On the Relationship Between Protein-DNA Interactions and Circadian Gene Expression in Mouse Liver

Virtually every cell in our body tracks daily time using an endogenous mechanism called the circadian clock, which has a period of about 24 hours. This timing system offers a selective advantage by enabling organisms to anticipate periodic changes in environmental conditions, such as light-dark cycles. As a consequence, most behavioral and physiological processes including sleep/wake patterns, body temperature and blood pressure are influenced and thus occur at the appropriate time of the day. Such molecular oscillators have been identified almost over the full tree of life, ranging from bacteria to human, as well as insects, fungi and plants. In the mouse liver, the circadian system has been involved in several processes such as glucose homeostasis, cholesterol biosynthesis and gating of the cell cycle. At the molecular level, the circadian clock uses interlocked feedback loops in which the heterodimeric transcription factor BMAL1/CLOCK in mammals or CLK/CYC in flies, plays a central role. In this transcriptional regulatory network, BMAL1/CLOCK activates via E-box motifs transcription of several repressors: the PER and CRY proteins, which repress their own activation by BMAL1/CLOCK, and the nuclear receptor REV-ERB??, which negatively regulates the transcription of the Bmal1 gene. BMAL1/CLOCK also drives rhythmic transcription of PAR-bZIP transcription factors such as DBP/HLF/TEF, which allow for phase-specific expression of many metabolic genes in the liver. While there is prominent control of physiological and behavioral functions by the circadian clock, the detailed links between circadian regulators and downstream targets are poorly known. Thus the main goal of this research was to apply combined experimental and computational methods to further elucidate these interactions, both in flies and in mammals. Identifying the targets of the heterodimeric CLK/CYC (or BMAL1/CLOCK in mammals) basic-helix-loop-helix (bHLH) transcription factor poses challenges and it has been difficult to decipher its specific sequence affinity beyond a canonical E-box motif. We used a comparative genomics approach to identify and model CLK/CYC bound enhancers. The presence of two highly conserved tandem E-box motifs (E1-E2) was detected among CLK/CYC targets genes in flies. A probabilistic model was derived from these sequences and validated with functional genomics datasets. A phylogenetic analysis showed that this motif is evolutionarily conserved among mammals, fishes and insects. Subsequently, we developed computational and experimental methods aimed at defining the target genes of BMAL1/CLOCK in a complex tissue such as the mouse liver. To this end, we mapped all DNA-binding sites of BMAL1 during one circadian cycle using chromatin immunoprecipitation combined with deep sequencing (ChIP-Seq). As a control experiment, we performed a ChIP-Seq experiment for the heterodimeric partner, CLOCK. We developed a novel deconvolution-based method for optimal detection of binding sites from ChIP-Seq data and implemented a bioinformatics pipeline to establish the relationship between transcription factor binding and mRNA expression. Our computational analysis revealed widespread daily rhythms in DNA binding, with maximum levels peaking at midday. In the list of candidate targets, genes belonging to the core circadian clockwork stood out as the most strongly bound, often showing multiple binding sites. In addition, BMAL1 targets were highly enriched in genes involved in carbohydrate and lipid metabolism, cancer pathways, and also in transcription factors, in particular nuclear receptors. Thus, our results show that BMAL1 directly controls precise processes such as glucose homeostasis and, at the same time, uses other transcriptional regulators to spread the circadian signal to a large number of indirect targets. Additionally, we characterized the DNA-binding specificity of BMAL1 using a combination of biochemical and bioinformatics methods. Genomic sequence analysis of the sites using Hidden Markov Models (HMMs) identified E-boxes and tandem E-box (E1-E2) consensus elements. Interestingly, we found that strongly bound BMAL1 sites exhibit high phylogenetic conservation among placental mammals, which was even more pronounced in core circadian genes. Electromobility shift assays showed that E1-E2 sites are bound by a dimer of BMAL1/CLOCK heterodimers with a spacing-dependent cooperative interaction, a finding that was further validated in transactivation assays. A highly significant fraction of BMAL1 target genes showed cyclic mRNA expression profiles with a phase distribution delayed by about 4 hours compared to the binding. Importantly, sites with E1-E2 elements showed tighter phase distribution in mRNA accumulation. These analyses and experiments revealed the importance of tandem E1-E2 elements, which may favor strong binding and precise timing of daily gene expression. Finally, analyzing the temporal profiles of BMAL1 binding, precursor mRNA and mature mRNA levels showed how transcriptional and posttranscriptional regulation contribute differentially to circadian expression phase. Taken together, our interdisciplinary approach clearly established BMAL1/CLOCK's primary function as a master regulator of the core circadian oscillator, while demonstrating also its direct contribution to a variety of output programs. Thus our results may help to better understand metabolic diseases linked to dysfunctions of the circadian clockwork. Moreover, a more precise description of the clock-controlled liver functions might be also useful in chronotherapy in order to improve effectiveness and tolerance of drugs by treating patients at the right moment of the day.


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