Estradiol-mediated Gene Signatures in Hippocampal Memory

项目摘要 性类固醇激素17 β-雌二醇（E2）在调节神经元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进行了数十年的研究 神经调节作用，关于哪些特定的基因靶点由E2调节仍是未知的 有助于其对神经元功能和记忆的影响。这部分是由于缺乏实验可行性， 作为研究E2对细胞类型特异性转录调控的必要工具， 特异性E2敏感基因，以前没有。在这里，我建议通过使用 单核RNA和ATAC测序，这将允许全面和公正的鉴定， 在单细胞水平上，两种性别中受E2调节的基因靶点（目的1）。这种方法有利于 现有的研究很少检查性类固醇激素的转录调控，这是使用脑匀浆 含有用于测序实验的多种细胞类型。虽然这些工作提供了信息， E2通过其协调转录控制的受体在多种不同的细胞中表达 类型因此，在单个细胞水平上，更高的分辨率接近10 X snRNA-seq和snATAC-seq 对于理解雌激素对基因表达的无数调节是必要的。的另一个限制 以前的研究一直无法直接操纵E2敏感基因靶点， 这种表达变化对记忆形成的要求。这项建议将克服这些限制 通过使用基于CRISPR/dCas 9的尖端表观遗传编辑方法来测试E2作用的要求， 在特定的基因位点介导其有益的记忆效应（目的2）。最后，尽管循环水平 E2是神经内分泌学研究的热点，E2的生物合成也发生在成人局部 通过芳香化酶（Cyp 19 a1）调节的过程。Aim 3将使用 CRISPR/dCas 9方法双向调节神经元中特异性芳香化酶表达，以测试 神经元特异性E2合成在记忆形成中的作用。总的来说，这些实验将提供很多 需要深入了解E2如何作为两性神经功能和记忆的关键调节剂。

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.