Sperm chromatin accessibility's involvement in the intergenerational effects of stress hormone receptor activation

Dexamethasone is a stress hormone receptor agonist used widely in clinics. We and others previously showed that paternal administration of dexamethasone in mice affects the phenotype of their offspring. The substrate of intergenerational transmission of environmentally induced effects often involves changes in sperm RNA, yet other epigenetic modifications in the germline can be affected and are also plausible candidates. First, we tested the involvement of altered sperm RNAs in the transmission of dexamethasone induced phenotypes across generations. We did this by injecting sperm RNA into naïve fertilized oocytes, before performing metabolic and behavioral phenotyping of the offspring. We observed phenotypic changes in discordance with those found in offspring generated by in vitro fertilization using sperm from dexamethasone exposed males. Second, we investigated the effect of dexamethasone on chromatin accessibility using ATAC sequencing and found significant changes at specific genomic features and gene regulatory loci. Employing q-RT-PCR, we show altered expression of a gene in the tissue of offspring affected by accessibility changes in sperm. Third, we establish a correlation between specific DNA modifications and stress hormone receptor activity as a likely contributing factor influencing sperm accessibility. Finally, we independently investigated this dependency by genetically reducing thymine-DNA glycosylase levels and observing concomitant changes at the level of chromatin accessibility and stress hormone receptor activity. Overall design: Comparative gene expression profiling analysis of RNA-seq data for mESC dpending on genotype.

地塞米松（Dexamethasone）是一种临床广泛应用的应激激素受体激动剂。此前我们与其他研究团队均证实，对小鼠父本施用地塞米松会对子代表型产生影响。环境诱导的表型效应的代际传递底物通常涉及精子RNA的改变，而生殖细胞内的其他表观遗传修饰同样可能发生异常，亦属于颇具潜力的候选调控机制。其一，我们验证了精子RNA异常是否参与地塞米松诱导的表型跨代传递：将精子RNA注射至未经过实验处理的受精卵中，随后对子代开展代谢与行为表型分析。我们观测到的表型变化，与使用地塞米松暴露雄鼠精子经体外受精获得的子代所呈现的表型并不相符。其二，我们通过ATAC测序（ATAC sequencing）探究了地塞米松对染色质开放状态的影响，发现其在特定基因组特征区域与基因调控位点上存在显著变化。借助定量实时反转录PCR（q-RT-PCR），我们证实：受精子染色质开放状态改变影响的子代组织中，某一基因的表达水平发生了异常。其三，我们明确了特定DNA修饰与应激激素受体活性之间存在相关性，这一相关性或为影响精子染色质开放状态的潜在调控因素。最后，我们通过遗传手段降低胸腺嘧啶DNA糖苷酶（thymine-DNA glycosylase）的表达水平，独立验证了这一依赖关系，并同步观测到染色质开放状态与应激激素受体活性的相应变化。整体实验设计：基于不同基因型小鼠胚胎干细胞（mouse embryonic stem cell, mESC）的RNA测序数据，开展比较基因表达谱分析。