Molecular Regulation of Circadian Chromatin.

Circadian rhythms are generated by transcriptional negative feedback loops and require histone modifications and chromatin remodeling to ensure appropriate timing and amplitude of clock gene expression. Circadian modifications to histones are important for transcriptional initiation and feedback inhibition serving as signaling platform for chromatin-remodeling enzymes. Current models indicate circadian-regulated facultative heterochromatin (CRFH) is a conserved mechanism at clock genes in Neurospora, Drosophila, and mice. CRFH consists of antiphasic rhythms in activating and repressive modifications generating chromatin states that cycle between transcriptionally permissive and nonpermissive. There are rhythms in histone H3 lysine 9 and 27 acetylation (H3K9ac and H3K27ac) and histone H3 lysine 4 methylation (H3K4me) during activation; while deacetylation, histone H3 lysine 9 methylation (H3K9me) and heterochromatin protein 1 (HP1) are hallmarks of repression. ATP-dependent chromatin-remodeling enzymes control accessibility, nucleosome positioning/occupancy, and nuclear organization. In Neurospora, the rhythm in facultative heterochromatin is mediated by the frequency (frq) natural antisense transcript (NAT) qrf. While in mammals, histone deacetylases (HDACs), histone H3 lysine 9 methyltransferase (KMT1/SUV39), and components of nucleosome remodeling and deacetylase (NuRD) are part of the nuclear PERIOD complex (PER complex). Genomics efforts have found relationships among rhythmic chromatin modifications at clock-controlled genes (ccg) revealing circadian control of genome-wide chromatin states. There are also circadian clock-regulated lncRNAs with an emerging function that includes assisting in chromatin dynamics. In this review, we explore the connections between circadian clock, chromatin remodeling, lncRNAs, and CRFH and how these impact rhythmicity, amplitude, period, and phase of circadian clock genes.

BMAL1 associates with chromosome ends to control rhythms in TERRA and telomeric heterochromatin.

The circadian clock and aging are intertwined. Disruption to the normal diurnal rhythm accelerates aging and corresponds with telomere shortening. Telomere attrition also correlates with increase cellular senescence and incidence of chronic disease. In this report, we examined diurnal association of White Collar 2 (WC-2) in Neurospora and BMAL1 in zebrafish and mice and found that these circadian transcription factors associate with telomere DNA in a rhythmic fashion. We also identified a circadian rhythm in Telomeric Repeat-containing RNA (TERRA), a lncRNA transcribed from the telomere. The diurnal rhythm in TERRA was lost in the liver of Bmal1-/- mice indicating it is a circadian regulated transcript. There was also a BMAL1-dependent rhythm in H3K9me3 at the telomere in zebrafish brain and mouse liver, and this rhythm was lost with increasing age. Taken together, these results provide evidence that BMAL1 plays a direct role in telomere homeostasis by regulating rhythms in TERRA and heterochromatin. Loss of these rhythms may contribute to telomere erosion during aging.

Age modulates liver responses to asparaginase-induced amino acid stress in mice.

Asparaginase is an amino acid-depleting agent used to treat blood cancers. Metabolic complications due to asparaginase affect liver function in humans. To examine how the liver response to asparaginase changes during maturity to adulthood, here we treated juvenile (2-week), young adult (8-week), and mature adult (16-week) mice with drug or excipient for 1 week and conducted RNA-Seq and functional analyses. Asparaginase reduced body growth and liver mass in juveniles but not in the adult animals. Unbiased exploration of the effect of asparaginase on the liver transcriptome revealed that the integrated stress response (ISR) was the only molecular signature shared across the ages, corroborating similar eukaryotic initiation factor 2 phosphorylation responses to asparaginase at all ages. Juvenile livers exhibited steatosis and iron accumulation following asparaginase exposure along with a hepatic gene signature indicating that asparaginase uniquely affects lipid, cholesterol, and iron metabolism in juvenile mice. In contrast, asparaginase-treated adult mice displayed greater variability in liver function, which correlated with an acute-phase inflammatory response gene signature. Asparaginase-exposed adults also had a serine/glycine/one-carbon metabolism gene signature in liver that corresponded with reduced circulating glycine and serine levels. These results establish the ISR as a conserved response to asparaginase-mediated amino acid deprivation and provide new insights into the relationship between the liver transcriptome and hepatic function upon asparaginase exposure.

Histone H3 lysine 4 methyltransferase is required for facultative heterochromatin at specific loci.

