Prenatal arsenic exposure alters transcriptional, post-transcriptional and post-translational programming of the glucocorticoid system in a sexually dimorphic manner

项目摘要 发育过程中接触无机砷与几种有害的健康影响有关，包括 认知能力下降，精神障碍发生率增加。更改了对 糖皮质激素受体(GR)可以解释砷的许多长期后果。而当 砷暴露对表观基因组的影响已经在癌症研究的背景下进行了研究，其影响 这种毒物在发育中的大脑中的作用，特别是当它与表观遗传学有关时，还不是很清楚。 在我们之前的工作中，我们发现产前暴露于50 ppb砷(PAE)的雄性后代减少了 GR蛋白和mRNA在脑内的表达。雌性PAE小鼠对砷诱导的改变具有抵抗力 尽管GR(NR3C1)基因表达升高，但GR蛋白仍有表达。在评估表观遗传机制时，我们发现PAE 受影响的组蛋白3、裂解酶4三甲基化(H3K4me3)水平以及几个非编码RNA(miRs和 IncRNA)以性别依赖的方式。这一竞争性更新的目标是证明 转录、转录后和翻译后调节导致PAE性别依赖效应 分子、生理和行为水平。 特定目标1将测试PAE改变控制组蛋白修饰的假设 应激相关基因在胎儿发育过程中以性别特异性的方式转录。我们会 评估组蛋白翻译后修饰(HPTM)、H3K4 ME3/H3K27 ME3、组蛋白编写器和擦除器， 并通过Western和序列芯片-qPCR技术对相关基因进行分析。我们将通过以下方式确认功能链接 在胚胎第12.5天(E12.5)药理学改变特定的组蛋白标记。 特定目标2将检验PAE改变转录后调控的假说 性别胎儿发育过程中应激相关基因表达水平的研究 具体的方式。这些实验将使用qPCR和miR-RIP靶向GR来评估miR的表达 信令系统。我们将在E12.5处使用LNA microRNA模拟和LNA-AntimiR确认一个功能链接。 具体目标3将检验PAE改变压力翻译后调节的假设-- 在性别胎儿发育过程中通过长非编码RNA(LncRNA)的相关蛋白质- 具体的方式。这些实验将评估生长停滞特异性5(Gas-5)在E12-18的表达。我们 将使用LNA-GAPmer和LNA-ANtimir方法确认与GR系统的功能链接。 特定目标4将测试PAE诱导的转录， 成人转录后和/或翻译后变化与功能结局 动物。这些实验将评估目标1-3中确定的干预措施对砷诱导的影响。 缺陷：皮质酮反应、学习和抑郁。

Project Summary Developmental exposure to inorganic arsenic has been associated with several adverse health effects, including reduction in cognitive performance and increased rates of psychiatric disorders. Altered programming of the glucocorticoid receptor (GR) could account for many of the long-lasting consequences of arsenic. While the impact of arsenic exposure on the epigenome has been studied in the context of cancer research, the influence of this toxicant in the developing brain, particularly as it relates to epigenetics, is not well understood. In our previous work, we found that male offspring exposed to 50 ppb arsenic prenatally (PAE) had decreased expression of both GR protein and mRNA in brain. Female PAE mice were resistant to arsenic induced changes in GR protein despite elevations in GR (Nr3c1) mRNA. In assessing epigenetic mechanisms, we found PAE affected levels of histone3 lysine4 trimethylation (H3K4me3) as well as several noncoding RNAs (miRs and lncRNA) in a sex-dependent manner. The goal of this competing renewal is to demonstrate that changes in transcriptional, posttranscriptional and posttranslational regulation result in PAE sex-dependent effects at a molecular, physiological and behavioral level. Specific Aim 1 will test the hypothesis that PAE alters histone modifications that control the transcription of stress-related genes during fetal development in a sex-specific manner. We will assess histone posttranslational modifications (HPTMs), H3K4 me3/ H3K27 me3, histone writers and erasers, and associated genes by western and sequential ChIP-qPCR techniques. We will confirm a functional link by pharmacologically altering specific histone marks at embryonic day 12.5 (E12.5). Specific Aim 2 will test the hypothesis that PAE alters the posttranscriptional regulation of stress-related mRNA levels through micro-RNAs (miRs) during fetal development in a sex- specific manner. These experiments will assess miR expression using qPCR and miR-RIP to target the GR signaling system. We will confirm a functional link using LNA microRNA mimics and LNA-antimiR at E12.5. Specific Aim 3 will test the hypothesis that PAE alters the posttranslational regulation of stress- related proteins through long noncoding RNA (lncRNA) during fetal development in a sex- specific manner. These experiments will assess growth arrest-specific 5 (Gas-5) expression at E12-18. We will confirm a functional link to the GR system using LNA-GAPmer and LNA–antimir approaches. Specific Aim 4 will test the mechanistic link between PAE-induced transcriptional, posttranscriptional and/or posttranslational changes and the functional outcomes in the adult animal. These experiments will assess the impact of interventions identified in Aims 1-3 on arsenic-induced deficits: corticosterone response, learning and depression.