Fig. 3.

TNF-α suppresses E-box-mediated transcription of clock genes. (A) Schematic representation of the luciferase reporter genes used. mPer1-luc (3-kb promoter fragment) and mPer3-luc (1.7-kb promoter fragment) contain promoter sequences upstream of their genes in the pGL3basic vector. pBmal1-luc is regulated by a 1-kb promoter fragment and a 1-kb sequence of the 3′UTR from mBmal1 gene. E54-TK consists of the three E-boxes of the mouse Per1 gene and their immediate flanking sequences in front of a TK promoter. DBP-Ebox857-luc contains one of four E-boxes of the Dbp promoter regulating the activity of a SV40 promoter. (B) Native clock gene promoters bearing an E-box are affected by TNF-α. Percent luciferase expression of mPer1-luc, mPer3-luc, and pBmal1-luc transfected NIH 3T3 cells after treatment with 10 ng/ml TNF-α overnight compared with untreated controls (100% luciferase expression) is shown. (C) E-boxes of mPer1 are affected by TNF-α treatment. TNF-α significantly reduces E54-TK-dependent luciferase expression but not pGL3-TK lacking E-boxes and flanking sequences as shown by raw data (RLU, relative light units; Left) and percent inhibition (Right). (D) The E-box of Dbp gene at position +857 is repressed by TNF-α even when CLOCK and BMAL1 are overexpressed. Cotransfection with CLOCK and BMAL1 leads to four to five times higher relative luciferase activity of 857wt compared with the mutated E-box vector (857mut) indicating that the interaction of CLOCK:BMAL1 with the E-box is functional. Luciferase activity is efficiently suppressed by overnight treatment with TNF-α as shown by raw data (Left) and percent inhibition (Right). TNF-α leads to a higher repression in 857wt than in 857mut. For all assays, mean ± SD of triplicates from one representative experiment of three; independent sample t test.