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

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 GATAfamily 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

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

Combinatorial Control of Light Induced Chromatin Remodeling and Gene Activation in <i>Neurospora</i>

<div><p>Light is an important environmental cue that affects physiology and development of <i>Neurospora crassa</i>. 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.</p></div

FF7 interacts weakly with SUB1 and co-regulates light-inducible and non light-inducible genes.

<p><b>A-B</b>. Western blots showing co-immunoprecipitation (co-IP) of <b>(A)</b> SUB1 with FF7_FLAG-HIS and <b>(B)</b> FF7_FLAG-HIS with SUB1. FLAG antibody was used for FF7_FLAG-HIS IP and α-SUB1 antibody was used for SUB1 IP. The asterisks (*) indicate cross-reactions of the FLAG antibody. <b>C</b>. FF7 binding motifs identified by MEME. The top 200 binding sites identified by FF7 ChIP-seq were used for the motif analysis. The upper motif is found in 117 / 200 binding sites whereas the lower motif is found in 36 / 200 binding sites. <b>D</b>. Occurrence of the major FF7 motif at FF7 binding sites. The grey area shows the occupancy of FF7 binding sites determined by ChIP-seq. The red line shows the occurrence of the FF7 binding motif “t/c AAGCG c/a”. <b>E</b>. Wig file showing MNase-WC2, SUB1 and FF7 ChIP-seq signals at the <i>rds1</i> promoter. Numbers on the ChIP-seq panels correspond the maximum coverage shown in the wig file. <b>F</b>. Venn-diagram showing the overlap between SUB1, WC2 and FF7 ChIP-seq signals. <b>G</b>. Heat-map showing light-inducible genes with significantly lower RNA levels in Δ<i>sub1</i> and in Δ<i>ff7</i> strains in comparison to <i>wt</i>. <b>H</b>. Wig file (left panel) showing the nucleosome position and occupancy at the <i>rds1</i> promoter in <i>wt</i> and Δ<i>ff7</i> strains in the dark and after light-exposure. The MNase-WC2 ChIP-seq (blue) is shown below the nucleosome signals. Numbers on the ChIP-seq panels show the maximum coverage shown in the wig file. ChIP-PCR analysis (right panel) of H2A occupancy at the binding sites of WCC and SUB1 at <i>rds1</i> promoter in the dark and 20 min after light-exposure (± SEM, n = 4). a<i>ctin</i> DNA was used for normalization. w<i>t</i> dark level was set to 1. <b>I</b>. Nucleosome occupancy at binding sites of WCC (n = 92) and in Δ<i>ff7</i> in dark (dotted lines) and 20 min after light-exposure (solid lines).</p

Light- and WCC-dependent nucleosome eviction is transcription independent.

<p><b>A</b>. Line graphs showing the averaged nucleosome occupancy in transcribed genes and promoters of all annotated <i>Neurospora</i> genes (n = 9733) in <i>wt</i>, Δ<i>sub1</i> and Δ<i>wc2</i> strains in dark (red) and 20 min (blue) after light-exposure of cultures. The center of the +1 nucleosome (nucleosome overlapping the annotated transcription start site) was used for alignment of sequence coverage of MNase-resistant fragments >100bp. <b>B</b>. Wig file showing the nucleosome position and occupancy at the <i>rds1</i> promoter in <i>wt</i>, Δ<i>sub1</i> and Δ<i>wc2</i> strains in the dark and after light-exposure. MNase-WC2 ChIP-seq (blue) is shown below the nucleosome signals. Numbers on the ChIP-seq panels show the maximum read coverage shown in the wig file. <b>C</b>. ChIP-PCR analysis showing H2A occupancy in the dark and 20 min after light exposure at the binding sites of WCC and SUB1 in the <i>rds1</i> promoter. ChIP was performed by immunoprecipitation with H2A antibody (± SEM, n = 4). a<i>ctin</i> gene was used for normalization. <i>wt</i> dark level was set to 1. <b>D</b>. Transcription-independent light-induced nucleosome eviction at WCC binding sites (BS). Four examples (wig files) of nucleosome position and occupancy at WCC BS in <i>wt</i>, Δ<i>sub1</i> and Δ<i>wc2</i> strains are shown. WCC binding (TAP-WC2 ChIP-seq) is shown above the nucleosome signals. The positions of GATC motifs are shown in the lower panels. Numbers on the ChIP-seq panels indicate the maximum nucleosome coverage shown in the Wig file. Regions used for ChIP-PCR analysis are indicated by black lines. <b>E</b>. ChIP-PCR analysis showing H2A occupancy in the regions shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005105#pgen.1005105.g004" target="_blank">Fig. 4D</a>. Occupancy of H2A was determined by immunoprecipitation with H2A antibody (± SEM, n = 4). a<i>ctin</i> gene was used for normalization. w<i>t</i> dark level was set to 1.</p

RCO-1 and RCM-1 regulate light-dependent gene transcription and photoadaptation.

<p>Quantitative RT-PCR experiments were performed to measure the relative accumulation of mRNA in mycelia of the wild type, a strain with a deletion of <i>rco-1</i> or a strain with the <i>rcm-1</i>^RIP allele grown at 22°C for 48 h in the dark and exposed to white light (1 W/m² blue light) for different times. The plots show the average and standard error of the mean of the relative mRNA accumulation in three independent experiments. The results from each PCR for each gene were normalized to the corresponding PCR for <i>tub-2</i> to correct for sampling errors and normalized to those obtained with the wild type after exposure to 30 min of light.</p