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 b