BACKGROUND: Histone H3 lysine 4 tri-methylation (H3K4me3) and histone H3 lysine 9 tri-methylation (H3K9me3) are widely perceived to be opposing and often mutually exclusive chromatin modifications. However, both are needed for certain light-activated genes in Neurospora crassa (Neurospora), including frequency (frq) and vivid (vvd). Except for these 2 loci, little is known about how H3K4me3 and H3K9me3 impact and contribute to light-regulated gene expression. RESULTS: In this report, we performed a multi-dimensional genomic analysis to understand the role of H3K4me3 and H3K9me3 using the Neurospora light response as the system. RNA-seq on strains lacking H3 lysine 4 methyltransferase (KMT2/SET-1) and histone H3 lysine 9 methyltransferase (KMT1/DIM-5) revealed some light-activated genes had altered expression, but the light response was largely intact. Comparing these 2 mutants to wild-type (WT), we found that roughly equal numbers of genes showed elevated and reduced expression in the dark and the light making the environmental stimulus somewhat ancillary to the genome-wide effects. ChIP-seq experiments revealed H3K4me3 and H3K9me3 had only minor changes in response to light in WT, but there were notable alterations in H3K4me3 in Δkmt1/Δdim-5 and H3K9me3 in Δkmt2/Δset-1 indicating crosstalk and redistribution between the modifications. Integrated analysis of the RNA-seq and ChIP-seq highlighted context-dependent roles for KMT2/SET1 and KMT1/DIM-5 as either co-activators or co-repressors with some overlap as co-regulators. At a small subset of loci, H3K4 methylation is required for H3K9me3-mediated facultative heterochromatin including, the central clock gene frequency (frq). Finally, we used sequential ChIP (re-ChIP) experiment to confirm Neurospora contains K4/K9 bivalent domains. CONCLUSIONS: Collectively, these data indicate there are obfuscated regulatory roles for H3K4 methylation and H3K9 methylation depending on genome location with some minor overlap and co-dependency.

Nutrition Risk is Associated with Leukocyte Telomere Length in Middle-Aged Men and Women with at Least One Risk Factor for Cardiovascular Disease.

Poor diet quality has been associated with several age-related chronic conditions, but its relationship to telomere length, a biological marker of cellular aging, is unclear. The purpose of this cross-sectional study was to determine whether overall diet quality was associated with relative leukocyte telomere length (rLTL) in a sample (n = 96) of nonsmoking middle-aged adults in Appalachia with at least one risk factor for cardiovascular disease. Diet quality was assessed using the Healthy Eating Index (HEI-2015), the alternate Mediterranean diet score (aMed), and the Dietary Screening Tool (DST). Peripheral rLTL was measured by quantitative real-time polymerase chain reaction. The associations between potentially confounding sociodemographic, lifestyle and health-related factors and the first and fourth rLTL quartile groups were examined using Chi-square or Fisher's Exact tests or logistic regression. The relationships between diet quality index scores and rLTL as a continuous variable were analyzed using simple linear regression and multivariate linear models, analogous to linear covariance analyses. The rLTL ranged from 0.46 to 1.49 (mean ± SEM was 1.02 ± 0.18). Smoking history, income level, and cardiovascular health (Life's Simple 7) were associated with the lowest and highest quartiles of rLTL and were used as covariates. In adjusted and unadjusted models, participants considered "at nutrition risk" by the DST were more likely to have shorter rLTL than those "not at risk or at potential risk" (p = 0.004). However, there was no evidence that adherence to the 2015⁻2020 Dietary Guidelines for Americans or to a Mediterranean diet was associated with rLTL in this sample. Intervention studies are needed to determine if improving the diet quality of those at nutrition risk results in reduced telomere attrition over time.

Long non-coding RNAs have age-dependent diurnal expression that coincides with age-related changes in genome-wide facultative heterochromatin.

