BCL6 inhibits STAT3 transcription via 2 upstream binding sites. (A) Schematic representation of the human STAT3 promoter region showing 5 potential BCL6 binding sites, location of ChIP primers, and the 3-kb fragment contained in the wt STAT3 luciferase reporter construct. (B) EMSA analysis of BCL6 binding to the 5 candidate BCL6 sites. The sequences for these 5 sites are shown together with the 10-bp core of the consensus BCL6-binding site FB20. (+) and (−) indicate the top or bottom strand of the STAT3 promoter region. Underlines indicate sequences identical to those in the consensus. In the top panel, the 20-bp FB20 probe was ³²P labeled and used in the assay with nuclear extracts of Ly1 cells. In the bottom panel, all 5 candidate BCL6 sites and FB20 were labeled and subjected to EMSA analysis with or without the use of excess, cold FB20 probe in competition. (C) BCL6 represses transcription from the 3-kb wt STAT3 reporter in BCL6-negative Mutu III cells. Based on site-directed mutagenesis, repression by BCL6 was attributed to both sites B and D. The activity of each reporter in the absence of BCL6 was normalized to 100. Error bars represent SD.