Reconstitution of an intact clock reveals mechanisms of circadian timekeeping.

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.

A Combined Computational and Genetic Approach Uncovers Network Interactions of the Cyanobacterial Circadian Clock.

UNLABELLED: Two-component systems (TCS) that employ histidine kinases (HK) and response regulators (RR) are critical mediators of cellular signaling in bacteria. In the model cyanobacterium Synechococcus elongatus PCC 7942, TCSs control global rhythms of transcription that reflect an integration of time information from the circadian clock with a variety of cellular and environmental inputs. The HK CikA and the SasA/RpaA TCS transduce time information from the circadian oscillator to modulate downstream cellular processes. Despite immense progress in understanding of the circadian clock itself, many of the connections between the clock and other cellular signaling systems have remained enigmatic. To narrow the search for additional TCS components that connect to the clock, we utilized direct-coupling analysis (DCA), a statistical analysis of covariant residues among related amino acid sequences, to infer coevolution of new and known clock TCS components. DCA revealed a high degree of interaction specificity between SasA and CikA with RpaA, as expected, but also with the phosphate-responsive response regulator SphR. Coevolutionary analysis also predicted strong specificity between RpaA and a previously undescribed kinase, HK0480 (herein CikB). A knockout of the gene for CikB (cikB) in a sasA cikA null background eliminated the RpaA phosphorylation and RpaA-controlled transcription that is otherwise present in that background and suppressed cell elongation, supporting the notion that CikB is an interactor with RpaA and the clock network. This study demonstrates the power of DCA to identify subnetworks and key interactions in signaling pathways and of combinatorial mutagenesis to explore the phenotypic consequences. Such a combined strategy is broadly applicable to other prokaryotic systems. IMPORTANCE: Signaling networks are complex and extensive, comprising multiple integrated pathways that respond to cellular and environmental cues. A TCS interaction model, based on DCA, independently confirmed known interactions and revealed a core set of subnetworks within the larger HK-RR set. We validated high-scoring candidate proteins via combinatorial genetics, demonstrating that DCA can be utilized to reduce the search space of complex protein networks and to infer undiscovered specific interactions for signaling proteins in vivo Significantly, new interactions that link circadian response to cell division and fitness in a light/dark cycle were uncovered. The combined analysis also uncovered a more basic core clock, illustrating the synergy and applicability of a combined computational and genetic approach for investigating prokaryotic signaling networks.

Combinatorial control of light induced chromatin remodeling and gene activation in Neurospora.

Light is an important environmental cue that affects physiology and development of Neurospora crassa. The light-sensing transcription factor (TF) WCC, which consists of the GATA-family TFs WC1 and WC2, is required for light-dependent transcription. SUB1, another GATA-family TF, is not a photoreceptor but has also been implicated in light-inducible gene expression. To assess regulation and organization of the network of light-inducible genes, we analyzed the roles of WCC and SUB1 in light-induced transcription and nucleosome remodeling. We show that SUB1 co-regulates a fraction of light-inducible genes together with the WCC. WCC induces nucleosome eviction at its binding sites. Chromatin remodeling is facilitated by SUB1 but SUB1 cannot activate light-inducible genes in the absence of WCC. We identified FF7, a TF with a putative O-acetyl transferase domain, as an interaction partner of SUB1 and show their cooperation in regulation of a fraction of light-inducible and a much larger number of non light-inducible genes. Our data suggest that WCC acts as a general switch for light-induced chromatin remodeling and gene expression. SUB1 and FF7 synergistically determine the extent of light-induction of target genes in common with WCC but have in addition a role in transcription regulation beyond light-induced gene expression.

Dawn- and dusk-phased circadian transcription rhythms coordinate anabolic and catabolic functions in Neurospora.

