title: Runx1 binds as a dimeric complex to overlapping Runx1 sites within a palindromic element in the human GM-CSF enhancer.
abstract: Runx1 is a developmentally regulated transcription factor that is essential for haemopoiesis. Runx1 can bind as a monomer to the core consensus sequence TGTGG, but binds more efficiently as a hetero-dimer together with the non-DNA binding protein CBFβ as a complex termed core binding factor (CBF). Here, we demonstrated that CBF can also assemble as a dimeric complex on two overlapping Runx1 sites within the palindromic sequence TGTGGCTGCCCACA in the human granulocyte macrophage colony-stimulating factor enhancer. Furthermore, we demonstrated that binding of Runx1 to the enhancer is rigidly controlled at the level of chromatin accessibility, and is dependent upon prior induction of NFAT and AP-1, which disrupt a positioned nucleosome in this region. We employed in vivo footprinting to demonstrate that, upon activation of the enhancer, both sites are efficiently occupied. In vitro binding assays confirmed that two CBF complexes can bind this site simultaneously, and transfection assays demonstrated that both sites contribute significantly to enhancer function. Computer modelling based on the Runx1/CBFβ/DNA crystal structure further revealed that two molecules of CBF could potentially bind to this class of palindromic sequence as a dimeric complex in a conformation whereby both Runx1 and CBFβ within the two CBF complexes are closely aligned.
authors.affiliation: Experimental Haematology, Leeds Institute of Molecular Medicine, University of Leeds, St James's University Hospital, Leeds LS9 7TF, UK.
authors.forename: Sarion R
authors.initials: SR
authors.lastname: Bowers
authors.forename: Fernando J
authors.initials: FJ
authors.lastname: Calero-Nieto
authors.forename: Stephanie
authors.initials: S
authors.lastname: Valeaux
authors.forename: Narcis
authors.initials: N
authors.lastname: Fernandez-Fuentes
authors.forename: Peter N
authors.initials: PN
authors.lastname: Cockerill
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D050676:Core Binding Factor Alpha 2 Subunit / Q000737:chemistry* / Q000378:metabolism; D050658:Core Binding Factor beta Subunit / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D004742:Enhancer Elements, Genetic; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D055029:Inverted Repeat Sequences; D051379:Mice; D008958:Models, Molecular
chemical_list: D050676:Core Binding Factor Alpha 2 Subunit; D050658:Core Binding Factor beta Subunit; C493728:RUNX1 protein, human; D016178:Granulocyte-Macrophage Colony-Stimulating Factor