Fig. 5

NPAS3 physically regulates the VGF promoter. a UCSC genome browser data used to identify potential NPAS3::ARNT binding sites proximal to the VGF locus. Accessed from http://genome.ucsc.edu. Top: ConTra v3.0 data indicating the location of predicted ARNT binding sites based on position weight matrices accessed from the JASPAR database. Middle: ENCODE ARNT ChIP-seq data indicating peaks proximal to the VGF locus. Bottom: relative localization of ChIP primers and promoter constructs used to assess regulation of this locus by NPAS3 and ARNT. VGF primers indicate regions amplified corresponding to Distal element (black line), Intron 1 (red line) and Promoter region (green line). b Luciferase data demonstrating that NPAS3 and ARNT regulate expression of the luciferase (luc2) reporter driven by a construct including 797 bp proximal to the transcription start site of VGF. 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, whiskers indicate data extremes. Data are representative of three biological replicates. *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