Abstract
RUNX2 is a transcription factor that is first expressed in early osteoblast-lineage cells and represents a primary determinant of osteoblastogenesis. While numerous target genes are regulated by RUNX2, little is known of sites on the genome occupied by RUNX2 or of the gene networks that are controlled by these sites. To explore this, we conducted a genome-wide analysis of the RUNX2 cistrome in both pre-osteoblastic MC3T3-E1 cells (POB) and their mature osteoblast progeny (OB), characterized the two cistromes and assessed their relationship to changes in gene expression. We found that although RUNX2 was widely bound to the genome in POB cells, this binding profile was reduced upon differentiation to OBs. Numerous sites were lost upon differentiation, new sites were also gained; many sites remained common to both cell states. Additional features were identified as well including location relative to potential target genes, abundance with respect to single genes, the frequent presence of a consensus TGTGGT RUNX2 binding motif, co-occupancy by C/EBPβ and the presence of a typical epigenetic histone enhancer signature. This signature was changed quantitatively following differentiation. While RUNX2 binding sites were associated extensively with adjacent genes, the distal nature of the majority of these sites prevented assessment of whether they represented direct targets of RUNX2 action. Changes in gene expression, however, revealed an abundance of genes that contained RUNX2 binding sites and were regulated in concert. These studies establish a basis for further analysis of the role of RUNX2 activity and its function during osteoblast lineage maturation.
Highlights
RUNX2 is indispensable for mature osteoblast differentiation and function
We show through ChIP-seq analysis that while the RUNX2 cistrome is extensive in pre-osteoblastic MC3T3-E1 cells (POB), it undergoes significant restriction upon differentiation to osteoblast progeny (OB)
RUNX2 Cistrome Analysis in POBs and in Vitro Differentiated OBs—The MC3T3-E1 cells (POBs) used in this study were initially validated for osteoblast differentiation following a 15 day treatment with ascorbic acid and -glycerophosphate (OBs) using Von Kossa and Alizarin Red staining procedures that verify appropriate differentiation, matrix secretion, nodule formation, and calcification indicative of mature osteoblasts
Summary
RUNX2 is indispensable for mature osteoblast differentiation and function. Results: During osteogenic differentiation, RUNX2 along with C/EBP bind throughout the genome near genes that are essential for the phenotype. Changes in gene expression revealed an abundance of genes that contained RUNX2 binding sites and were regulated in concert These studies establish a basis for further analysis of the role of RUNX2 activity and its function during osteoblast lineage maturation. As with most programs of differentiation, this complex cellular progression from the mesenchymal stem cell (MSC) is driven by unique spatial and temporal changes in gene expression This process is dynamically programmed at the level of the genome via both genetic and epigenetic determinants, and initiated via activation of distinct signaling pathways (2, 4 –7). It is apparent that in addition to SOX9, RUNX2, and OSX, osteoblast differentiation and function are regulated by the temporal and overlapping expression of a myriad of additional transcription factors that include MSX2, TWIST1, SATB2, NFATc1, ATF4, STAT1, C/EBP isoforms, and multiple nuclear receptors as well [2]. Changes in RUNX2 binding are noted at genes whose activities are both increased and decreased during the process of differentiation
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