Fig. 6: PER2 promoter binds to transcription factor ATF3 to activate PER2 transcription.

A Venn diagram showed intersection of transcription factors regulating PER2 from three databases, with binding rankings in order of ATF3, RUNX1, and IRF1. The PER2 promoter starts 2000 bp before the transcription start site. B IHC assay of PER2 and ATF3 expression in OSCC tissues and Pearson correlations between PER2 and ATF3 protein expression (n = 26, scale bars = 50 μm, ANT adjacent normal tissue). C Western blotting of PER2 and ATF3 protein expression in CAL27, TSCCA and SCC25 cells, and Pearson correlations between expression of PER2 and ATF3 proteins. D ChIP assay showed significantly enhanced binding of ATF3 to the PER2 promoter in OE-ATF3-SCC25 cells compared with Vector-SCC25 cells. E Dual-luciferase reporter gene assay to detect regulation of the PER2 promoter by OE-ATF3. F Analysis of hTFtarget database showed that ATF3 has five binding sites (−89 ~ −79, −257 ~ −249, −808 ~ −795, −1050 ~ −1042, and −1100 ~ −1089) with the PER2 promoter, and the TSS in the figure denotes the transcription start site. G Construction of truncated domain#1 (−1300 to 0), domain#2 (−1000 to 0), and domain#3 (−700 to 0) and wild-type (WT) PER2 promoter nucleotide sequences. H Dual-luciferase reporter gene assay to detect regulation by ATF3 binding of the WT PER2 promoter sequence and the three segment truncations. I Prediction based on the JASPAR database of the possible base sequence of ATF3 in the PER2 promoter sequence (GCTGAGGTCAGC), and analysis of the conservation of each base position in this sequence. Each column corresponds to one base position, and each base position consists of a stack of bases at that position, and the greater the total height of the stack of bases (denoted as bits, i.e., the value of vertical coordinates), the greater the conservation of bases at that position. J Schematic representation of the putative ATF3 binding site in the PER2 promoter and point mutation. Arrows indicate the direction of PER2 sequence transcription direction; blue bases show the predicted binding site (i.e., the WT sequence GCTGAGGTCAGC); and red bases indicate the mutated (Mut) binding site (i.e., Mut sequence AAAAAAAAAAAA). K Dual-luciferase reporter gene assay to examine the role of ATF3 in regulating the nucleotide sequence of the PER2 promoter in the WT and Mut groups. All data represent three replicate independent experiments. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.