Genome-Wide and Phase-Specific DNA-Binding Rhythms of BMAL1 Control Circadian Output Functions in Mouse Liver
(A) BMAL1-bound E1-E2 sites in the second intron of Dbp (Dbp-I2) and at the promoter of Per2 show an increased BMAL1/CLOCK transactivation compared to either the E2-mutated (E1-mE2) version or the 10-bp spacer version (sp10) (Student's one-tailed t test, p<0.05, n = 3). Empty vector (Prom[−]) is shown as a negative control. Wild-type (WT) levels are set to 100%. Error bars represent the standard error of the mean. (B) Mutation in the Dbp-I2 sequence shows that an E1 is needed for robust BMAL1/CLOCK transactivation. The wild-type version was compared to E1 mutated (mE1-E2), both E1 and E2 mutated (mE1-mE2), E2 replaced by E1 (E1-E1), and E1 replaced by E2 (E2-E2). All mutated versions have reduced activity compared to wild-type (p<0.005, n = 4), with the exception of E1-E1, which shows a level of transactivation similar to that of wild-type. (C) Modifying the spacer length of the Dbp-I2 tandem E-boxes from 4 bp to 20 bp shows a spacer preference at 6–8 bp, but also at 17 bp, which corresponds to a full helical turn of the DNA. Indeed, sp4, sp10, and sp20 have a significantly reduced activity compared to sp7 (p<0.01, n = 4).