BACKGROUND: Disrupted diurnal rhythms cause accelerated aging and an increased incidence in age-related disease and morbidity. The circadian clock governs cell physiology and metabolism by controlling transcription and chromatin. The goal of this study is to further understand the mechanism of age-related changes to circadian chromatin with a focus on facultative heterochromatin and diurnal non-coding RNAs. RESULTS: We performed a combined RNA-seq and ChIP-seq at two diurnal time-points for three different age groups to examine the connection between age-related changes to circadian transcription and heterochromatin in neuronal tissue. Our analysis focused on uncovering the relationships between long non-coding RNA (lncRNA) and age-related changes to histone H3 lysine 9 tri-methylation (H3K9me3), in part because the Period (Per) complex can direct facultative heterochromatin and models of aging suggest age-related changes to heterochromatin and DNA methylation. Our results reveal that lncRNAs and circadian output change dramatically with age, but the core clock genes remain rhythmic. Age-related changes in clock-controlled gene (ccg) expression indicate there are age-dependent circadian output that change from anabolic to catabolic processes during aging. In addition, there are diurnal and age-related changes in H3K9me3 that coincide with changes in transcription. CONCLUSIONS: The data suggest a model where some age-related changes in diurnal expression are partially attributed to age-related alterations to rhythmic facultative heterochromatin. The changes in heterochromatin are potentially mediated by changes in diurnal lncRNA creating an interlocked circadian-chromatin regulatory network that undergoes age-dependent metamorphosis.

Role of activating transcription factor 4 in the hepatic response to amino acid depletion by asparaginase.

The anti-leukemic agent asparaginase activates the integrated stress response (ISR) kinase GCN2 and inhibits signaling via mechanistic target of rapamycin complex 1 (mTORC1). The study objective was to investigate the protective role of activating transcription factor 4 (ATF4) in controlling the hepatic transcriptome and mediating GCN2-mTORC1 signaling during asparaginase. We compared global gene expression patterns in livers from wildtype, Gcn2 -/-, and Atf4 -/- mice treated with asparaginase or excipient and further explored selected responses in livers from Atf4 +/- mice. Here, we show that ATF4 controls a hepatic gene expression profile that overlaps with GCN2 but is not required for downregulation of mTORC1 during asparaginase. Ingenuity pathway analysis indicates GCN2 independently influences inflammation-mediated hepatic processes whereas ATF4 uniquely associates with cholesterol metabolism and endoplasmic reticulum (ER) stress. Livers from Atf4 -/- or Atf4 +/- mice displayed an amplification of the amino acid response and ER stress response transcriptional signatures. In contrast, reduction in hepatic mTORC1 signaling was retained in Atf4 -/- mice treated with asparaginase. CONCLUSIONS: GCN2 and ATF4 serve complementary roles in the hepatic response to asparaginase. GCN2 functions to limit inflammation and mTORC1 signaling whereas ATF4 serves to limit the amino acid response and prevent ER stress during amino acid depletion by asparaginase.

The Arcuate Estrogen-Regulated Transcriptome: Estrogen Response Element-Dependent and -Independent Signaling of ERα in Female Mice.

To influence energy homeostasis and reproduction, 17ß-estradiol (E2) controls the arcuate nucleus (ARC) through multiple receptor-mediated mechanisms, but primarily via estrogen receptor (ER) α, which signals through both estrogen response element (ERE)-dependent and -independent mechanisms. To determine ERα-mediated, ERE-dependent, and ERE-independent E2 signaling in the ARC, we examined the differential regulation of the mouse arcuate transcriptome by E2 using three mice genotypes: (1) wild-type, (2) ERα knock-in/knockout (ERE-independent mechanisms), and (3) total ERα knockout (ERα-independent mechanisms). Females were ovariectomized and injected with oil or E2, and RNA sequencing on the ARC was used to identify E2-regulated genes in each genotype. Our results show that E2 regulates numerous genes involved in cell signaling, cytoskeleton structure, inflammation, neurotransmission, neuropeptide production, and transcription. Furthermore, ERE-independent signaling regulates ARC genes expressed in kisspeptin neurons and transcription factors that control the hypothalamic/pituitary/gonadal axis. Interestingly, a few genes involved in mitochondrial oxidative respiration were regulated by E2 through ERα-independent signaling. A comparison within oil- and E2-treated females across the three genotypes suggests that genes involved in cell growth and proliferation, extracellular matrices, neuropeptides, receptors, and transcription are differentially expressed across the genotypes. Comparing with previously published chromatin immunoprecipitation sequencing analysis, we found that ERE-independent regulation in the ARC is mainly mediated by tethering of ERα, which is consistent with previous findings. We conclude that the mouse arcuate estrogen-regulated transcriptome is regulated by multiple receptor-mediated mechanisms to modulate the central control of energy homeostasis and reproduction, including novel E2-responsive pathways.

The Suf Iron-Sulfur Cluster Biosynthetic System Is Essential in Staphylococcus aureus, and Decreased Suf Function Results in Global Metabolic Defects and Reduced Survival in Human Neutrophils.

