Cooperation between bHLH transcription factors and histones for DNA access.

The basic helix-loop-helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members1. Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2,3). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry-exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A-H2B acidic patch4, the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes5-7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors.

Tissue-specific BMAL1 cistromes reveal that rhythmic transcription is associated with rhythmic enhancer-enhancer interactions.

The mammalian circadian clock relies on the transcription factor CLOCK:BMAL1 to coordinate the rhythmic expression of thousands of genes. Consistent with the various biological functions under clock control, rhythmic gene expression is tissue-specific despite an identical clockwork mechanism in every cell. Here we show that BMAL1 DNA binding is largely tissue-specific, likely because of differences in chromatin accessibility between tissues and cobinding of tissue-specific transcription factors. Our results also indicate that BMAL1 ability to drive tissue-specific rhythmic transcription is associated with not only the activity of BMAL1-bound enhancers but also the activity of neighboring enhancers. Characterization of physical interactions between BMAL1 enhancers and other cis-regulatory regions by RNA polymerase II chromatin interaction analysis by paired-end tag (ChIA-PET) reveals that rhythmic BMAL1 target gene expression correlates with rhythmic chromatin interactions. These data thus support that much of BMAL1 target gene transcription depends on BMAL1 capacity to rhythmically regulate a network of enhancers.

TRITHORAX-dependent arginine methylation of HSP68 mediates circadian repression by PERIOD in the monarch butterfly.

Transcriptional repression drives feedback loops that are central to the generation of circadian (∼24-h) rhythms. In mammals, circadian repression of circadian locomotor output cycles kaput, and brain and muscle ARNT-like 1 (CLOCK:BMAL1)-mediated transcription is provided by a complex formed by PERIOD (PER) and CRYPTOCHROME (CRY) proteins. PER initiates transcriptional repression by binding CLK:BMAL1, which ultimately results in their removal from DNA. Although PER's ability to repress transcription is widely recognized, how PER binding triggers repression by removing CLK:BMAL1 from DNA is not known. Here, we use the monarch butterfly as a model system to address this problem because it harbors a simplified version of the CLK:BMAL1-activated circadian clock present in mammals. We report that an intact CLOCK mouse exon 19 homologous region (CLKe19r) and the histone methyltransferase TRITHORAX (TRX) are both necessary for monarch CLK:BMAL1-mediated transcriptional activation, CLK-PER interaction, and PER repression. Our results show that TRX catalytic activity is essential for CLK-PER interaction and PER repression via the methylation of a single arginine methylation site (R45) on heat shock protein 68 (HSP68). Our study reveals TRX and HSP68 as essential links between circadian activation and PER-mediated repression and suggests a potential conserved clock function for HSPs in eukaryotes.

Lack of food intake during shift work alters the heart transcriptome and leads to cardiac tissue fibrosis and inflammation in rats.

BACKGROUND: Many epidemiological studies revealed that shift work is associated with an increased risk of a number of pathologies, including cardiovascular diseases. An experimental model of shift work in rats has additionally been shown to recapitulate aspects of metabolic disorders observed in human shift workers, including increased fat content and impaired glucose tolerance, and used to demonstrate that restricting food consumption outside working hours prevents shift work-associated obesity and metabolic disturbance. However, the way distinct shift work parameters, such as type of work, quantity, and duration, affect cardiovascular function and the underlying mechanisms, remains poorly understood. Here, we used the rat as a model to characterize the effects of shift work in the heart and determine whether they can be modulated by restricting food intake during the normal active phase. RESULTS: We show that experimental shift work reprograms the heart cycling transcriptome independently of food consumption. While phases of rhythmic gene expression are distributed across the 24-h day in control rats, they are clustered towards discrete times in shift workers. Additionally, preventing food intake during shift work affects the expression level of hundreds of genes in the heart, including genes encoding components of the extracellular matrix and inflammatory markers found in transcriptional signatures associated with pressure overload and cardiac hypertrophy. Consistent with this, the heart of shift worker rats not eating during work hours, but having access to food outside of shift work, exhibits increased collagen 1 deposition and displays increased infiltration by immune cells. While maintaining food access during shift work has less effects on gene expression, genes found in transcriptional signatures of cardiac hypertrophy remain affected, and the heart of shift worker rats exhibits fibrosis without inflammation. CONCLUSIONS: Together, our findings unraveled differential effects of food consumption on remodeled transcriptional profiles of the heart in shift worker rats. They also provide insights into how shift work affects cardiac function and suggest that some interventions aiming at mitigating metabolic disorders in shift workers may have adverse effects on cardiovascular diseases.

