Genome-Wide and Phase-Specific DNA-Binding Rhythms of BMAL1 Control Circadian Output Functions in Mouse Liver
(A) Cumulative distribution of BMAL1 site positions relative to the closest annotated Ensembl TSSs show that 40% of all sites are within 1 kb of a TSS. (B) Histogram of BMAL1 site positions, showing that BMAL1 sites cluster near TSSs, with a maximal density near −100 bp. No clustering is found near gene 3′ ends. (C) Positioning of BMAL1 sites near coding genes. Sites were assigned according to the following definitions: promoter (site is within ±2 kb of an annotated TSS), upstream (−10 kb to −2 kb), gene (+2 kb to the polyadenylation site), downstream (polyadenylation site to +10 kb), and other (not in any of the four previous classes). The fractions expected from the respective sizes of the classes in the genome show that BMAL1 sites are mostly overrepresented in promoters and depleted inside genes. (D) Number of sites in close proximity (<10 kb) to annotated features, including non-coding RNAs (ncRNAs). These are split in micro RNAs (miRNAs) and others (long intergenic non-coding RNAs, small nucleolar RNAs, ribosomal RNAs, small nuclear RNAs, and miscellaneous RNAs). Middle column: fraction of sites from each category; last column: background fraction from the Ensembl annotation. The vast majority of sites (83%) are near genes expressed in liver (see Materials and Methods). (E) Strong BMAL1 binding correlates with high phylogenetic conservation. Sites are ranked according to the number of tags at their peak binding, and all Ensembl TSSs are shown as controls. In the window of ±50 bp around each site, the maximal value of the placental mammals PhastCons conservation score is used. PhastCons score ranges from 0 (no conservation) to 1 (perfect conservation). (F) Mean PhastCons conservation score for three classes of sites: 41 sites in RCGs (defined in Figure 1C), proximal sites (within 1 kb of an annotated Ensembl feature, 709 sites), and distal sites (1,340 sites). All categories are significantly more conserved than control regions (+500 bp downstream of each site). ***, p<1×10−6, Student's t test.