Figure 4. Chromatin inhibits transcriptional activation by Pdx-1.

Schematic representations of the insulin reporters used are shown at the top. The reporters contain tandem insulin 1 A4/A3 elements (or the same elements containing mutations in the Pdx-1 binding sequences) placed upstream of a minimal promoter driving the luciferase gene. The reporters were either used directly (naked reporter) or reconstituted as chromatin with histone octamers (chromatinized reporter) and subjected to transcription reactions in vitro using HeLa nuclear extract with or without 100 ng Pdx-1. To ensure that transcriptional initiation was measured, reactions were subjected to 5’ RACE, followed by quantitation using real-time PCR. To highlight the differences in transcription observed after adding Pdx-1, data are expressed as transcript levels relative to a reaction containing only HeLa nuclear extract (defined as 1). Data represent the mean ± S.E. of three independent in vitro transcription reactions.