Genome-wide discovery of the daily transcriptome, DNA regulatory elements and transcription factor occupancy in the monarch butterfly brain.

The Eastern North American monarch butterfly, Danaus plexippus, is famous for its spectacular seasonal long-distance migration. In recent years, it has also emerged as a novel system to study how animal circadian clocks keep track of time and regulate ecologically relevant daily rhythmic activities and seasonal behavioral outputs. However, unlike in Drosophila and the mouse, little work has been undertaken in the monarch to identify rhythmic genes at the genome-wide level and elucidate the regulation of their diurnal expression. Here, we used RNA-sequencing and Assay for Transposase-Accessible Chromatin (ATAC)-sequencing to profile the diurnal transcriptome, open chromatin regions, and transcription factor (TF) footprints in the brain of wild-type monarchs and of monarchs with impaired clock function, including Cryptochrome 2 (Cry2), Clock (Clk), and Cycle-like loss-of-function mutants. We identified 217 rhythmically expressed genes in the monarch brain; many of them were involved in the regulation of biological processes key to brain function, such as glucose metabolism and neurotransmission. Surprisingly, we found no significant time-of-day and genotype-dependent changes in chromatin accessibility in the brain. Instead, we found the existence of a temporal regulation of TF occupancy within open chromatin regions in the vicinity of rhythmic genes in the brains of wild-type monarchs, which is disrupted in clock deficient mutants. Together, this work identifies for the first time the rhythmic genes and modes of regulation by which diurnal transcription rhythms are regulated in the monarch brain. It also illustrates the power of ATAC-sequencing to profile genome-wide regulatory elements and TF binding in a non-model organism for which TF-specific antibodies are not yet available.

CLOCK:BMAL1 is a pioneer-like transcription factor.

The mammalian circadian clock relies on the master genes CLOCK and BMAL1 to drive rhythmic gene expression and regulate biological functions under circadian control. Here we show that rhythmic CLOCK:BMAL1 DNA binding promotes rhythmic chromatin opening. Mechanisms include CLOCK:BMAL1 binding to nucleosomes and rhythmic chromatin modification; e.g., incorporation of the histone variant H2A.Z. This rhythmic chromatin remodeling mediates the rhythmic binding of other transcription factors adjacent to CLOCK:BMAL1, suggesting that the activity of these other transcription factors contributes to the genome-wide CLOCK:BMAL1 heterogeneous transcriptional output. These data therefore indicate that the clock regulation of transcription relies on the rhythmic regulation of chromatin accessibility and suggest that the concept of pioneer function extends to acute gene regulation.

Regulation of circadian clock transcriptional output by CLOCK:BMAL1.

The mammalian circadian clock relies on the transcription factor CLOCK:BMAL1 to coordinate the rhythmic expression of 15% of the transcriptome and control the daily regulation of biological functions. The recent characterization of CLOCK:BMAL1 cistrome revealed that although CLOCK:BMAL1 binds synchronously to all of its target genes, its transcriptional output is highly heterogeneous. By performing a meta-analysis of several independent genome-wide datasets, we found that the binding of other transcription factors at CLOCK:BMAL1 enhancers likely contribute to the heterogeneity of CLOCK:BMAL1 transcriptional output. While CLOCK:BMAL1 rhythmic DNA binding promotes rhythmic nucleosome removal, it is not sufficient to generate transcriptionally active enhancers as assessed by H3K27ac signal, RNA Polymerase II recruitment, and eRNA expression. Instead, the transcriptional activity of CLOCK:BMAL1 enhancers appears to rely on the activity of ubiquitously expressed transcription factors, and not tissue-specific transcription factors, recruited at nearby binding sites. The contribution of other transcription factors is exemplified by how fasting, which effects several transcription factors but not CLOCK:BMAL1, either decreases or increases the amplitude of many rhythmically expressed CLOCK:BMAL1 target genes. Together, our analysis suggests that CLOCK:BMAL1 promotes a transcriptionally permissive chromatin landscape that primes its target genes for transcription activation rather than directly activating transcription, and provides a new framework to explain how environmental or pathological conditions can reprogram the rhythmic expression of clock-controlled genes.

Nascent-seq indicates widespread cotranscriptional RNA editing in Drosophila.

