Understanding Circadian Output Networks in Neurospora Crassa

The Neurospora crassa circadian clock is based on a highly regulated molecular negative feedback loop, similar to molecular clocks in all eukaryotes. A core component of the N. crassa molecular clock is the White Collar complex (WCC), composed of the blue light photoreceptor WC-1 and its partner WC-2. The WCC serves as a master regulator that controls light signaling, and the precise timing of target gene expression. Up to 40% of the eukaryote genome is under the control of the clock at the level of transcript abundance, but the molecular links between the core oscillator and downstream target genes, as well as the mechanisms controlling the phase of rhythmic gene expression, are not understood. Using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq), about 400 binding sites for the WCC were identified throughout the N. crassa genome. We found that 24 transcription factors (TFs) were significantly enriched among the direct WCC target genes. As expected for genes that are controlled by the WCC, the first-tier TFs are both clock- and light-regulated. These data led to the hypothesis that the WCC functions to control rhythms in TFs, which in turn control rhythmicity and phase of downstream target genes and processes. To test this hypothesis, the first-tier TF ADV-1 (Arrested Development-1) was investigated in detail to characterize the downstream circadian genetic network. ADV-1 target genes were identified using ChIP- and RNA-seq, and as expected many ADV-1 downstream target genes were light-responsive and/or clock-controlled. An enrichment for ADV-1 target genes involved in cell fusion, a process that is critical for normal vegetative and sexual development in N. crassa, provided a rationale for the observed developmental defects in ADV-1 deletion cells, and suggested that cell fusion is clock-controlled. Importantly, this work revealed that the transduction of time-of-day information through ADV-1 to its downstream targets is more complex than anticipated. Specifically, I show that deletion of ADV-1 does not always lead to predicted changes in rhythmic gene expression and/or phase, suggesting that ADV-1 functions in combination with other first-tier TFs to control rhythmicity. In support of this idea, genome-wide binding profiles of all of the first-tier TFs uncovered complex feedback and feed forward regulation involving ADV-1. Thus, my data revealed that in order to fully understand how the clock signals phase information to downstream targets, we need to go beyond the candidate gene approach, and instead develop computational models from our TF ChIP-seq and rhythmic transcriptome data to model how time of day information is transduced in the molecular circadian output gene network. Predictions of the model can then be validated using ADV-1 deletion cells alone, or in combination with deletion of other first-tier TFs in the network, with the goal of deriving design principles that define conserved aspects of the circadian output network in all eukaryotes, and important in human health. To test this hypothesis, the first-tier TF ADV-1 (Arrested Development-1) was investigated in detail to characterize the downstream circadian genetic network. ADV-1 target genes were identified using ChIP- and RNA-seq, and as expected many ADV-1 downstream target genes were light-responsive and/or clock-controlled. An enrichment for ADV-1 target genes involved in cell fusion, a process that is critical for normal vegetative and sexual development in N. crassa, provided a rationale for the observed developmental defects in ADV-1 deletion cells, and suggested that cell fusion is clock- controlled. Importantly, this work revealed that the transduction of time-of-day information through ADV-1 to its downstream targets is more complex than anticipated. Specifically, I show that deletion of ADV-1 does not always lead to predicted changes in rhythmic gene expression and/or phase, suggesting that ADV-1 functions in combination with other first-tier TFs to control rhythmicity. In support of this idea, genome-wide binding profiles of all of the first-tier TFs uncovered complex feedback and feed forward regulation involving ADV-1. Thus, my data revealed that in order to fully understand how the clock signals phase information to downstream targets, we need to go beyond the candidate gene approach, and instead develop computational models from our TF ChIP-seq and rhythmic transcriptome data to model how time of day information is transduced in the molecular circadian output gene network. Predictions of the model can then be validated using ADV-1 deletion cells alone, or in combination with deletion of other first-tier TFs in the network, with the goal of deriving design principles that define conserved aspects of the circadian output network in all eukaryotes, and important in human health

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

The Neurospora Transcription Factor ADV-1 Transduces Light Signals and Temporal Information to Control Rhythmic Expression of Genes Involved in Cell Fusion

Light and the circadian clock have a profound effect on the biology of organisms through the regulation of large sets of genes. Toward understanding how light and the circadian clock regulate gene expression, we used genome-wide approaches to identify the direct and indirect targets of the light-responsive and clock-controlled transcription factor ADV-1 in Neurospora crassa. A large proportion of ADV-1 targets were found to be light- and/or clock-controlled, and enriched for genes involved in development, metabolism, cell growth, and cell fusion. We show that ADV-1 is necessary for transducing light and/or temporal information to its immediate downstream targets, including controlling rhythms in genes critical to somatic cell fusion. However, while ADV-1 targets are altered in predictable ways in Δadv-1 cells in response to light, this is not always the case for rhythmic target gene expression. These data suggest that a complex regulatory network downstream of ADV-1 functions to generate distinct temporal dynamics of target gene expression relative to the central clock mechanism