Chromatin accessibility in <i>Bmal1</i>^-/- mice at ZT6 is generally similar as in the Wild-Type (WT) mice but is lower at BMAL1 sites.

<p>A. The <i>Rev-erbα</i> (left) and <i>Gsk3a</i> (right) promoters. DNase I signal (in red) is strongly reduced in <i>Bmal1</i>^-/- mice at sites bound by CLOCK:BMAL1 in WT mice (BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signal in blue) in the <i>Rev-erbα</i> promoter but is similar in WT and <i>Bmal1</i>^-/- mice at the <i>Gsk3a</i> promoter that are not bound by BMAL1. The vertical scale is the same for all three DNase I tracks, as well as for both BMAL1 ChiP-seq tracks. Wild-type ZT18 signals are lower (about half) than at ZT6 in both genes but not as low as in the <i>Bmal1</i>^-/- mice. B. Comparison of DNase I signals at ZT6 in <i>Bmal1</i>^-<b>/-</b> versus WT mice. All DNase I hypersensitive sites (DHSs) overlapping BMAL1 ChIP-seq peaks in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001069#pbio.2001069.ref017" target="_blank">17</a>] are shown (<i>n</i> = 1,555). The dashed lines indicate 4-fold difference. C. Boxplots showing DNase I intensity at the same sites as in B, at peak (ZT6) and trough (ZT18) activities of BMAL1 in the WT, and at ZT6 in <i>Bmal1</i>^-/- mice for all BMAL1-binding sites (green), BMAL1 sites with an associated expression phase between ZT2 and ZT10 (orange), and with a tandem E-box (grey). All pairwise comparisons (within the same color) between either ZT6 versus ZT18 or ZT6 versus ZT6 <i>Bmal1</i>^-/- are significant (<i>p</i> < 0.001). D–E. Same as B–C but using overlap with USF1 ChIP-seq peaks [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001069#pbio.2001069.ref074" target="_blank">74</a>] to select DHSs (<i>n</i> = 1,705).</p

BMAL1 footprints indicate temporally changing protein–DNA complexes, consistent with binding of a heterotetramer to DNA.

<p>A. Genomic profiles of DNase I cuts around double E-boxes with a spacer of 6 bp (E1-E2 sp6). We selected <i>n</i> = 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 <i>Bmal1</i>^<i>-/-</i>, only one E-box appears occupied. B. Width (left-side <i>y</i>-axis, green) of the protected region in WT and in <i>Bmal1</i>^<i>-/-</i> mice for E1-E2 sp6 motifs occupied by BMAL1. Fraction of predicted occupied sites is shown in blue (right-side <i>y</i>-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.</p

Distal DNase I Hypersensitive Sites (DHSs) help identify diurnally active transcription regulators.

<p>A. Scheme of the linear model to infer active transcription regulators: transcription factor (TF) motifs in DHSs within a symmetric window around active transcription start sites (TSSs) are used to explain diurnal rhythms in transcription. B. Fraction of explained temporal variance (deviance ratio) in RNA polymerase II (Pol II) loading (at the TSS of all actives genes) for WT and <i>Bmal1</i>^-/- mice, in function of the window size (radius) for DHS inclusion, shows a maximum at around 50 kb. Here, <i>α</i> = 0 was used in the glmnet (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001069#sec015" target="_blank">Materials and methods</a>). C–D. Inferred TF motif activities for WT and in <i>Bmal1</i>^-/- mice shown with amplitudes (distance from center) and peak times (clockwise, ZT0 at the top) using a window size of 50 kb. All 819 (WT) and 629 (<i>Bmal1</i>^-/-) motifs (overlap is 427) with nonzero activities are shown. Note though that most activities are very small and cluster in the center. Certain families of TFs are indicated in colors (full results are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001069#pbio.2001069.s018" target="_blank">S4 Table</a>). Radial scale for activities is arbitrary but comparable in C and D. E. Quantification of western blots for pCREB (Ser 133 phosphorylation) and CREB in WT and <i>Bmal1</i>^<i>-</i>/- genotypes (log₂ (pCREB/CREB)). Nuclear extracts from four independent livers were harvested every 2 h. Both genotypes showed a significant oscillation (<i>p</i> < 0.05, harmonic regression) of the mean signal from the four mice. Though the peak time in <i>Bmal1</i>^-/- mice is delayed by 1.8 h, the comparison of the rhythm in the two genotypes was not significant (<i>p</i> = 0.49, Chow test). Individual blots are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001069#pbio.2001069.s007" target="_blank">S7 Fig</a>.</p