The RNA editing enzyme ADAR chemically modifies adenosine (A) to inosine (I), which is interpreted by the ribosome as a guanosine. Here we assess cotranscriptional A-to-I editing in Drosophila by isolating nascent RNA from adult fly heads and subjecting samples to high throughput sequencing. There are a large number of edited sites within nascent exons. Nascent RNA from an ADAR-null strain was also sequenced, indicating that almost all A-to-I events require ADAR. Moreover, mRNA editing levels correlate with editing levels within the cognate nascent RNA sequence, indicating that the extent of editing is set cotranscriptionally. Surprisingly, the nascent data also identify an excess of intronic over exonic editing sites. These intronic sites occur preferentially within introns that are poorly spliced cotranscriptionally, suggesting a link between editing and splicing. We conclude that ADAR-mediated editing is more widespread than previously indicated and largely occurs cotranscriptionally.

A new twist on clock protein phosphorylation: a conformational change leads to protein degradation.

Progressive phosphorylation of circadian clock proteins is a hallmark of time-keeping. In this issue of Molecular Cell, Querfurth et al. (2011) demonstrate that phosphorylation of Neurospora FRQ induces a conformational change, which can account for its temporally gated degradation.

Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression.

CLOCK (CLK) is a master transcriptional regulator of the circadian clock in Drosophila. To identify CLK direct target genes and address circadian transcriptional regulation in Drosophila, we performed chromatin immunoprecipitation (ChIP) tiling array assays (ChIP-chip) with a number of circadian proteins. CLK binding cycles on at least 800 sites with maximal binding in the early night. The CLK partner protein CYCLE (CYC) is on most of these sites. The CLK/CYC heterodimer is joined 4-6 h later by the transcriptional repressor PERIOD (PER), indicating that the majority of CLK targets are regulated similarly to core circadian genes. About 30% of target genes also show cycling RNA polymerase II (Pol II) binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. CLK has specific targets in different tissues, implying that important CLK partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation.

Dynamic PER repression mechanisms in the Drosophila circadian clock: from on-DNA to off-DNA.

Transcriptional feedback loops are central to the generation and maintenance of circadian rhythms. In animal systems as well as Neurospora, transcriptional repression is believed to occur by catalytic post-translational events. We report here in the Drosophila model two different mechanisms by which the circadian repressor PERIOD (PER) inhibits CLOCK/CYCLE (CLK/CYC)-mediated transcription. First, PER is recruited to circadian promoters, which leads to the nighttime decrease of CLK/CYC activity. This decrease is proportional to PER levels on DNA, and PER recruitment probably occurs via CLK. Then CLK is released from DNA and sequestered in a strong, approximately 1:1 PER-CLK off-DNA complex. The data indicate that the PER levels bound to CLK change dynamically and are important for repression, first on-DNA and then off-DNA. They also suggest that these mechanisms occur upstream of post-translational events, and that elements of this two-step mechanism likely apply to mammals.

A role for microRNAs in the Drosophila circadian clock.

Little is known about the contribution of translational control to circadian rhythms. To address this issue and in particular translational control by microRNAs (miRNAs), we knocked down the miRNA biogenesis pathway in Drosophila circadian tissues. In combination with an increase in circadian-mediated transcription, this severely affected Drosophila behavioral rhythms, indicating that miRNAs function in circadian timekeeping. To identify miRNA-mRNA pairs important for this regulation, immunoprecipitation of AGO1 followed by microarray analysis identified mRNAs under miRNA-mediated control. They included three core clock mRNAs-clock (clk), vrille (vri), and clockworkorange (cwo). To identify miRNAs involved in circadian timekeeping, we exploited circadian cell-specific inhibition of the miRNA biogenesis pathway followed by tiling array analysis. This approach identified miRNAs expressed in fly head circadian tissue. Behavioral and molecular experiments show that one of these miRNAs, the developmental regulator bantam, has a role in the core circadian pacemaker. S2 cell biochemical experiments indicate that bantam regulates the translation of clk through an association with three target sites located within the clk 3' untranslated region (UTR). Moreover, clk transgenes harboring mutated bantam sites in their 3' UTRs rescue rhythms of clk mutant flies much less well than wild-type CLK transgenes.

Nascent-Seq analysis of Drosophila cycling gene expression.