Staphylococcus aureus remains a causative agent for morbidity and mortality worldwide. This is in part a result of antimicrobial resistance, highlighting the need to uncover novel antibiotic targets and to discover new therapeutic agents. In the present study, we explored the possibility that iron-sulfur (Fe-S) cluster synthesis is a viable antimicrobial target. RNA interference studies established that Suf (sulfur mobilization)-dependent Fe-S cluster synthesis is essential in S. aureus We found that sufCDSUB were cotranscribed and that suf transcription was positively influenced by sigma factor B. We characterized an S. aureus strain that contained a transposon inserted in the intergenic space between sufC and sufD (sufD*), resulting in decreased transcription of sufSUB Consistent with the transcriptional data, the sufD* strain had multiple phenotypes associated with impaired Fe-S protein maturation. They included decreased activities of Fe-S cluster-dependent enzymes, decreased growth in media lacking metabolites that require Fe-S proteins for synthesis, and decreased flux through the tricarboxylic acid (TCA) cycle. Decreased Fe-S cluster synthesis resulted in sensitivity to reactive oxygen and reactive nitrogen species, as well as increased DNA damage and impaired DNA repair. The sufD* strain also exhibited perturbed intracellular nonchelated Fe pools. Importantly, the sufD* strain did not exhibit altered exoprotein production or altered biofilm formation, but it was attenuated for survival upon challenge by human polymorphonuclear leukocytes. The results presented are consistent with the hypothesis that Fe-S cluster synthesis is a viable target for antimicrobial development.

The frequency natural antisense transcript first promotes, then represses, frequency gene expression via facultative heterochromatin.

The circadian clock is controlled by a network of interconnected feedback loops that require histone modifications and chromatin remodeling. Long noncoding natural antisense transcripts (NATs) originate from Period in mammals and frequency (frq) in Neurospora. To understand the role of NATs in the clock, we put the frq antisense transcript qrf (frq spelled backwards) under the control of an inducible promoter. Replacing the endogenous qrf promoter altered heterochromatin formation and DNA methylation at frq. In addition, constitutive, low-level induction of qrf caused a dramatic effect on the endogenous rhythm and elevated circadian output. Surprisingly, even though qrf is needed for heterochromatic silencing, induction of qrf initially promoted frq gene expression by creating a more permissible local chromatin environment. The observation that antisense expression can initially promote sense gene expression before silencing via heterochromatin formation at convergent loci is also found when a NAT to hygromycin resistance gene is driven off the endogenous vivid (vvd) promoter in the Δvvd strain. Facultative heterochromatin silencing at frq functions in a parallel pathway to previously characterized VVD-dependent silencing and is needed to establish the appropriate circadian phase. Thus, repression via dicer-independent siRNA-mediated facultative heterochromatin is largely independent of, and occurs alongside, other feedback processes.

The histone H3 lysine 9 methyltransferase DIM-5 modifies chromatin at frequency and represses light-activated gene expression.

The transcriptional program controlling the circadian rhythm requires coordinated regulation of chromatin. Characterization of the chromodomain helicase DNA-binding enzyme CHD1 revealed DNA methylation in the promoter of the central clock gene frequency (frq) in Neurospora crassa. In this report, we show that the DNA methylation at frq is not only dependent on the DNA methyltransferase DIM-2 but also on the H3K9 methyltransferase DIM-5 and HP1. Histone H3 lysine 9 trimethylation (H3K9me3) occurs at frq and is most prominent 30 min after light-activated expression. Strains lacking dim-5 have an increase in light-induced transcription, and more White Collar-2 is found associated with the frq promoter. Consistent with the notion that DNA methylation assists in establishing the proper circadian phase, loss of H3K9 methylation results in a phase advance suggesting it delays the onset of frq expression. The dim-5 deletion strain displays an increase in circadian-regulated conidia formation on race tubes and there is a synthetic genetic interaction between dim-5 and ras-1(bd). These results indicate DIM-5 has a regulatory role in muting circadian output. Overall, the data support a model where facultative heterochromatic at frq serves to establish the appropriate phase, mute the light response, and repress circadian output.

Regulated DNA methylation and the circadian clock: implications in cancer.

