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Effects of gene-by-environment interaction on the methylome of mice deficient for tryptophan hydroxylase 2 [MBD-seq]

ABSTRACT: Purpose: Investigation of effects of early adversity in mice deficient for brain serotonin on the methylome Methods: Following materal separation in early childhood and behavioural screening in adulthood, 4 moth old males, either wildtype or carrying a hetero- or homozygous knockout of the thryptophan hyroxylase 2 gene, were sacrificed, brains extracted, the amygdalae of both hemispheres dissected, blended and separated in two protions. From one of these portions DNA was processed by an external company, i.e. Nxt-Dx (Ghent, Belgium). DNA was sheared by sonication, followed by methyl binding domain (MBD) based capture, leading to an enrichment of methylated fragments. Subsequently, library preparation was performed followed by multiplexed paired-end sequencing with a read-length of 50 bp and an approximate output of 20 million reads/sample on the Illumina HiSeq4000 platform. Reads were mapped to the Mus musculus GRCm38.p5 genome using Bowtie2 (v2.1.0) software in end-to-end & sensitive-mode. After mapping, coverage peaks were generated using MACS 14 peak caller (Zhang et al. 2008). Results: Either Tph2 genotype, early adversity as well as their interaction resulted in unique profiles of differentially enriched methylated peaks as well as expressed RNA. As expected, the effect sizes and significances were rather subtle and the overlap between top hits of methylome and transcriptome were marginal. Conclusion: Tph2, MS and their interaction induced changes in gene expression and DNA methylation, within a physiological range. The observed changes by MS seemed furthermore strongly influenced by the Tph2 genotype, ergo brain 5-HT availability.

ORGANISM(S): Mus musculus

PROVIDER: GSE110329 | GEO | 2019/05/06

REPOSITORIES: GEO

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Project description:Purpose: Investigation of effects of early adversity in mice deficient for serotonin transporter on H3K4me3 enrichment in the context of differential susceptibility Methods: Following prenatal stress and behavioural screening in adulthood, 3 month old females, either wildtype or carrying a heterozygous knockout of the serotonin transporter gene, were sacrificed, brains extracted, the hippocampi of both hemispheres dissected, blended and separated in two protions. For one of these portions the material was split again in two portions, of which one was fixed using 1% PFA, and was subsequently processed using a standard Chromatin immuno precipitation protocol. Following ChIP, the samples were processed by Nxt-Dx Belgium. Library preparation was performed using the NEBNext Ultra II DNA Library prep kit for Illumina (NEB, Ipswitch, Massatchusettes, USA). Subsequently, the whole IP material was subjected to ends prep and ligation of Illumina adaptors. A clean up of the adaptor-ligated DNA with AMPure XP beads (Beckman Coulter) was performed without size selection. The eluted material was subjected to enrichment PCR (14 cycles) with the NEBNext Index primers, and a last clean up with AMPure XP beads followed. The quality of the final libraries was checked on a Bioanalyzer 2100 DNA 1000 chip (Agilent, Santa Clara, CA, USA). The concentration was determined by performing qPCR on the samples using a dilution of PhiX index3 as standard. The concentration of all indexed samples was adjusted to 10 nM and samples were pooled for sequencing. Sequencing was performed on an Illumina HiSeq4000 (read-length of 50 bp with 25-30 million reads/sample, paired-end). Results: dependent on the serotonin transporter genotype and behaviourally determined susceptibility to early life adversity, animals displayed distinct H3K4me3 enrichment. As expected, the effect sizes and significances were rather subtle. Conclusion: the epigenetic regulation by prenatal stress seems to be modulated by the serotonin transporter genotype

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Effects of gene-by-environment interaction on the methylome of mice deficient for tryptophan hydroxylase 2 [MBD-seq]

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