Rhythmic mRNA expression is a hallmark of circadian biology and has been described in numerous experimental systems including mammals. A small number of core clock gene mRNAs and a much larger number of output mRNAs are under circadian control. The rhythmic expression of core clock genes is regulated at the transcriptional level, and this regulation is important for the timekeeping mechanism. However, the relative contribution of transcriptional and post transcriptional regulation to global circadian mRNA oscillations is unknown. To address this issue in Drosophila, we isolated nascent RNA from adult fly heads collected at different time points and subjected it to high-throughput sequencing. mRNA was isolated and sequence din parallel. Some genes had cycling nascent RNAs with no cycling mRNA, caused,most likely, by light-mediated read-through transcription. Most genes with cycling mRNAs had significant nascent RNA cycling amplitudes, indicating a prominent role for circadian transcriptional regulation. However, a considerable fraction had higher mRNA amplitudes than nascent RNA amplitudes. The same comparison for core clock gene mRNAs gives rise to a qualitatively similar conclusion. The data therefore indicate a significant quantitative contribution of post transcriptional regulation to mRNA cycling.

Cotranscriptional splicing efficiency differs dramatically between Drosophila and mouse.

Spliceosome assembly and/or splicing of a nascent transcript may be crucial for proper isoform expression and gene regulation in higher eukaryotes. We recently showed that cotranscriptional splicing occurs efficiently in Drosophila, but there are not comparable genome-wide nascent splicing data from mammals. To provide this comparison, we analyze a recently generated, high-throughput sequencing data set of mouse liver nascent RNA, originally studied for circadian transcriptional regulation. Cotranscriptional splicing is approximately twofold less efficient in mouse liver than in Drosophila, i.e., nascent intron levels relative to exon levels are ∼0.55 in mouse versus 0.25 in the fly. An additional difference between species is that only mouse cotranscriptional splicing is optimal when 5'-exon length is between 50 and 500 bp, and intron length does not correlate with splicing efficiency, consistent with exon definition. A similar analysis of intron and exon length dependence in the fly is more consistent with intron definition. Contrasted with these differences are many similarities between the two systems: Alternatively annotated introns are less efficiently spliced cotranscriptionally than constitutive introns, and introns of single-intron genes are less efficiently spliced than introns from multi-intron genes. The most striking common feature is intron position: Cotranscriptional splicing is much more efficient when introns are far from the 3' ends of their genes. Additionally, absolute gene length correlates positively with cotranscriptional splicing efficiency independently of intron location and position, in flies as well as in mice. The gene length and distance effects indicate that more "nascent time" gives rise to greater cotranscriptional splicing efficiency in both systems.

Circadian regulation of stereotypic chromatin conformations at enhancers.

Cooperation between the circadian transcription factor (TF) CLOCK:BMAL1 and other TFs at cis-regulatory elements (CREs) is critical to daily rhythms of transcription. Yet, the modalities of this cooperation are unclear. Here, we analyzed the co-binding of multiple TFs on single DNA molecules in mouse liver using single molecule footprinting (SMF). We found that SMF reads clustered in stereotypic chromatin states that reflect distinguishable organization of TFs and nucleosomes, and that were remarkably conserved between all samples. DNA protection at CLOCK:BMAL1 binding motif (E-box) varied between CREs, from E-boxes being solely bound by CLOCK:BMAL1 to situations where other TFs competed with CLOCK:BMAL1 for E-box binding. SMF also uncovered CLOCK:BMAL1 cooperative binding at E-boxes separated by 250 bp, which structurally altered the CLOCK:BMAL1-DNA interface. Importantly, we discovered multiple nucleosomes with E-boxes at entry/exit sites that were removed upon CLOCK:BMAL1 DNA binding, thereby promoting the formation of open chromatin states that facilitate DNA binding of other TFs and that were associated with rhythmic transcription. These results demonstrate the utility of SMF for studying how CLOCK:BMAL1 and other TFs regulate stereotypical chromatin states at CREs to promote transcription.

Manipulation of Rhythmic Food Intake in Mice Using a Custom-Made Feeding System.

