title: Epigenetic landscape during osteoblastogenesis defines a differentiation-dependent Runx2 promoter region.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405-0068, USA. Electronic address: phillip.tai@med.uvm.edu.
authors.forename: Phillip W L
authors.initials: PW
authors.lastname: Tai
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405-0068, USA. Electronic address: hai.wu@uvm.edu.
authors.forename: Hai
authors.initials: H
authors.lastname: Wu
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405-0068, USA. Electronic address: jonathan.a.gordon@uvm.edu.
authors.forename: Jonathan A R
authors.initials: JA
authors.lastname: Gordon
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655-0002, USA. Electronic address: troy.whitfield@umassmed.edu.
authors.forename: Troy W
authors.initials: TW
authors.lastname: Whitfield
authors.affiliation: Department of Cell and Developmental Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655-0002, USA. Electronic address: rasim.barutcu@umassmed.edu.
authors.forename: A Rasim
authors.initials: AR
authors.lastname: Barutcu
authors.affiliation: Mayo Clinic, 200 First Street SW, Rochester, MN 55905-0001, USA. Electronic address: vanwijnen.andre@mayo.edu.
authors.forename: André J
authors.initials: AJ
authors.lastname: van Wijnen
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405-0068, USA. Electronic address: Jane.Lian@med.uvm.edu.
authors.forename: Jane B
authors.initials: JB
authors.lastname: Lian
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405-0068, USA. Electronic address: Gary.Stein@med.uvm.edu.
authors.forename: Gary S
authors.initials: GS
authors.lastname: Stein
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405-0068, USA. Electronic address: Janet.Stein@med.uvm.edu.
authors.forename: Janet L
authors.initials: JL
authors.lastname: Stein
mesh_terms: D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics*; D044127:Epigenesis, Genetic; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010012:Osteogenesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020033:Protein Isoforms / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D015533:Transcriptional Activation
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D020033:Protein Isoforms; C493716:Runx2 protein, mouse; D008156:Luciferases
keywords: DNase hypersensitivity; Gene regulation; Histone modification; Osteoblast differentiation; Runx2-P1