Runx2 is a developmentally regulated gene in vertebrates and is essential for bone formation and skeletal homeostasis. The induction of runx2-P1 isoform transcripts is a hallmark of early osteoblastogenesis. Although previous in vitro studies have defined a minimal Runx2-P1 promoter sequence with well-characterized functional elements, several lines of evidence suggest that transcription of the Runx2-P1 isoform relies on elements that extend beyond the previously defined P1 promoter boundaries. In this study, we examined Runx2-P1 transcriptional regulation in a cellular in vivo context during early osteoblastogenesis of MC3T3-E1 cultures and BMSCs induced towards the bone lineage by multi-layered analysis of the Runx2-P1 gene promoter using the following methodologies: 1) sequence homology among several mammalian species, 2) DNaseI hypersensitivity coupled with massively parallel sequencing (DNase-seq), and 3) chromatin immunoprecipitation of activating histone modifications coupled with massively parallel sequencing (ChIP-seq). These epigenetic features have allowed the demarcation of boundaries that redefine the minimal Runx2-P1 promoter to include a 336-bp sequence that mediates responsiveness to osteoblast differentiation. We also find that an additional level of control is contributed by a regulatory region in the 5'-UTR of Runx2-P1.
[Show abstract][Hide abstract] ABSTRACT: Three-dimensional organization of chromatin is fundamental for transcriptional regulation. Tissue-specific transcriptional
programs are orchestrated by transcription factors and epigenetic regulators. The RUNX2 transcription factor is required for
differentiation of precursor cells into mature osteoblasts. Although organization and control of the bone-specific Runx2-P1 promoter have been studied extensively, long-range regulation has not been explored. In this study, we investigated higher-order
organization of the Runx2-P1 promoter during osteoblast differentiation. Mining the ENCODE database revealed interactions between Runx2-P1 and Supt3h promoters in several non-mesenchymal human cell lines. Supt3h is a ubiquitously expressed gene located within the first intron of Runx2. These two genes show shared synteny across species from humans to sponges. Chromosome conformation capture analysis in the
murine pre-osteoblastic MC3T3-E1 cell line revealed increased contact frequency between Runx2-P1 and Supt3h promoters during differentiation. This increase was accompanied by enhanced DNaseI hypersensitivity along with RUNX2 and
CTCF binding at the Supt3h promoter. Furthermore, interplasmid-3C and luciferase reporter assays showed that the Supt3h promoter can modulate Runx2-P1 activity via direct association. Taken together, our data demonstrate physical proximity between Runx2-P1 and Supt3h promoters, consistent with their syntenic nature. Importantly, we identify the Supt3h promoter as a potential regulator of the bone-specific Runx2-P1 promoter.
Nucleic Acids Research 08/2014; 42(16). DOI:10.1093/nar/gku712 · 9.11 Impact Factor
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