Figure 3

Lef-1 affects the activity of human SLUG promoter " in vitro " and binds it " in vivo ". (A) The SLUG promoter region under investigation is reported. The positions of putative Lef/Tcf binding sites are enclosed by rectangles and are compared with those recently investigated by others. Positions of PCR primers used in ChIP experiments are also reported. (B) The DNA construct containing the human SLUG promoter region was cloned into upstream of the firefly luciferase (LUC) reporter gene. hOBs and SaOS-2 osteoblast-like cells were transfected with the pGL3-SLUG Luc reporter vector containing the sequence from +1 to -982 of the human SLUG promoter (pGL3-SLUG 982 bp), in the absence (-) or presence (+) of 2.5 μg of hLef-1 expression plasmid. The results of reporter gene assays were normalized with protein concentration and β-gal activity for transfection efficiency and the data are represented as ratios of luciferase units to β-galactosidase units. MCF7 breast cancer cell line was used as negative control. All experiments were performed in triplicate and the average of the ratio of the reporter activity + SEM is shown. * = p < 0.05. (C) Recruitment of Lef1/TCF transcription factors to the human SLUG promoter is demonstrated by "in vivo" chromatin immunoprecipitation (ChIP) binding assays. Soluble chromatin was prepared from hOBs and immunoprecipitated with the indicated specific antibodies against Lef-1, TCF-1, and TCF-4. The associations of the transcription factors to bound precipitated DNA were monitored on the human SLUG promoter regions 1, 2 and 3 by PCR with the primers indicated in the scheme. Input represents a positive control using the starting material (0.2%) prior to immunoprecipitation. Representative agarose gels are shown.