Fig. 7

NPAS3 and ARNT physically regulate the TXNIP locus. a UCSC genome browser data used to identify potential NPAS3::ARNT binding sites proximal to the TXNIP locus. Accessed from http://genome.ucsc.edu. Top: ConTra v3.0 predicted ARNT binding sites in the promoter, 3′UTR and intron 1 of TXNIP based on position weight matrices accessed from the JASPAR database. Middle: ENCODE ARNT ChIP-seq data indicating peaks proximal to the TXNIP locus. Bottom: relative localization of ChIP primers and promoter constructs used to assess regulation of this locus by NPAS3 and ARNT. TXNIP primers indicate regions amplified corresponding to Distal element (black line), Exon 1 (red line) and Promoter region (green line). Red asterisks indicate variants in promoter construct relative to wild-type sequence. b Luciferase data demonstrating that NPAS3 and ARNT regulate expression of reporter luciferase (luc2) driven by a construct including 867 bp proximal to the transcription start site of TXNIP. RLU = relative luciferase units, the ratio of firefly luciferase and renilla luciferase to normalize for transfection efficiency. Top and bottom of each box indicate the 25th and 75th centile, internal bar indicates the median. Data are presented for two biological replicates performed in each low- and high-glucose conditions. *p < 0.05, **p < 0.01, ***p < 0.001. c, d Western blots demonstrating expression of NPAS3 and ARNT constructs. e HaloCHIP PCR data demonstrating that NPAS3 interacts with the promoter region of VGF relative to other regions of the locus assayed. f HaloCHIP PCR data demonstrating ARNT signal at all regions tested at the VGF locus. HaloCHIP results are representative of three replicates performed