Genomic occupancy of HLH, AP1 and Runx2 motifs within a nuclease sensitive site of the Runx2 gene

Runt-related transcription factors are principal developmental regulators that control lineage commitment and cell type-specificity in diverse species. Null mutations in each of the three known runt-related transcription factor (Runx) genes in mouse causes dramatic tissue-specific phenotypes. Mutations and/or deregulation of the corresponding human genes are linked to familial diseases and genetic predispositions related to cancer and distinct abnormalities in tissue-formation (Blyth et al., 2005; Ito, 2008). While Runx proteins may functionally compensate each other, the unique phenotypes of mice with Runx null mutations are attributable to tissue- and developmental stage-specific activation of transcription. The Runx2 gene is prominently transcribed in the mesenchymal lineage to support normal development of bone and cartilage in vivo which accounts for the observed skeletal phenotypes of mice with Runx2 mutations that render mesenchymal cells Runx2 deficient (Komori et al., 1997; Otto et al., 1997; Choi et al., 2001; Lengner et al., 2002; Jeong et al., 2008; Lou et al., 2009; Zhang et al., 2009a; Liu et al., 2011). Runx2 transcription, as the initial rate-limiting step in its expression, is exquisitely regulated by a multitude of developmental signals, regulatory promoter elements and cognate transcription factors (Lian et al., 2006; Franceschi et al., 2007; Marie, 2008; Long, 2012). The Runx2 gene is expressed from two promoters (P1 and P2), and the upstream P1 promoter supports osteoblast-specific gene transcription. Of note, polymorphisms in the P1 and P2 promoter have been correlated with bone mineral density and viral integration sites are capable of ectopic activation of Runx2 gene transcription in non-osseous cell types (Stewart et al., 2002; Doecke et al., 2006; Lee et al., 2009). The P1 promoter is autoregulated through at least seven Runx binding sites (Drissi et al., 2000, 2002b) and responds to steroid hormones (Tou et al., 2001; Drissi et al., 2002a), BMPs (Xiao et al., 2001; Tou et al., 2003; Lee et al., 2005) and WNTs (Gaur et al., 2005). The P1 promoter is controlled by several homeodomain proteins including AP1 (Drissi et al., 2002a), Dlx5 (Gaur et al., 2005), Nkx3.2 (Lengner et al., 2005), HoxA10 (Hassan et al., 2007), SP1 and Ets proteins (Zhang et al., 2009b), Hif2α (Tamiya et al., 2008), C/EBPβ (Wiper-Bergeron et al., 2007; Henriquez et al., 2011) and NF-1 related proteins (Zambotti et al., 2002). Regulation of Runx2 gene transcription by this cohort of primary DNA binding proteins occurs within the context of nucleosomal organization and higher order chromatin structure that together modulate accessibility of transcription factors to gene promoters. The Runx2 gene, which is in throughout the osteogenic lineage, and the osteocalcin (OC) gene, which is transcriptionally activated at the maturation stage of osteoblast differentiation, together represent two versatile and intensively studied models for understanding transcriptional control during osteogenesis (Lian et al., 2004; Montecino et al., 2008). Activation of OC gene expression occurs concomitant with creation of nuclease hypersensitive sites, increased acetylation of histone H3 and H4, as well as specific binding of multiple transcription factors including Runx2 (Javed et al., 1999; Shen et al., 2002, 2003; Hassan et al., 2004). Studies using rat osteosarcoma cells and trans-differentiated mouse myoblasts have revealed SWI/SNF dependent changes in the chromatin organization of the Runx2 P1 promoter (Cruzat et al., 2009). In this study, we established key parameters of chromatin fine-structure of the Runx2 gene promoter in mouse osteoblasts in which the Runx2 P1 promoter is naturally activated. We define histone modifications, nuclease hypersensitive sites and two genomic protein/DNA interactions that mediate transcriptional regulation of Runx2 gene expression. Our key finding is that the Runx2 P1 promoter contains two stable genomic protein-DNA interaction domains that may transcriptionally control the multiple physiological activities of Runx2 during skeletal development and bone formation in vivo.