Interaction of EWSR1-DDIT3 and potential C/EBP-binding sites within Opn and Col11a2 promoters <i>in vivo</i>.

<p>Transient ChIP assays using an antibody against the N-terminal FLAG epitope or normal IgG. C3H10T1/2 cells were transfected with Opn (<b>A</b>) or Col11a2 (<b>B</b>) luciferase reporter plasmids plus FLAG-tagged expression vectors. Promoter DNA fragments containing potential C/EBP-binding sites (open box) were immunoprecipitated with EWSR1-DDIT3 when wild-type Opn (pGL3-Opn) and Col11a2 (pGL3-Col11a2) promoter constructs were analyzed. Either deleting (EWSR1-DDIT3 del LZ, broken line) or mutating (EWSR1-DDIT3 mut LZ, filled box) the LZ dimer forming domain of EWSR1-DDIT3 or mutating potential C/EBP-binding sites within Opn (inverted CCAAT box, pGL3-Opn mut, filled box) and Col11a2 (half C/EBP site, pGL3-Col11a2 mut, filled box) promoters eliminated the protein–DNA interaction. As indicated by the arrows, the forward PCR primers are promoter sequence-specific primers and locate upstream to the potential C/EBP-binding site (open box), while the reverse PCR primer pGL3 is a plasmid-specific primer.