Rhythmic CLOCK-BMAL1 binding to multiple E-box motifs drives circadian Dbp transcription and chromatin transitions

Abstract

Mammalian circadian rhythms are based on transcriptional and post-translational feedback loops. Essentially, the activity of the transcription factors BMAL1 (also known as MOP3) and CLOCK is rhythmically counterbalanced by Period (PER) and Cryptochrome (CRY) proteins to govern time of day-dependent gene expression 1 . Here we show that circadian regulation of the mouse albumin D element-binding protein (Dbp) gene involves rhythmic binding of BMAL1 and CLOCK and marked daily chromatin transitions. Thus, the Dbp transcription cycle is paralleled by binding of BMAL1 and CLOCK to multiple extra-and intragenic E boxes, acetylation of Lys9 of histone H3, trimethylation of Lys4 of histone H3 and a reduction of histone density. In contrast, the antiphasic daily repression cycle is accompanied by dimethylation of Lys9 of histone H3, the binding of heterochromatin protein 1a and an increase in histone density. The rhythmic conversion of transcriptionally permissive chromatin to facultative heterochromatin relies on the presence of functional BMAL1-CLOCK binding sites.Circadian rhythms are based on cell-autonomous oscillators that maintain gene expression with a period length of about a day even in the absence of external timing cues 2,3 . In mammals, the rhythmgenerating molecular circuitry is thought to rely on the opposing effects of transcriptional activators and repressors that generate a negative feedback loop [1][2][3] . Briefly, the PAS domain helix-loop-helix proteins BMAL1 and CLOCK stimulate transcription of Cry and Per via binding to E-box motifs present in these genes. Once the repressor proteins CRY and PER reach a critical concentration, they form complexes with the CLOCK-BMAL heterodimer and thereby attenuate the transactivation potential of these transcription factors. As a consequence, Per and Cry transcription is reduced, PER and CRY accumulation decreases below the concentration required for autorepression and a new transcription cycle of PER and CRY genes can ensue.The transcription factor DBP shows high-amplitude circadian oscillations 4,5 , and we found that Dbp transcription reaches its maximum and minimum at Zeitgeber time (ZT) 7 and ZT19, respectively (Fig. 1a), where ZT0 and ZT12 are the times when lights are switched on and off, respectively. Putative circadian regulatory regions containing E-box motifs have previously been mapped in the Dbp locus through the identification of DNase I-hypersensitive sites 6 , and genetic experiments have suggested that at least some of these might serve as binding sites for BMAL1 and CLOCK 6,7 . To examine whether these sites indeed bind CLOCK and BMAL1 in vivo, we have used a chromatin immunoprecipitation (ChIP) technique that we developed for the analysis of solid tissues (see Methods). For simplicity, the Dbp gene was subdivided into nine B800-bp segments according to the previously established map of DNase I hypersensitivity sites. ChIP experiments, performed with liver chromatin at 2-h intervals around the clock, suggested that both CLOCK and BMAL1 bind to ...

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“…2f) was performed as previously described 61 . Primers for the E-box-containing region in the mouse Ho-1 promoter were: 5 -TGCAACAGCAGCGAGAAC-3 (forward), 5 -GAAACTC TGGAGGCGACTG-3 (reverse) and 5 -FAM-CCACCACGTGACCCGCGTAC-TAMRA-3 (probe).…”

“…Cross-linking, chromatin preparation and chromatin immunoprecipitation were performed as described in ref. 61, with modifications. Cells were sonicated on ice for 15 s five times at a 50% setting, then centrifuged for 10 min (16,000g).…”

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“…Using the circadian transcription factor albumin D-site binding protein (Dbp) gene as a model system, Stratmann et al (2010) uncovered a mechanism that partially uncouples the expression of this gene from the circadian oscillator. The Dbp gene has been identified as a direct target for rhythmic BMAL1 and CLOCK binding (Kiyohara et al, 2008;Ripperger and Schibler, 2006;Ripperger et al, 2000;Yamaguchi et al, 2000). In their study, Stratmann et al (2010) have identified a special function of a BMAL1 binding site in the promoter region of the Dbp gene to adjust the phase of its expression according to the photoperiod.…”

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