Mechanisms of bisphenol A induced epigenomic alterations in the mammalian germline

项目摘要/摘要 双酚A普遍存在于许多消费品中。人类每天接触到双酚A，超过90% 在接受调查的个人中，他们的尿液中含有可检测到的BPA水平。肥胖、代谢紊乱和 在人类和动物中，神经疾病一直与双酚A接触有关。在啮齿动物中，暴露于 F0孕鼠的BPA导致F2代肥胖表型，并在未暴露者中持续存在 F3和后续世代。这些观察表明，暴露于双酚A会导致表观遗传。 F1生殖系的变化，可在世代间和世代间遗传。然而，这些机制 通过它，这些表观遗传改变被建立、维持并传递给后代 很大程度上仍未被勘探。为了解决这个问题，我们将怀孕的雌性小鼠暴露在BPA中 胚胎7-13天，当胎儿的生殖系被重新编程时。我们发现暴露在双酚A中 导致肥胖表型，通过母体和父系传播至少4代。精子 来自BPA暴露小鼠肥胖后代的含有新的染色质可访问部位 与CTCF和几个核激素受体的新结合部位相对应的未暴露对照。 重要的是，这些新的位点存在于F2-F5小鼠的精子中，这表明它们直接或间接地 对肥胖的表型负责。总之，这些结果表明，由 双酚A可以通过雄性生殖系进行跨代传播。然而，我们不知道这些是如何 更改最初是在重新编程期间在生殖系中获得的，或者在不存在的情况下如何保持这些更改 双酚A最初的刺激方案。在这里，我们提出实验来研究双酚A是如何引起表观遗传变化的。 在早期生殖系细胞中建立和维持。目标1中提议的工作将确定新的 发育过程中不同时间点双酚A暴露对转录因子结合位点的影响 原始生殖细胞(PGCs)、成熟原细胞和精原干细胞(SSCs)，并检查 Tf结合的变化是否与这些细胞的转录组的变化有关。在目标2中，我 将研究PGCs中Tf结合的变化与DNA甲基化、组蛋白变化的关系 在雄性生殖系分化的整个过程中，新的Tf位点上的组蛋白变异和修饰。目标3 将分析在没有表观遗传改变的情况下F2代种系的维持情况 双酚A暴露，以及TF结合和DNA甲基化变化之间的时间关系和 染色质结构。从拟议的研究中获得的知识将首次揭示双酚A是如何- 在胚胎生殖系的表观遗传重编程过程中建立了诱导的表观遗传改变 维持在后代的雄性生殖细胞中。这些研究将具有非常重要的意义 了解内分泌诱导表型跨代遗传的机制 破坏化学物质(EDCs)，这是设计干预措施扭转这些影响的第一步。

PROJECT SUMMARY/ABSTRACT BPA is ubiquitously found in many consumer products. Humans are exposed to BPA daily and more than 90% of surveyed individuals have detectable levels of BPA in their urine. Obesity, metabolic disorders, and neurological disorders have been associated with BPA exposure in humans and animals. In rodents, exposure to BPA of F0 pregnant females results in obesity phenotypes in the F2 generation that persist in the unexposed F3 and subsequent generations. These observations suggest that exposure to BPA induces epigenetic alterations in the F1 germline that can be inherited inter- and transgenerationally. However, the mechanisms by which these epigenetic alterations are established, maintained, and transmitted to subsequent generations remain largely unexplored. To address this question, we exposed pregnant mouse females to BPA during embryonic days 7-13, when the germline of the fetus is being reprogrammed. We find that exposure to BPA leads to an obese phenotype that is transmitted maternally and paternally for at least 4 generations. Sperm from the obese progeny of BPA-exposed mice contain new chromatin accessible sites not present in unexposed controls corresponding to new binding sites for CTCF and several nuclear hormone receptors. Importantly, these new sites are present in sperm of F2-F5 mice, suggesting that they are directly or indirectly responsible for the obesity phenotype. Together, these results suggest that epigenetic alterations induced by BPA can be transmitted transgenerationally by the male germline. However, we do not know how these alterations are initially acquired in the germline during reprograming or how they are maintained in the absence of the original stimulus by BPA. Here we propose experiments to study how BPA-induced epigenetic changes are established and maintained in early germ line cells. The work proposed in Aim 1 will identify new transcription factor (TF) binding sites induced by BPA exposure at different time points during the development of the Primordial Germ Cells (PGCs), prospermatogonia, and Spermatogonial Stem Cell (SSCs), and examine whether the changes in TF binding are associated with changes in the transcriptome of these cells. In Aim 2, I will study the relationship between changes in TF binding in PGCs and alterations of DNA methylation, histone modifications, and histone variants at new TF sites throughout the differentiation of the male germline. Aim 3 will analyze the maintenance of epigenetic alterations in the germline of the F2 generation in the absence of BPA exposure, and the temporal relationship between TF binding and changes in DNA methylation and chromatin structure. The knowledge gained from the proposed studies will reveal for the first time how BPA- induced epigenetic alterations are established during epigenetic reprogramming in the embryonic germline and maintained in the male germ cells of subsequent generations. These studies will be highly significant in understanding the mechanisms of transgenerational inheritance of epiphenotypes induced by endocrine disrupting chemicals (EDCs), a first step towards designing interventions to reverse these effects.