BACKGROUND: Circadian clocks control rhythmic expression of a large number of genes in coordination with the 24 hour day-night cycle. The mechanisms generating circadian rhythms, their amplitude and circadian phase are dependent on a transcriptional network of immense complexity. Moreover, the contribution of post-transcriptional mechanisms in generating rhythms in RNA abundance is not known. RESULTS: Here, we analyzed the clock-controlled transcriptome of Neurospora crassa together with temporal profiles of elongating RNA polymerase II. Our data indicate that transcription contributes to the rhythmic expression of the vast majority of clock-controlled genes (ccgs) in Neurospora. The ccgs accumulate in two main clusters with peak transcription and expression levels either at dawn or dusk. Dawn-phased genes are predominantly involved in catabolic and dusk-phased genes in anabolic processes, indicating a clock-controlled temporal separation of the physiology of Neurospora. Genes whose expression is strongly dependent on the core circadian activator WCC fall mainly into the dawn-phased cluster while rhythmic genes regulated by the glucose-dependent repressor CSP1 fall predominantly into the dusk-phased cluster. Surprisingly, the number of rhythmic transcripts increases about twofold in the absence of CSP1, indicating that rhythmic expression of many genes is attenuated by the activity of CSP1. CONCLUSIONS: The data indicate that the vast majority of transcript rhythms in Neurospora are generated by dawn and dusk specific transcription. Our observations suggest a substantial plasticity of the circadian transcriptome with respect to the number of rhythmic genes as well as amplitude and phase of the expression rhythms and emphasize a major role of the circadian clock in the temporal organization of metabolism and physiology.

Metabolic compensation of the Neurospora clock by a glucose-dependent feedback of the circadian repressor CSP1 on the core oscillator.

Conidial separation 1 (CSP1) is a global transcription repressor. It is expressed under control of the white collar complex (WCC), the core transcription factor of the circadian clock of Neurospora. Here we report that the length of the circadian period decreases with increasing glucose concentrations in csp1 mutant strains, while the period is compensated for changes in glucose concentration in wild-type strains. Glucose stimulated CSP1 expression. Overexpression of CSP1 caused period lengthening and, eventually, complete dampening of the clock rhythm. We show that CSP1 inhibits expression of the WHITE COLLAR 1 (WC1) subunit of the WCC by repressing the wc1 promoter. Glucose-dependent repression of wc1 transcription by CSP1 compensated for the enhanced translation of WC1 at high glucose levels, resulting in glucose-independent expression of the WCC and, hence, metabolic compensation that maintained a constant circadian period. Thus, the negative feedback of CSP1 on WC1 expression constitutes a molecular pathway that coordinates energy metabolism and the circadian clock.

A global circadian repressor controls antiphasic expression of metabolic genes in Neurospora.

The white-collar complex (WCC), the core transcription factor of the circadian clock of Neurospora, activates morning-specific expression of the transcription repressor CSP1. Newly synthesized CSP1 exists in a transient complex with the corepressor RCM1/RCO1 and the ubiquitin ligase UBR1. CSP1 is rapidly hyperphosphorylated and degraded via UBR1 and its ubiquitin conjugase RAD6. Genes controlled by CSP1 are rhythmically expressed and peak in the evening (i.e., in antiphase to morning-specific genes directly controlled by WCC). Rhythmic expression of these second-tier genes depends crucially on phosphorylation and rapid turnover of CSP1, which ensures tight coupling of CSP1 abundance and function to the circadian activity of WCC. Negative feedback of CSP1 on its own transcription buffers the amplitude of CSP1-dependent oscillations against fluctuations of WCC activity. CSP1 predominantly regulates genes involved in metabolism. It controls ergosterol synthesis and fatty acid desaturases and thereby modulates the lipid composition of membranes.

Transcription factors in light and circadian clock signaling networks revealed by genomewide mapping of direct targets for neurospora white collar complex.

Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.

Activity of the circadian transcription factor White Collar Complex is modulated by phosphorylation of SP-motifs.

Posttranslational modifications, particularly phosphorylation, regulate activity, stability and localization of proteins in circadian clocks, thereby contributing to a stable oscillation with a period of approximately 24h. The White Collar Complex (WCC) is the central transcription factor of the circadian clock of Neurospora crassa. Its activity is regulated in a circadian manner by rhythmic phosphorylation, mediated by the clock protein Frequency (FRQ). Here we present purification of TAP-tagged WCC and identification of novel phosphorylation sites of WC-1 and WC-2, all of which appear to be proline directed. Exchange of a single WC-2 serine residue (S433) to alanine or aspartate affects WCC-dependent transcription and circadian period, suggesting an important role of WC-2 S433 phosphorylation for WCC activity and circadian timing.