FIG. 3.

Luman binds and activates transcription from the second half-site of ERSE-II. (A) Dual luciferase assays were performed as described in the legend for Fig. 2. In all the half-site mutants of ERSE and ERSE-II, the nucleotides A, C, G, and T were substituted for C, A, T, and G, respectively. (B) In vitro binding of Luman to the second half-site of ERSE-II by EMSA. Mutant sequences of the two half-sites of ERSE-II are underlined. Equal amounts of purified GST (G) and GST-Luman (L) proteins were incubated with the indicated double-stranded probes labeled with ³²P and separated on a 4% nondenaturing polyacrylamide gel electrophoresis gel. (C) Direct binding of Luman to the Herp promoter as demonstrated by ChIP assay. Top, schematic diagram of the human Herp promoter, with positions of the primer pair used in this ChIP assay indicated. Bottom, 293 cells were transfected with plasmid pcDNA3.1 or pcFLAG-Luman and then cross-linked by formaldehyde. Chromatin was immunoprecipitated with the indicated antibodies. Purified precipitates or input DNA was analyzed by PCR using primers specific for Herp (−193/−15) or the control OPR150 (−311/−28) and Smad6 (−186/+63) promoters. PCR products were subjected to gel electrophoresis and visualized by ethidium bromide staining.