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Network dynamics mediate circadian clock plasticity

ABSTRACT: Purpose: Alteration in light dark cycle leads to changes in daily behavior in mammals. We have previously demonstrated that different light:dark cycle lengths result in dynamic changes in DNA methylation within the suprachiasmatic nuclei (SCN) that globally alter transcription of both clock genes and non-clock genes. The goal of this study was to re-examine DNA methylation in the SCN separately for dorsal and ventral regions. Method: Six-week-old wild-type mice (C57BL/6Cr1) were entrained to light:dark cycles either 22, 24, or 26 hours in length (T22, T24, or T26) for six weeks. After 6 weeks, we recorded wheel-running behavior in constant darkness. After 3 days in DD, we used Clocklab software to predict time of activity onset for each mouse. Brains were isolated at the circadian time CT4 and then sliced immediately using vibratome, one coronal SCN slice of ~250μM was collected from the middle part of the SCN. Using ophthalmic knife vSCN and dSCN sub-regions were separated. To get enough DNA, a pool of vSCN and dSCN was prepared twice from 5 mice from each T-cycle. Genomic DNA was extracted from SCN slices by overnight Proteinase K digestion, followed by phenol-chloroform and ethanol precipitation. After RNase treatment, genomic DNA was sonicated to generate small fragments (average of 300bp), using a Covaris sonicator (BioRuptor). DNA immunoprecipitation was performed using Epiquik™ MeDIP Ultra Kit (EPIGENTEK™), following manufacturer's instructions. The methylated enriched DNA was then used for library preparation using Accel-NGS™ DNA Library Kits (Swift Biosciences), following manufacturer's instructions. Quality check, library preparation and sequencing were performed by Fasteris (CH). Results: 13-21 x 10^6 reads were obtained from each meDIP library. Quality control was performed on the fasta raw sequence data using FastQC.MeDIP-seq data were aligned to the mm9 mouse genome build using the Burrows-Wheeler Alignment tool (BWA) (Li and Durbin, 2009). Mapped reads were than analyzed using the MEDIPS package (Lienhard et al., 2014). Methylation score was calculated for each region of 500bp across the genome. CpG density-dependent normalization was incorporated. Pairwise correlation analysis of the genome wide coverage profile between MEDIP replicate samples was performed using the MEDIPS.correlation function. Pearson correlation coefficient r varied between 0.85 and 0.92, indicating good reproducibility of MEDIP-seq signal. Differential methylation analysis was performed using MEDIPS in 500bp widows excluding regions with less than 8 unique mapped reads. DMR were than identified by filtering for a widows associated with a p ≤0.001. DMR were than mapped using the web based chip-seq tool “ChIPseek” (Chen et al., 2014). Different sets of genes showed methylation changes in dorsal vs. ventral regions. Interestingly, far greater changes were observed in vSCN than in dSCN.

ORGANISM(S): Mus musculus

PROVIDER: GSE89255 | GEO | 2016/10/28

SECONDARY ACCESSION(S): PRJNA350842

REPOSITORIES: GEO

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Project description: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.

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.

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Network dynamics mediate circadian clock plasticity

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