Rhythmic gene expression is a hallmark of the circadian rhythm and is essential for driving the rhythmicity of biological functions at the appropriate time of day. Studies over the last few decades have shown that rhythmic food intake (i.e., the time at which organisms eat food during the 24 h day), significantly contributes to the rhythmic regulation of gene expression in various organs and tissues throughout the body. The effects of rhythmic food intake on health and physiology have been widely studied ever since and have revealed that restricting food intake for 8 h during the active phase attenuates metabolic diseases arising from a variety of obesogenic diets. These studies often require the use of controlled methods for timing the delivery of food to animals. This manuscript describes the design and use of a low-cost and efficient system, built in-house for measuring daily food consumption as well as manipulating rhythmic food intake in mice. This system entails the use of affordable raw materials to build cages suited for food delivery, following a user-friendly handling procedure. This system can be used efficiently to feed mice on different feeding regimens such as ad libitum, time-restricted, or arrhythmic schedules, and can incorporate a high-fat diet to study its effect on behavior, physiology, and obesity. A description of how wild-type (WT) mice adapt to the different feeding regimens is provided.

Circadian transcription contributes to core period determination in Drosophila.

The Clock-Cycle (CLK-CYC) heterodimer constitutes a key circadian transcription complex in Drosophila. CYC has a DNA-binding domain but lacks an activation domain. Previous experiments also indicate that most of the transcriptional activity of CLK-CYC derives from the glutamine-rich region of its partner CLK. To address the role of transcription in core circadian timekeeping, we have analyzed the effects of a CYC-viral protein 16 (VP16) fusion protein in the Drosophila system. The addition of this potent and well-studied viral transcriptional activator (VP16) to CYC imparts to the CLK-CYC-VP16 complex strongly enhanced transcriptional activity relative to that of CLK-CYC. This increase is manifested in flies expressing CYC-VP16 as well as in S2 cells. These flies also have increased levels of CLK-CYC direct target gene mRNAs as well as a short period, implicating circadian transcription in period determination. A more detailed examination of reporter gene expression in CYC-VP16-expressing flies suggests that the short period is due at least in part to a more rapid transcriptional phase. Importantly, the behavioral effects require a period (per) promoter and are therefore unlikely to be merely a consequence of generally higher PER levels. This indicates that the CLK-CYC-VP16 behavioral effects are a consequence of increased per transcription. All of this also suggests that the timing of transcriptional activation and not the activation itself is the key event responsible for the behavioral effects observed in CYC-VP16-expressing flies. The results taken together indicate that circadian transcription contributes to core circadian function in Drosophila.

Clock-controlled rhythmic transcription: is the clock enough and how does it work?

Circadian clocks regulate the rhythmic expression of thousands of genes underlying the daily oscillations of biological functions. Here, we discuss recent findings showing that circadian clock rhythmic transcriptional outputs rely on additional mechanisms than just clock gene DNA binding, which may ultimately contribute to the plasticity of circadian transcriptional programs.

Rhythmic Food Intake Drives Rhythmic Gene Expression More Potently than the Hepatic Circadian Clock in Mice.

Every mammalian tissue exhibits daily rhythms in gene expression to control the activation of tissue-specific processes at the most appropriate time of the day. Much of this rhythmic expression is thought to be driven cell autonomously by molecular circadian clocks present throughout the body. By manipulating the daily rhythm of food intake in the mouse, we here show that more than 70% of the cycling mouse liver transcriptome loses rhythmicity under arrhythmic feeding. Remarkably, core clock genes are not among the 70% of genes losing rhythmic expression, and their expression continues to exhibit normal oscillations in arrhythmically fed mice. Manipulation of rhythmic food intake also alters the timing of key signaling and metabolic pathways without altering the hepatic clock oscillations. Our findings thus demonstrate that systemic signals driven by rhythmic food intake significantly contribute to driving rhythms in liver gene expression and metabolic functions independently of the cell-autonomous hepatic clock.

Clk post-transcriptional control denoises circadian transcription both temporally and spatially.

The transcription factor CLOCK (CLK) is essential for the development and maintenance of circadian rhythms in Drosophila. However, little is known about how CLK levels are controlled. Here we show that Clk mRNA is strongly regulated post-transcriptionally through its 3' UTR. Flies expressing Clk transgenes without normal 3' UTR exhibit variable CLK-driven transcription and circadian behaviour as well as ectopic expression of CLK-target genes in the brain. In these flies, the number of the key circadian neurons differs stochastically between individuals and within the two hemispheres of the same brain. Moreover, flies carrying Clk transgenes with deletions in the binding sites for the miRNA bantam have stochastic number of pacemaker neurons, suggesting that this miRNA mediates the deterministic expression of CLK. Overall our results demonstrate a key role of Clk post-transcriptional control in stabilizing circadian transcription, which is essential for proper development and maintenance of circadian rhythms in Drosophila.