furthermore, the long noncoding RNA LncRNA-ANCR targets EZH2, and this inhibition of EZH2 supports expression of RUNX2 (60).…”

Epigenetic Plasticity Drives Adipogenic and Osteogenic Differentiation of Marrow-derived Mesenchymal Stem Cells

“…The epigenetic marks in osteoblastic cells (15,16,21) and in adipocyte cell culture models (22) have revealed distinct patterns for gene expression in terminally differentiated cell types. Previous research has determined that the histone-modifying enzymes responsible for many of these patterns, including KDM5B, EZH2, and others, play a clear role in skeletal development and differentiation of the MSC through modulation of RUNX2 (23,24).…”

Epigenetic Changes During Mechanically Induced Osteogenic Lineage Commitment

“…While these areas are often responsible for controlling gene expression [34], many other potential regions of gene expression control exist. Nonpromoter regions such as upstream regions (À1 to À20 kb from transcription start site), introns, exons, and intergenic regions have been demonstrated to bind Runx2 during osteogenic differentiation [35]. Finally, we were limited to evaluating a few target genes, but many more genes are expressed during the course of osteogenic and adipogenic differentiation.…”