Transcriptional regulatory logic of the diurnal cycle in the mouse liver
Fig 6
BMAL1 footprints indicate temporally changing protein–DNA complexes, consistent with binding of a heterotetramer to DNA.
A. Genomic profiles of DNase I cuts around double E-boxes with a spacer of 6 bp (E1-E2 sp6). We selected n = 249 E1-E2 sp6 motifs overlapping a BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) peak and show the average of profiles for loci classified as bound by the mixture model (posterior probability >0.5). At ZT6, we observed that nucleotides around both E-boxes are protected. In contrast, at ZT18, the width of the protected region is reduced by approximately half, with the second E-box no longer protected from digestion. The signals are anchored to the motif position. Orientation of sites and signals is according to the best match to the E1-E2 sp6 motif. In Bmal1-/-, only one E-box appears occupied. B. Width (left-side y-axis, green) of the protected region in WT and in Bmal1-/- mice for E1-E2 sp6 motifs occupied by BMAL1. Fraction of predicted occupied sites is shown in blue (right-side y-axis). C. Two views of the 3-D computational model of the CLOCK:BMAL1 heterotetramer showing two heterodimers of CLOCK:BMAL1 occupying an E1-E2 sp6 site. The two heterodimers are shown in green and blue, while darker green and darker blue correspond to BMAL1 and lighter colors to CLOCK proteins. Information content along the DNA strands is shown in grey with highly constrained nucleotides of the motif in red. D. Zoom on the interacting residuals on the PAS-B domain of CLOCK implicated in the heterotetramer formation.