Effects of gene-by-environment interaction on the H3K4me3 profile in the light of differential susceptibility

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 Overall design: H3K4me3 enrichment affected in serotonin transporter deficient or wildtype offspring of prenatal stress that do or do not succumb to the adverse effects of prenatal stress, i.e. vulnerable or resilient offspring [contributor] Nxt-Dx (Ghent, Belgium)

研究目的：探究5-羟色胺转运体（serotonin transporter）缺陷小鼠的早期不良经历，在差异易感性背景下对组蛋白H3赖氨酸4三甲基化（H3K4me3）富集水平的影响。
实验方法：首先对小鼠进行产前应激处理，并在其成年后开展行为筛选。随后选取3月龄雌性小鼠，分为野生型与5-羟色胺转运体基因杂合敲除型两组，对其实施安乐死并取出脑组织，解剖分离双侧海马体，经匀浆后分为两份；其中一份再次分为两子份，一份采用1%多聚甲醛（PFA）固定，随后按照标准染色质免疫共沉淀（Chromatin immunoprecipitation, ChIP）流程进行处理。ChIP完成后，样本交由比利时Nxt-Dx公司进行后续处理。文库构建采用适配Illumina平台的NEBNext Ultra II DNA文库制备试剂盒（NEB，美国马萨诸塞州伊普斯维奇）。随后对全部免疫沉淀（IP）产物进行末端修复及Illumina接头连接，使用AMPure XP磁珠（贝克曼库尔特）对连接有接头的DNA进行纯化，该步骤未进行片段大小筛选。将洗脱产物与NEBNext索引引物进行富集PCR扩增（14个循环），随后再次使用AMPure XP磁珠进行纯化。最终文库的质量通过Agilent 2100生物分析仪DNA 1000芯片（安捷伦，美国加利福尼亚州圣克拉拉）进行检测，浓度通过定量聚合酶链式反应（qPCR）测定，以PhiX索引3标准品作为稀释参照。将所有索引化样本的浓度均调整至10 nM，混合后进行测序。测序在Illumina HiSeq4000平台上完成，采用双端测序模式，读长为50 bp，每个样本的测序读段数为2500万至3000万。
研究结果：根据5-羟色胺转运体基因型以及行为学判定的早期不良经历易感性，小鼠呈现出不同的H3K4me3富集水平。正如预期，效应量与显著性水平均相对微弱。
研究结论：产前应激所介导的表观遗传调控似乎受到5-羟色胺转运体基因型的调控。
实验整体设计：产前应激子代中，出现或未出现产前应激不良影响（即易感型或抵抗型）的5-羟色胺转运体缺陷型或野生型小鼠，其H3K4me3富集水平存在差异。
贡献方：比利时根特Nxt-Dx公司