Since the cloning and discovery of DNA methyltransferases (DNMT), there has been a growing interest in DNA methylation, its role as an epigenetic modification, how it is established and removed, along with the implications in development and disease. In recent years, it has become evident that dynamic DNA methylation accompanies the circadian clock and is found at clock genes in Neurospora, mice and cancer cells. The relationship among the circadian clock, cancer and DNA methylation at clock genes suggests a correlative indication that improper DNA methylation may influence clock gene expression, contributing to the etiology of cancer. The molecular mechanism underlying DNA methylation at clock loci is best studied in the filamentous fungi, Neurospora crassa, and recent data indicate a mechanism analogous to the RNA-dependent DNA methylation (RdDM) or RNAi-mediated facultative heterochromatin. Although it is still unclear, DNA methylation at clock genes may function as a terminal modification that serves to prevent the regulated removal of histone modifications. In this capacity, aberrant DNA methylation may serve as a readout of misregulated clock genes and not as the causative agent. This review explores the implications of DNA methylation at clock loci and describes what is currently known regarding the molecular mechanism underlying DNA methylation at circadian clock genes.

A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile.

Cloning by homologous recombination (HR) in Saccharomyces cerevisiae is an extremely efficient and cost-effective alternative to other methods of recombinant DNA technologies. Unfortunately, it is incompatible with all the various specialized plasmids currently used in microbiology and biomedical research laboratories, and is therefore, not widely adopted. In an effort to dramatically improve the versatility of yeast gap-repair cloning and make it compatible with any DNA plasmid, we demonstrate that by simply including a yeast-cloning cassette (YCC) that contains the 2-micron origin of replication (2µm ori) and the ura3 gene for selection, multiple DNA fragments can be assembled into any DNA vector. We show this has almost unlimited potential by building a variety of plasmid for different uses including: recombinant protein production, epitope tagging, site-directed mutagenesis, and expression of fluorescent fusion proteins. We demonstrate the use in a variety of plasmids for use in microbial systems and even demonstrate it can be used in a vertebrate model. This method is remarkably simple and extremely efficient, plus it provides a significant cost saving over commercially available kits.

Circadian rhythms synchronize mitosis in Neurospora crassa.

The cell cycle and the circadian clock communicate with each other, resulting in circadian-gated cell division cycles. Alterations in this network may lead to diseases such as cancer. Therefore, it is critical to identify molecular components that connect these two oscillators. However, molecular mechanisms between the clock and the cell cycle remain largely unknown. A model filamentous fungus, Neurospora crassa, is a multinucleate system used to elucidate molecular mechanisms of circadian rhythms, but not used to investigate the molecular coupling between these two oscillators. In this report, we show that a conserved coupling between the circadian clock and the cell cycle exists via serine/threonine protein kinase-29 (STK-29), the Neurospora homolog of mammalian WEE1 kinase. Based on this finding, we established a mathematical model that predicts circadian oscillations of cell cycle components and circadian clock-dependent synchronized nuclear divisions. We experimentally demonstrate that G1 and G2 cyclins, CLN-1 and CLB-1, respectively, oscillate in a circadian manner with bioluminescence reporters. The oscillations of clb-1 and stk-29 gene expression are abolished in a circadian arrhythmic frq(ko) mutant. Additionally, we show the light-induced phase shifts of a core circadian component, frq, as well as the gene expression of the cell cycle components clb-1 and stk-29, which may alter the timing of divisions. We then used a histone hH1-GFP reporter to observe nuclear divisions over time, and show that a large number of nuclear divisions occur in the evening. Our findings demonstrate the circadian clock-dependent molecular dynamics of cell cycle components that result in synchronized nuclear divisions in Neurospora.

Aging well with a little wine and a good clock.

Age-related decline in mammalian circadian rhythm has been recognized for decades, but the underlying molecular mechanisms have remained elusive. In this issue of Cell, Chang and Guarente use brain-specific SIRT1 knockout mice and transgenic mice overexpressing SIRT1 to develop an enticing model for how SIRT1 helps maintain the robustness of the aging circadian clock.

Methylation of histone H3 on lysine 4 by the lysine methyltransferase SET1 protein is needed for normal clock gene expression.

The circadian oscillator controls time-of-day gene expression by a network of interconnected feedback loops and is reset by light. The requisite for chromatin regulation in eukaryotic transcription necessitates temporal regulation of histone-modifying and chromatin-remodeling enzymes for proper clock function. CHD1 is known to bind H3K4me3 in mammalian cells, and Neurospora CHD1 is required for proper regulation of the frequency (frq) gene. Based on this, we examined a strain lacking SET1 to determine the role of H3K4 methylation in clock- and light-mediated frq regulation. Expression of frq was altered in strains lacking set1 under both circadian- and light-regulated gene expression. There is a delay in the phasing of H3K4me3 relative to the peak in frq expression. White Collar 2 (WC-2) association with the frq promoter persists longer in Δset1, suggesting a more permissible chromatin state. Surprisingly, SET1 is required for DNA methylation in the frq promoter, indicating a dependence on H3K4me for DNA methylation. The data support a model where SET1 is needed for proper regulation by modulating chromatin at frq.

