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Circadian behavior is light-reprogrammed by plastic DNA methylation (sequencing)

ABSTRACT: The timing of daily “circadian” behavior can be highly variable among different individuals, and twin studies suggest that about half of this variability is environmentally controlled. Similar plasticity can be seen in mice exposed to an altered lighting environment – for example, 22-hour days instead of 24-hour ones – which stably alters the genetically determined period of circadian behavior for months. The mechanisms mediating these environmental influences are unknown. Here, we show that transient exposure of mice to such lighting stably alters global transcription in the suprachiasmatic nucleus of the hypothalamus (the SCN, the “master clock” tissue determining circadian behavior in mammals). We have also showed that, these changes in transcription are due to change in DNA methylation in the SCN. Indeed, genome-wide methylation profiling revealed global alterations in promoter DNA methylation in the SCN. Importantly, infusion of a methyltransferase inhibitor to the SCN during 22-hour days suppressed period changes. We also found that these behavioral and DNA methylation changes are reversible upon entrainment to 24-hours days. We conclude that the SCN utilizes DNA methylation as a mechanism to drive circadian clock plasticity. Methods: First, 16 mice 5 to 6 weeks old were exposed to DD for 7 days in order to measure their FRP. Then, mice were separated into 2 groups of 8 and entrained to ST and NT cycle. After 4 weeks we recorded wheel-running behavior in constant darkness. After 5 days in DD, we used Clocklab software to predict time of activity onset for each mouse. Brains were isolated at CT4 and sliced with a microtome using a fresh HBSS medium (thickness 250 uM) and the SCN was cut out with the aid of a binocular microscope and stored immediately at –80°C. Total RNA was extracted using the SurePrep™ Nuclear or Cytoplasmic RNA Purification Kit (Fisher Scientific) according to the manufacturer's protocol. A total of 500 ng of RNA was used for subsequent steps. These steps included DNAase treatment, polyA selection, depletion of ribosomal RNA, overall quality check, and library preparation, and were performed by Fasteris (CH). Libraries were prepared according to standard Illumina protocols. sequence quality, read mapping and data analysis: all steps were done using the GeneProf platform. Briefly, the sequencing reads of each library were first quality checked for read length and nucleotide composition, and then submitted for mapping to the mouse genome refrence sequence (Ensemble 58 mouse Genes, NCBIM37 assembly) using the alignment tool TopHat. Results: Entrainment to non-24 hours light/dark cycle induces a global changes in the SCN transcriptome.

ORGANISM(S): Mus musculus

PROVIDER: GSE54124 | GEO | 2014/01/16

SECONDARY ACCESSION(S): PRJNA235222

REPOSITORIES: GEO

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Project description:The timing of daily M-bM-^@M-^\circadianM-bM-^@M-^] behavior can be highly variable among different individuals, and twin studies suggest that about half of this variability is environmentally controlled. Similar plasticity can be seen in mice exposed to an altered lighting environment M-bM-^@M-^S for example, 22-hour days instead of 24-hour ones M-bM-^@M-^S which stably alters the genetically determined period of circadian behavior for months. The mechanisms mediating these environmental influences are unknown. Here, we show that transient exposure of mice to such lighting stably alters global transcription in the suprachiasmatic nucleus of the hypothalamus (the SCN, the M-bM-^@M-^\master clockM-bM-^@M-^] tissue determining circadian behavior in mammals). We have also showed that, these changes in transcription are due to change in DNA methylation in the SCN. Indeed, genome-wide methylation profiling revealed global alterations in promoter DNA methylation in the SCN. Importantly, infusion of a methyltransferase inhibitor to the SCN during 22-hour days suppressed period changes. We also found that these behavioral and DNA methylation changes are reversible upon entrainment to 24-hours days. We conclude that the SCN utilizes DNA methylation as a mechanism to drive circadian clock plasticity. Methods: First, 16 mice 5 to 6 weeks old were exposed to DD for 7 days in order to measure their FRP. Then, mice were separated into 2 groups of 8 and entrained to ST and NT cycle. After 4 weeks we recorded wheel-running behavior in constant darkness. After 5 days in DD, we used Clocklab software to predict time of activity onset for each mouse. Brains were isolated at CT4 and sliced with a microtome using a fresh HBSS medium (thickness 250 uM) and the SCN was cut out with the aid of a binocular microscope and stored immediately at M-bM-^@M-^S80M-BM-0C. Total RNA was extracted using the SurePrepM-bM-^DM-" Nuclear or Cytoplasmic RNA Purification Kit (Fisher Scientific) according to the manufacturer's protocol. A total of 500 ng of RNA was used for subsequent steps. These steps included DNAase treatment, polyA selection, depletion of ribosomal RNA, overall quality check, and library preparation, and were performed by Fasteris (CH). Libraries were prepared according to standard Illumina protocols. sequence quality, read mapping and data analysis: all steps were done using the GeneProf platform. Briefly, the sequencing reads of each library were first quality checked for read length and nucleotide composition, and then submitted for mapping to the mouse genome refrence sequence (Ensemble 58 mouse Genes, NCBIM37 assembly) using the alignment tool TopHat. Results: Entrainment to non-24 hours light/dark cycle induces a global changes in the SCN transcriptome. SCN mRNA profile of mice entrained to normal and short T-cycle using RNAseq technology.

Project description:Seasonal daylength, or circadian photoperiod, is a pervasive environmental signal that profoundly influences physiology and behavior. In mammals, the central circadian clock resides in the suprachiasmatic nuclei (SCN) of the hypothalamus where it receives retinal input and synchronizes, or entrains, organismal physiology and behavior to the prevailing light cycle. The process of entrainment induces sustained plasticity in the SCN, but the molecular mechanisms underlying SCN plasticity are incompletely understood. Entrainment to different photoperiods persistently alters the timing, waveform, period, and light resetting properties of the SCN clock and its driven rhythms. To elucidate novel molecular mechanisms of photoperiod plasticity, we performed RNAseq on whole SCN dissected from mice raised in Long (LD 16:8) and Short (LD 8:16) photoperiods. Fewer rhythmic genes were detected in Long photoperiod and in general the timing of gene expression rhythms was advanced 4-6 hours. However, a few genes showed significant delays, including Gem. There were significant changes in the expression clock-associated gene Timeless and in SCN genes related to light responses, neuropeptides, GABA, ion channels, and serotonin. Particularly striking were differences in the expression of the neuropeptide signaling genes Prokr2 and Cck, as well as convergent regulation of the expression of three SCN light response genes, Dusp4, Rasd1, and Gem. Transcriptional modulation of Dusp4 and Rasd1, and phase regulation of Gem, are compelling candidate molecular mechanisms for plasticity in the SCN light response through their modulation of the critical NMDAR-MAPK/ERK-CREB/CRE light signaling pathway in SCN neurons. Modulation of Prokr2 and Cck may critically support SCN neural network reconfiguration during photoperiodic entrainment. Our findings identify the SCN light response and neuropeptide signaling gene sets as rich substrates for elucidating novel mechanisms of photoperiod plasticity. Data is also available on our corresponding website, where users can search and view the expression and rhythmic properties of genes across these photoperiod conditions.

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Circadian behavior is light-reprogrammed by plastic DNA methylation (sequencing)

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