title: Genomic occupancy of HLH, AP1 and Runx2 motifs within a nuclease sensitive site of the Runx2 gene.
abstract: Epigenetic mechanisms mediating expression of the Runt-related transcription factor Runx2 are critical for controlling its osteogenic activity during skeletal development. Here, we characterized bona fide regulatory elements within 120 kbp of the endogenous bone-related Runx2 promoter (P1) in osteoblasts by genomic DNase I footprinting and chromatin immuno-precipitations (ChIPs). We identified a ~10 kbp genomic domain spanning the P1 promoter that interacts with acetylated histones H3 and H4 reflecting an open chromatin conformation in MC3T3 osteoblasts. This large chromatin domain contains a single major DNaseI hypersensitive (DHS) region that defines a 0.4 kbp "basal core" promoter. This region encompasses two endogenous genomic protein/DNA interaction sites (i.e., footprints at Activating Protein 1 [AP1], E-box and Runx motifs). Helix-Loop-Helix (HLH)/E-box occupancy and presence of the DHS region persists in several mesenchymal cell types, but AP1 site occupancy occurs only during S phase when Runx2 expression is minimal. Point-mutation of the HLH/E box dramatically reduces basal promoter activity. Our results indicate that the Runx2 P1 promoter utilizes two stable principal protein/DNA interaction domains associated with AP1 and HLH factors. These sites function together with dynamic and developmentally responsive sites in a major DHS region to support epigenetic control of bone-specific transcription when osteoblasts transition into a quiescent or differentiated state.
authors.affiliation: Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
authors.forename: Hayk
authors.initials: H
authors.lastname: Hovhannisyan
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Mohammad Q
authors.initials: MQ
authors.lastname: Hassan
authors.forename: Hai
authors.initials: H
authors.lastname: Wu
authors.forename: Carlotta
authors.initials: C
authors.lastname: Glackin
authors.forename: Jane B
authors.initials: JB
authors.lastname: Lian
authors.forename: Janet L
authors.initials: JL
authors.lastname: Stein
authors.forename: Martin
authors.initials: M
authors.lastname: Montecino
authors.forename: Gary S
authors.initials: GS
authors.lastname: Stein
authors.forename: Andre J
authors.initials: AJ
authors.lastname: van Wijnen
mesh_terms: D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology; D018257:Helix-Loop-Helix Motifs / Q000235:genetics* / Q000502:physiology; D006657:Histones / Q000378:metabolism; D008648:Mesoderm / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D054730:Protein Interaction Domains and Motifs / Q000235:genetics* / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism
chemical_list: D002843:Chromatin; D050659:Core Binding Factor Alpha 1 Subunit; D006657:Histones; C493716:Runx2 protein, mouse; D018808:Transcription Factor AP-1; D003850:Deoxyribonuclease I