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

Cross-Talk between the Cellular Redox State and the Circadian System in Neurospora

The circadian system is composed of a number of feedback loops, and multiple feedback loops in the form of oscillators help to maintain stable rhythms. The filamentous fungus Neurospora crassa exhibits a circadian rhythm during asexual spore formation (conidiation banding) and has a major feedback loop that includes the FREQUENCY (FRQ)/WHITE COLLAR (WC) -1 and -2 oscillator (FWO). A mutation in superoxide dismutase (sod)-1, an antioxidant gene, causes a robust and stable circadian rhythm compared with that of wild-type (Wt). However, the mechanisms underlying the functions of reactive oxygen species (ROS) remain unknown. Here, we show that cellular ROS concentrations change in a circadian manner (ROS oscillation), and the amplitudes of ROS oscillation increase with each cycle and then become steady (ROS homeostasis). The ROS oscillation and homeostasis are produced by the ROS-destroying catalases (CATs) and ROS-generating NADPH oxidase (NOX). cat-1 is also induced by illumination, and it reduces ROS levels. Although ROS oscillation persists in the absence of frq, wc-1 or wc-2, its homeostasis is altered. Furthermore, genetic and biochemical evidence reveals that ROS concentration regulates the transcriptional function of WCC and a higher ROS concentration enhances conidiation banding. These findings suggest that the circadian system engages in cross-talk with the cellular redox state via ROS-regulatory factors

Systems Biology of the Clock in Neurospora crassa

A model-driven discovery process, Computing Life, is used to identify an ensemble of genetic networks that describe the biological clock. A clock mechanism involving the genes white-collar-1 and white-collar-2 (wc-1 and wc-2) that encode a transcriptional activator (as well as a blue-light receptor) and an oscillator frequency (frq) that encodes a cyclin that deactivates the activator is used to guide this discovery process through three cycles of microarray experiments. Central to this discovery process is a new methodology for the rational design of a Maximally Informative Next Experiment (MINE), based on the genetic network ensemble. In each experimentation cycle, the MINE approach is used to select the most informative new experiment in order to mine for clock-controlled genes, the outputs of the clock. As much as 25% of the N. crassa transcriptome appears to be under clock-control. Clock outputs include genes with products in DNA metabolism, ribosome biogenesis in RNA metabolism, cell cycle, protein metabolism, transport, carbon metabolism, isoprenoid (including carotenoid) biosynthesis, development, and varied signaling processes. Genes under the transcription factor complex WCC ( = WC-1/WC-2) control were resolved into four classes, circadian only (612 genes), light-responsive only (396), both circadian and light-responsive (328), and neither circadian nor light-responsive (987). In each of three cycles of microarray experiments data support that wc-1 and wc-2 are auto-regulated by WCC. Among 11,000 N. crassa genes a total of 295 genes, including a large fraction of phosphatases/kinases, appear to be under the immediate control of the FRQ oscillator as validated by 4 independent microarray experiments. Ribosomal RNA processing and assembly rather than its transcription appears to be under clock control, suggesting a new mechanism for the post-transcriptional control of clock-controlled genes

Evaluation of DNA Damage in Eurasian Marsh Frogs (Pelophylax ridibundus) by Comet Assay for Determination of Possible Pollution in the Different Lakes in Central Anatolia, Turkey

In the present study, adult Eurasian marsh frogs, Pelophylax ridibundus, and water samples were collected from a reference lake and three water bodies in central Anatolia, Turkey, to evaluate the water for chemical pollutants and possible effects of pollutants on the DNA of frog erythrocytes by using a comet assay. The results for DNA damage parameters of the comet assay (total comet length, tail intensity, and olive tail moment) and their statistical analysis by ANOVA demonstrated that P. ridibundus and the comet assay together represent an useful approach for the early detection of polluted water bodies

Transcriptional interference by antisense RNA is required for circadian clock function.

Eukaryotic circadian oscillators consist of negative feedback loops that generate endogenous rhythmicities(1). Natural antisense RNAs are found in a wide range of eukaryotic organisms(2-5). Nevertheless, the physiological importance and mode of action of most antisense RNAs is not clear(6-9). frequency (frq) encodes a component of the Neurospora core circadian negative feedback loop which was thought to generate sustained rhythmicity(10). Transcription of qrf, the long non-coding frq antisense RNA, is light induced, and its level oscillates in antiphase to frq sense RNA(3). Here we show that qrf transcription is regulated by both light-dependent and -independent mechanisms. Light-dependent qrf transcription represses frq expression and regulates clock resetting. qrf expression in the dark, on the other hand, is required for circadian rhythmicity. frq transcription also inhibits qrf expression and surprisingly, drives the antiphasic rhythm of qrf transcripts. The mutual inhibition of frq and qrf transcription thus forms a double negative feedback loop that is interlocked with the core feedback loop. Genetic and mathematical modeling analyses indicate that such an arrangement is required for robust and sustained circadian rhythmicity. Moreover, our results suggest that antisense transcription inhibits sense expression by mediating chromatin modifications and premature transcription termination. Together, our results established antisense transcription as an essential feature in a circadian system and shed light on the importance and mechanism of antisense action