Epigenetic Mechanisms of Chronic Stress Action

项目总结： 这是新的R01授权的修订版，其特征是在 小鼠边缘脑区对早期生活应激(ELS)的反应，从而增加个体的 一生易受后续压力的影响。这项工作在过去的十年里得到了一个 然而，NIMH Conte Center，该中心现在已经“日落”了--它不能续签--因此，这个新的 建议继续这一原有的研究路线。我们的创新假设是ELS诱导稳定 “染色质疤痕”驱动基因表达的长期变化，然后介导下游变化 在成年期对慢性应激的细胞和回路功能以及最终的行为反应。我们集中精力 伏隔核(NAC)在大脑奖赏和动机中的中枢作用及经验研究 它在控制应力敏感性方面的核心作用。我们利用开放式、无偏见的蛋白质组学和下一步- 世代测序方法，首先确定最健壮和最重要的假定染色质疤痕 NAC以特定细胞类型的方式，然后在分子、细胞和 行为水平。重要的是，我们只调查那些在死后NAC中有效的机制 抑郁的人类。一个主要的焦点是H3K79me2(组蛋白H3的Lys79的二甲基化)，诱导 这是ELS在雄性和雌性小鼠的NAC中引起的最显著的组蛋白修饰。这个 控制H3K79me2的甲基转移酶(DOT1L)和去甲基酶(Kdm2b)在NAC中均被诱导 ELS，D2型中棘神经元(MSN)特有的效应。病毒介导的双向操作 DOT1L或Kdm2b选择性地在雄性和雌性小鼠的D2 NAC MSN中确立了它们所起的作用 基于快速行为筛查的ELS增加应激敏感性能力中的酶 化验。我们现在将压力易感性的测量扩展到许多更复杂的行为 具有更大翻译潜力的程序，并测试DOT1L抑制剂的能力，目前正在进行临床试验 某些癌症，在全身给药时逆转ELS诱导的应激敏感性，进一步促进 拟议研究的翻译潜力。此外，我们已经通过RNAseq展示了DOT1L 在D2 MSN中的过表达模拟ELS在该细胞中诱导的很大一部分基因表达变化 类型，而DOT1L击倒阻止ELS操作。我们现在将H3K79me2浓缩映射到全基因组 D2 MSN通过Cut&RUNseq鉴定ELS改变表达受其影响的基因网络 组蛋白标记。我们还将生成更完整的开放式蛋白质组和转录组数据集，以及 鉴定ELS在NAC中诱导的其他可能的染色质疤痕。大量初步数据， 例如，暗示H3K27me1是一种显著的染色质恐慌，在雄性小鼠的D1MSN中占主导地位 只有这样。总而言之，这项工作将描述驱动持续强化压力状态的新机制 并提供对成年后逆转这种易感性的新方法的洞察。

PROJECT SUMMARY: This is a revised version of a new R01 grant that characterizes stable epigenetic changes that are induced in mouse limbic brain regions in response to early life stress (ELS) which then increase an individual's susceptibility to subsequent stress for a lifetime. This work has been supported over the past ten years by an NIMH Conte Center, however, that Center has now “sun-setted”—it cannot be renewed—hence, this new proposal to continue this original line of research. Our innovative hypothesis is that ELS induces stable “chromatin scars” that drive long-lived changes in gene expression, which then mediate downstream changes in cell and circuit function and ultimately behavioral responses to chronic stress in adulthood. We concentrate on nucleus accumbens (NAc) based on its central role in brain reward and motivation and on empirical findings of its central role in controlling stress susceptibility. We utilize open-ended, unbiased proteomic and next- generation sequencing approaches to first identify the most robust and significant putative chromatin scars in NAc in a cell-type-specific manner and to then characterize those mechanisms at the molecular, cellular, and behavioral levels. Importantly, we investigate only those mechanisms that are validated in postmortem NAc of depressed humans. One major focus is H3K79me2 (dimethylation of Lys79 of histone H3), the induction of which is the most significant histone modification caused by ELS in NAc of both male and female mice. The methyltransferase (DOT1L) and demethylase (KDM2B) that control H3K79me2 are both induced in NAc by ELS, effects specific to D2-type medium spiny neurons (MSNs). Bidirectional viral-mediated manipulation of DOT1L or KDM2B selectively in D2 NAc MSNs of male and female mice establishes the role played by these enzymes in mediating the ability of ELS to increase stress susceptibility based on rapid behavioral screening assays. We will now extend measures of stress susceptibility to numerous, more sophisticated behavioral procedures with greater translational potential, and test the ability of a DOT1L inhibitor, now in clinical trials for certain cancers, to reverse ELS-induced stress susceptibility upon systemic administration, further promoting the translational potential of the proposed research. As well, we have shown by RNAseq that DOT1L overexpression in D2 MSNs mimics a large portion of gene expression changes induced by ELS in this cell type, while DOT1L knockdown blocks ELS action. We will now map H3K79me2 enrichment genome-wide in D2 MSNs by CUT&RUNseq to identify networks of genes whose altered expression by ELS is mediated by this histone mark. We will also generate more complete open-ended proteomic and transcriptomic datasets and characterize additional putative chromatin scars induced in NAc by ELS. Substantial preliminary data, for example, implicate H3K27me1 as a prominent chromatin scare that predominates in D1 MSNs of male mice only. Together, this work will characterize novel mechanisms that drive a persisting state of enhanced stress susceptibility and offer insight into new ways of reversing such susceptibility in adulthood.