CLOCK:BMAL1 is a pioneer-like transcription factor

Abstract

The mammalian circadian clock relies on the master genes CLOCK and BMAL1 to drive rhythmic gene expression and regulate biological functions under circadian control. Here we show that rhythmic CLOCK:BMAL1 DNA binding promotes rhythmic chromatin opening. Mechanisms include CLOCK:BMAL1 binding to nucleosomes and rhythmic chromatin modification; e.g., incorporation of the histone variant H2A.Z. This rhythmic chromatin remodeling mediates the rhythmic binding of other transcription factors adjacent to CLOCK:BMAL1, suggesting that the activity of these other transcription factors contributes to the genome-wide CLOCK:BMAL1 heterogeneous transcriptional output. These data therefore indicate that the clock regulation of transcription relies on the rhythmic regulation of chromatin accessibility and suggest that the concept of pioneer function extends to acute gene regulation.

Keywords: H2A.Z; MNase-seq; chromatin modifications; circadian rhythms; nucleosome occupancy; nucleosome positioning; regulation of transcription.

Figure 1.

CLOCK:BMAL1 promotes the rhythmic removal of nucleosomes at its DNA-binding sites. (A) Average nucleosome signal at the top 400 CLOCK:BMAL1 DNA-binding sites (±0.6 kb) in mouse livers during the light phase (ZT2, ZT6, and ZT10; green) and dark phase (ZT14, ZT18, and ZT22; red/orange) of wild-type mice and in Bmal1^−/− mice (average signal for six time points; black). (B) Effect of CLOCK:BMAL1 DNA-binding strength on the average nucleosome signal at CLOCK:BMAL1 DNA-binding sites (±75 bp), at times of high binding (ZT6 and ZT10; open circles) or low binding (ZT18 and ZT22; closed circles) to DNA in wild-type (black) and Bmal1^−/− (blue) mice. The 3217 CLOCK:BMAL1 peaks were equally distributed in 10 bins based on the strength of CLOCK:BMAL1 DNA binding. (C) Average nucleosome signal at the top 400 Rev-erbA DNA-binding sites (±1 kb) in mouse livers. (D–F) Average nucleosome signal at the top 25% of CLOCK:BMAL1 DNA-binding sites (±0.8 kb) located in gene bodies (D), intergenic regions (E), and TSSs (F) in mouse livers.

Figure 2.

CLOCK binds to DNA wrapped around nucleosomes. (A) CLOCK ChIP-seq signal on mononucleosome (i.e., mouse liver chromatin digested by MNase) at ZT22 (light blue; left) and ZT06 (dark blue; right) for the top 400 CLOCK:BMAL1 DNA-binding sites. The signal corresponds to the average of three independent ChIP-seq experiments. Input MNase-seq signal at the same binding sites is displayed for both time points (ZT22 [dark orange] and ZT06 [green]). (B) CLOCK ChIP-seq over input signal ratio on MNase-treated chromatin at ZT22 (light blue) and ZT06 (dark blue) at the top 400 CLOCK:BMAL1 DNA-binding sites. (C) CLOCK ChIP-seq over input signal ratio on sonicated chromatin at ZT22 (orange) and ZT06 (green) at the top 400 CLOCK:BMAL1 DNA-binding sites.

Figure 3.

Rhythmic CLOCK binding on DNA is associated with rhythmic H2A.Z signal at CLOCK:BMAL1 DNA-binding sites. (A) H2A.Z ChIP-seq over input signal ratio on MNase-treated chromatin in wild-type mice during the light phase (green) and dark phase (orange/red) and in Bmal1^−/− mice (average of six time points; black). Signal ratio is displayed at CLOCK:BMAL1 peaks located within gene bodies, intergenic regions, or TSSs. (B) Average H2A.Z ChIP-seq/input signal (top) and percentage of nucleosome signal decrease (bottom) at TSSs, intergenic regions, and gene bodies. (Left) Values in Bmal1^−/− mice. (Right) CLOCK:BMAL1-specific contribution (e.g., values above those observed in Bmal1^−/− mice).

Figure 4.

CLOCK:BMAL1-mediated rhythmic nucleosome removal promotes the rhythmic binding of transcription factors to DNA. (A) Coassociation between transcription factors in mouse livers. Thirty-one publicly available mouse liver ChIP-seq data sets were analyzed by pairs using the Genome Structure Correction statistic as previously described (Dunham et al. 2012). Black rectangles denote core clock genes and nuclear receptors (see the text for more details). (B) Percentage of overlap between 31 publicly available mouse liver ChIP-seq data sets. Black rectangles denote transcription factors that exhibit an overlap superior to 40% with core clock genes (see the text for more details). (C) Rhythmic nucleosome signal at a CLOCK:BMAL1 DNA-binding site located near HNF6 DNA-binding sites. Nucleosome signal is displayed for wild-type mice at time of high (average ZT6 and ZT10; green) or low (average ZT18 and ZT22; red) CLOCK:BMAL1 DNA binding. The HNF6 ChIP-seq signal from Faure et al. (2012) is shown in gray. Genomic locations of CLOCK:BMAL1 (blue) and HNF6 (black) consensus sequences are also displayed. (D) HNF6 ChIP-seq signal in mouse livers at ZT08 (white) and ZT20 (black) at several specific DNA-binding sites (n = 4 mice per time point). Values represent the average ± SEM. (**) P < 0.05; (***) P < 0.01. (E) Model illustrating a new mechanism by which CLOCK:BMAL1 regulates the expression of its target genes.