Ap-2δ binds to the Bhlhb4 and the Pou4f3 promoter.
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(a) Luc reporter plasmids were cotransfected with an Ap-2δ expression plasmid into Neuro2A cells. Ap-2δ activates native promoters of Bhlhb4 and Pou4f3 for more than 2.5-fold. The bars represent relative luciferase units normalized to total protein and show the mean ± s.e.m. of three independent transfections. Reporter-only transfections (pGl3-basic) were set at 1. p-values indicate significant differences from reporter-only transfected cells (student's t-test). (b) ChIP/qPCR assays in wild-type and HA-Ap-2δ transfected Neuro2a cells. (Top) Ap-2δ binds to the Bhlhb4 promoter 1.8 kb and 300 bp upstream of the TSS. (Bottom) Ap-2δ occupies the Pou4f3 promoter 2.8 kb upstream of the TSS. Bars represent mean abundance ± s.e.m. relative to histone H3 as determined from triplicate PCR measurements. p-values indicate significant differences from HA-Ap-2δ transfected Neuro2a cells precipitated with an anti-HA antibody compared to non-transfected Neuro2a cells or IgG treated lysates (student's-test) (c) (Top) EMSA was performed by incubating 32P-labeled, binding site-containing oligonucleotides with in vitro-translated Ap-2δ protein. The Ap-2δ/DNA complex is specifically supershifted with an α-Ap-2δ antibody and competed using an 80-fold excess of unlabeled oligonucleotide. (Bottom) EMSA was performed with an oligonucleotide containing the wildtype binding site of the Pou4f3 promoter (core sequence: GCC TGA GGG) or mutated forms (Pou mt1: GCC AGT GGG, Pou mt2: GCC GTT GGG). Upon mutation of the binding site, Ap-2δ binding is abolished.