Glucose decreases the ability of HNF4α to activate the transcription of the <i>NR2F2</i> proximal promoter and to bind its chromatin target.

<p>(A) INS-1 832/13 cells were transiently co-transfected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035810#pone.0035810-Perilhou2" target="_blank">[7]</a> with a luciferase reporter gene driven by various lengths of the <i>NR2F2</i> promoter, designated by their 5′ and 3′ end positions relative to the defined <i>NR2F2</i> gene transcription initiation site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035810#pone.0035810-Soosaar1" target="_blank">[25]</a> and a control vector expressing <i>Renilla</i> luciferase. Cells were cultured in the presence of either 5 mM (white bars) or 20 mM (black bars) glucose. Results were calculated from the ratio of luciferase/<i>Renilla</i> activity. Background expression was subtracted using the mean expression level of empty pGL3-basic. Means ± SEM of results obtained from at least three independent transfections performed in triplicate are shown. *Significant differences, P<0.05. (B) Comparison of mRNA levels of NR2F2 and HNF4α relative to those of cyclophilin determined by real-time RT-QPCR in INS-1 832/13 β-cells stimulated with 5 mM (white bars) or 20 mM glucose (black bars) for 24 h as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035810#pone.0035810-Perilhou1" target="_blank">[3]</a> (C) ChIP from INS-1 832/13 cells cultured in the presence of either 5 mM or 20 mM glucose. Targets for QPCR amplifications were the proximal <i>NR2F2</i> promoter containing the DR1 DNA binding site or a downstream coding region (negative control). Amount of target chromatin precipitated by the HNF4α-specific antibody relative to that precipitated by control IgG (mean of three independent experiments ± SEM). *Significant difference, P<0.05. (D) COS cells were transiently co-transfected with the −328/+873 construct and 50 ng of empty KS vector (white bars), or HNF4α (grey bars) expression vectors. Means ± SEM of results from at least three separate transfections performed in triplicate are shown. *Significant difference, P<0.05. (E) INS-1 832/13 cells were transiently co-transfected with the wild-type or mutated −328/+873 constructs containing the DR1 elements. Cells were cultured in the presence of either 5 mM (white bars) or 20 mM (black bars) glucose. Means ± SEM of results from ten independent transfections performed in triplicate are shown. *Statistically significant differences in percentage of expression (where value for 5 mM glucose is 100% relative to −328/+873 construct) at P<0.05.