CHD1 remodels chromatin and influences transient DNA methylation at the clock gene frequency.

Circadian-regulated gene expression is predominantly controlled by a transcriptional negative feedback loop, and it is evident that chromatin modifications and chromatin remodeling are integral to this process in eukaryotes. We previously determined that multiple ATP-dependent chromatin-remodeling enzymes function at frequency (frq). In this report, we demonstrate that the Neurospora homologue of chd1 is required for normal remodeling of chromatin at frq and is required for normal frq expression and sustained rhythmicity. Surprisingly, our studies of CHD1 also revealed that DNA sequences within the frq promoter are methylated, and deletion of chd1 results in expansion of this methylated domain. DNA methylation of the frq locus is altered in strains bearing mutations in a variety of circadian clock genes, including frq, frh, wc-1, and the gene encoding the frq antisense transcript (qrf). Furthermore, frq methylation depends on the DNA methyltransferase, DIM-2. Phenotypic characterization of Δdim-2 strains revealed an approximate WT period length and a phase advance of approximately 2 hours, indicating that methylation plays only an ancillary role in clock-regulated gene expression. This suggests that DNA methylation, like the antisense transcript, is necessary to establish proper clock phasing but does not control overt rhythmicity. These data demonstrate that the epigenetic state of clock genes is dependent on normal regulation of clock components.

Genetic and molecular characterization of a cryptochrome from the filamentous fungus Neurospora crassa.

In plants and animals, cryptochromes function as either photoreceptors or circadian clock components. We have examined the cryptochrome from the filamentous fungus Neurospora crassa and demonstrate that Neurospora cry encodes a DASH-type cryptochrome that appears capable of binding flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF). The cry transcript and CRY protein levels are strongly induced by blue light in a wc-1-dependent manner, and cry transcript is circadianly regulated, with a peak abundance opposite in phase to frq. Neither deletion nor overexpression of cry appears to perturb the free-running circadian clock. However, cry disruption knockout mutants show a small phase delay under circadian entrainment. Using electrophoretic mobility shift assays (EMSA), we show that CRY is capable of binding single- and double-stranded DNA (ssDNA and dsDNA, respectively) and ssRNA and dsRNA. Whole-genome microarray experiments failed to identify substantive transcriptional regulatory activity of cry under our laboratory conditions.

A high-density single nucleotide polymorphism map for Neurospora crassa.

We report the discovery and validation of a set of single nucleotide polymorphisms (SNPs) between the reference Neurospora crassa strain Oak Ridge and the Mauriceville strain (FGSC 2555), of sufficient density to allow fine mapping of most loci. Sequencing of Mauriceville cDNAs and alignment to the completed genomic sequence of the Oak Ridge strain identified 19,087 putative SNPs. Of these, a subset was validated by cleaved amplified polymorphic sequence (CAPS), a simple and robust PCR-based assay that reliably distinguishes between SNP alleles. Experimental confirmation resulted in the development of 250 CAPS markers distributed evenly over the genome. To demonstrate the applicability of this map, we used bulked segregant analysis followed by interval mapping to locate the csp-1 mutation to a narrow region on LGI. Subsequently, we refined mapping resolution to 74 kbp by developing additional markers, resequenced the candidate gene, NCU02713.3, in the mutant background, and phenocopied the mutation by gene replacement in the WT strain. Together, these techniques demonstrate a generally applicable and straightforward approach for the isolation of novel genes from existing mutants. Data on both putative and validated SNPs are deposited in a customized public database at the Broad Institute, which encourages augmentation by community users.

SIRT1 is a circadian deacetylase for core clock components.

The transcriptional activator CLOCK is a histone acetyltransferase that is required for the circadian expression of many genes. Asher et al. (2008) and Nakahata et al. (2008) now demonstrate that the NAD(+)-dependent enzyme SIRT1 functions as a histone deacetylase that counteracts the activity of CLOCK. These results broaden our understanding of the impact of cellular metabolism on the circadian system.