Estradiol-mediated Gene Signatures in Hippocampal Memory

项目摘要 性类固醇激素17雌二醇(17-estadiol，E_2)在调节神经生理学方面具有公认的作用， 改变基因表达，促进记忆形成。尽管对E2‘S的研究已有数十年 神经调节作用，关于哪些特定的基因靶点受E2调节仍不清楚 有助于其对神经功能和记忆的影响。这在一定程度上是由于缺乏实验可行性， 作为检测E2对细胞类型的特异性转录调控和靶向操作的必要工具 在特定的E2敏感基因中，以前没有得到过。在这里，我建议使用以下方法来克服这些障碍 单核RNA和ATAC测序，这将允许全面和公正地鉴定 雌二醇在单细胞水平上调节的基因靶点(目标1)。这种方法对 目前很少有研究检验性类固醇激素的转录调控，这种激素使用脑匀浆 包含用于测序实验的各种细胞类型。尽管这样的工作内容丰富，但 E2调节转录调控的受体在多种不同的细胞中表达 类型。因此，更高的分辨率接近于在单个细胞水平上的10倍SNRNA-SEQ和SNATAC-SEQ 了解雌激素对基因表达的全面调控将是必要的。另一个限制是 以前的研究一直无法直接操纵E2敏感的基因靶点来经验性地测试 这种表达变化对记忆形成的要求。这项提议将克服这些限制。 通过使用基于CRISPR/dCas9的尖端表观遗传编辑方法来测试E2作用的要求 在特定的基因位点上调节其有益的助记效应(目标2)。最后，尽管循环中的 E2一直是神经内分泌学研究的重点，成年局部也存在E2的生物合成 两性的大脑，通过芳香酶(CYP19A1)调节的过程。AIM 3将使用 CRISPR/dCas9方法双向调节神经元中特异性芳香酶的表达，以测试 神经元特异性E2合成在记忆形成中的作用。总体而言，这些实验将提供许多 需要深入了解雌二醇如何作为神经功能和记忆的关键调节器在两性中发挥作用。

Project Summary The sex-steroid hormone 17-estradiol (E2) has a well-established role in mediating neuronal physiology, altering gene expression, and facilitating memory formation. Despite decades of research on E2’s neuromodulatory effects, much remains unknown regarding which specific gene targets regulated by E2 contribute to its effects on neuronal function and memory. This is in part due to a lack of experimental feasibility, as the tools necessary for examining cell-type specific transcriptional regulation by E2, and targeted manipulation of specific E2-sensitive genes, were not previously available. Here, I propose to overcome these hurdles by using single-nuclei RNA- and ATAC-sequencing, which will allow for comprehensive and unbiased identification of gene targets regulated by E2 in both sexes at the single-cell level (Aim 1). This approach is advantageous to the few existing studies examining transcriptomic regulation of sex-steroid hormones, which use brain homogenates containing a variety of cell types for sequencing experiments. Although such work has been informative, the receptors through which E2 orchestrates transcriptional control are expressed in a wide variety of distinct cell types. As such, higher resolution approaches such 10X snRNA-seq and snATAC-seq at the individual cell level will be necessary to understand the full myriad of estrogenic regulation of gene expression. Another limitation of previous studies has been the inability to directly manipulate E2-sensitive gene targets to empirically test the requirement of such changes in expression on memory formation. This proposal will overcome such limitations by using cutting-edge CRISPR/dCas9-based epigenetic editing approaches to test the requirement of E2 action at specific gene loci in mediating its beneficial mnemonic effects (Aim 2). Finally, although circulating levels of E2 have been the focus of much neuroendocrinology research, E2 biosynthesis also occurs locally in the adult brain of both sexes, via a process regulated by the enzyme aromatase (Cyp19a1). Aim 3 will use a CRISPR/dCas9 approach to bidirectionally regulate aromatase expression specifically in neurons, to test the role of neuron-specific E2 synthesis in memory formation. Collectively, these experiments will provide much needed insight into how E2 acts as a key modulator of neuronal function and memory in both sexes.