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In mammals, the circadian clock coordinates cell physiological processes including inflammation. Recent studies suggested a crosstalk between these two pathways. However, the mechanism of how inflammation affects the clock is not well understood. Here, we investigated the role of the proinflammatory transcription factor NF-κB in regulating clock function. Using a combination of genetic and pharmacological approaches, we show that perturbation of the canonical NF-κB subunit RELA in the human U2OS cellular model altered core clock gene expression. While RELA activation shortened period length and dampened amplitude, its inhibition lengthened period length and caused amplitude phenotypes. NF-κB perturbation also altered circadian rhythms in the master suprachiasmatic nucleus (SCN) clock and locomotor activity behavior under different light/dark conditions. We show that RELA, like the clock repressor CRY1, repressed the transcriptional activity of BMAL1/CLOCK at the circadian E-box cis-element. Biochemical and biophysical analysis showed that RELA binds to the transactivation domain of BMAL1. These data support a model in which NF-kB competes with CRY1 and coactivator CBP/p300 for BMAL1 binding to affect circadian transcription. This is further supported by chromatin immunoprecipitation analysis showing that binding of RELA, BMAL1 and CLOCK converges on the E-boxes of clock genes. Taken together, these data support a significant role for NF-κB in directly regulating the circadian clock and highlight mutual regulation between the circadian and inflammatory pathways. 

Circadian clocks control gene expression to provide an internal representation of local time. We report reconstitution of a complete cyanobacterial circadian clock in vitro, including the central oscillator, signal transduction pathways, downstream transcription factor, and promoter DNA. The entire system oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of each component simultaneously without user intervention. We identified the molecular basis for loss of cycling in an arrhythmic mutant and explored fundamental mechanisms of timekeeping in the cyanobacterial clock. We find that SasA, a circadian sensor histidine kinase associated with clock output, engages directly with KaiB on the KaiC hexamer to regulate period and amplitude of the central oscillator. SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex and enhance rhythmicity of the oscillator, particularly under limiting concentrations of KaiB. Thus, the expanded in vitro clock reveals previously unknown mechanisms by which the circadian system of cyanobacteria maintains the pace and rhythmicity under variable protein concentrations. 

Multisite phosphorylation of the PERIOD 2 (PER2) protein is the key step that determines the period of the mammalian circadian clock. Previous studies concluded that an unidentified kinase is required to prime PER2 for subsequent phosphorylation by casein kinase 1 (CK1), an essential clock component that is conserved from algae to humans. These subsequent phosphorylations stabilize PER2, delay its degradation, and lengthen the period of the circadian clock. Here, we perform a comprehensive biochemical and biophysical analysis of mouse PER2 (mPER2) priming phosphorylation and demonstrate, surprisingly, that CK1δ/ε is indeed the priming kinase. We find that both CK1ε and a recently characterized CK1δ2 splice variant more efficiently prime mPER2 for downstream phosphorylation in cells than the well-studied splice variant CK1δ1. While CK1 phosphorylation of PER2 was previously shown to be robust to changes in the cellular environment, our phosphoswitch mathematical model of circadian rhythms shows that the CK1 carboxyl-terminal tail can allow the period of the clock to be sensitive to cellular signaling. These studies implicate the extreme carboxyl terminus of CK1 as a key regulator of circadian timing.