Gene expression plasticity of the mammalian brain circadian clock in response to photoperiod.

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.

Characterization of glucocorticoid-induced loss of DNA methylation of the stress-response gene Fkbp5 in neuronal cells.

Exposure to stress or glucocorticoids (GCs) is associated with epigenetic and transcriptional changes in genes that either mediate or are targets of GC signalling. FKBP5 (FK506 binding protein 5) is one such gene that also plays a central role in negative feedback regulation of GC signalling and several stress-related psychiatric disorders. In this study, we sought to examine how the mouse Fkbp5 gene is regulated in a neuronal context and identify requisite factors that can mediate the epigenetic sequelae of excess GC exposure. Mice treated with GCs were used to establish the widespread changes in DNA methylation (DNAm) and expression of Fkbp5 across four brain regions. Then two cell lines were used to test the persistence, decay, and functional significance of GC-induced methylation changes near two GC response elements (GREs) in the fifth intron of Fkbp5. We also tested the involvement of DNMT1, cell proliferation, and MeCP2 in mediating the effect of GCs on DNAm and gene activation. DNAm changes at some CpGs persist while others decay, and reduced methylation states are associated with a more robust transcriptional response. Importantly, the ability to undergo GC-induced DNAm loss is tied to DNMT1 function during cell division. Further, GC-induced DNAm loss is associated with reduced binding of MeCP2 at intron 5 and a physical interaction between the fifth intron and promoter of Fkbp5. Our results highlight several key factors at the Fkbp5 locus that may have important implications for GC- or stress-exposure during early stages of neurodevelopment.

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure.

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform. Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

Effect of Genotype and Maternal Affective Disorder on Intronic Methylation of FK506 Binding Protein 5 in Cord Blood DNA.

A single nucleotide polymorphism (SNP: rs1360780) in FKBP5 (FK506 Binding Protein 5) has been shown to interact with exposure to childhood adversity to promote loss of methylation and increase in gene expression in adults. We asked whether rs1360780 can influence FKBP5 intronic methylation in the context of exposure to maternal affective disorders in utero. Sixty cord blood DNA samples from the Boston Birth Cohort were genotyped at rs1360780 and studied for methylation changes as they relate to genotype and exposure to affective disorders during pregnancy. Linear regression was employed to contrast the risk (TT) genotype to the heterozygous (CT) and homozygous (CC) genotypes with adjustment for potential confounders. The recessive genotype (TT) was associated with increased methylation at multiple CpGs in the FKBP5 intron 5 region (p < 0.01). These findings were enhanced among cases exposed to maternal affective disorders (p = 0.02). A human cell line treated with cortisol showed that changes in intron 5 CpG methylation and FKBP5 expression were inversely associated. These findings suggest that rs1360780 can influence FKBP5 intronic methylation by acting in cis as a methylation quantitative locus and highlight the impact of genotypic risk on methylation in utero. Additionally, prenatal stress exposure compounded with the risk genotype may lead to a compensatory increase in methylation.