Egr1 is a sex-specific regulator of neuronal chromatin, synaptic plasticity, and behaviour [RNA-Seq]

Sex differences are found in brain structure and function across species, and across brain disorders in humans. The major source of brain sex differences is differential secretion of steroid hormones from the gonads across the lifespan. Specifically, ovarian hormones oestrogens and progesterone are known to dynamically change structure and function of the adult female brain, having a major impact on psychiatric risk. However, due to limited molecular studies in female rodents, very little is still known about molecular drivers of female-specific brain and behavioural plasticity. Here we show that overexpressing Egr1, a candidate oestrous cycle-dependent transcription factor, induces sex-specific changes in ventral hippocampal neuronal chromatin, gene expression, and synaptic plasticity, along with hippocampus-dependent behaviours. Importantly, Egr1 overexpression mimics the high-oestrogenic phase of the oestrous cycle, and affects behaviours in ovarian hormone-depleted females but not in males. We demonstrate that Egr1 opens neuronal chromatin directly across the sexes, although with limited genomic overlap. Our study not only reveals the first sex-specific chromatin regulator in the brain, but also provides functional evidence that this sex-specific gene regulation drives neuronal gene expression, synaptic plasticity, and anxiety- and depression-related behaviour. Our study exemplifies an innovative sex-based approach to studying neuronal gene regulation in order to understand sex-specific synaptic and behavioural plasticity and inform novel brain disease treatments. Overall design: Male and ovariectomized, or intact proestrus female C57BL/6J mice underwent stereotaxic surgery at 7 weeks of age and received either Egr1 overexpression (AAV9-hSYN-Egr1-IRES-eGFP-WPRE3), Egr1 knockdown (AAV9-hSYN-Egr1-shRNA-eGFP-WPRE), eGFP control (AAV9-hSYN-eGFP-WPRE), or scrambled shRNA control (AAV9-hSYN-scramble-eGFP-WPRE) AAV targeted to the ventral hippocampus to determine the sex-dependent effects of Egr1 overexpresssion or knockdown on the neurons in this brain region. Following 3 weeks of incubation, mice were sacrificed at 10-11 weeks of age and bilateral ventral hippocampi were flash frozen in liquid nitrogen. Nuclei were then extracted from frozen tissue and neuronal nuclei were obtained by FACS using an antibody targeting the neuronal nuclei-specific NeuN protein. Nuclear RNA was extracted from NeuN+ nulcei and RNA-seq libraries were prepared with the KAPA RNA HyperPrep kit. 150 bp, paired-end sequencing was performed in one lane of an S1 flow cell on the NovaSeq 6000 instrument, yielding 60-90 million reads per sample.

跨物种以及人类脑部疾病场景中，均存在大脑结构与功能的性别差异。这类脑性别差异的核心成因是生命周期内性腺分泌类固醇激素的差异化模式。具体而言，卵巢分泌的雌激素与孕酮可动态重塑成年雌性大脑的结构与功能，对精神疾病风险产生显著影响。然而，由于针对雌性啮齿类动物的分子研究相对匮乏，目前学界对雌性特异性脑与行为可塑性的分子驱动机制仍知之甚少。

本研究证实，过表达早期生长应答蛋白1（Egr1，一种依赖动情周期的候选转录因子），可诱导腹侧海马神经元的染色质开放状态、基因表达及突触可塑性出现性别特异性改变，并伴随海马依赖性行为的显著变化。值得注意的是，Egr1过表达可模拟动情周期的高雌激素阶段，仅在卵巢激素耗竭的雌性小鼠中影响相关行为，而对雄性小鼠无此类作用。

研究表明，尽管两性间染色质开放的基因组重叠度有限，Egr1可在雌雄个体中直接介导神经元染色质开放。本研究不仅首次揭示了大脑内的性别特异性染色质调控因子，还为性别特异性基因调控可驱动神经元基因表达、突触可塑性以及焦虑与抑郁相关行为提供了功能学证据。本研究展示了一种基于性别的创新性研究范式，用于探究神经元基因调控机制，以解析性别特异性突触与行为可塑性，并为新型脑部疾病治疗策略的开发提供参考。

实验设计：将雄性、去卵巢雌性以及完整动情前期雌性C57BL/6J小鼠于7周龄时接受立体定位手术，向腹侧海马定向注射以下腺相关病毒（AAV）载体：Egr1过表达载体（AAV9-hSYN-Egr1-IRES-eGFP-WPRE3）、Egr1敲低载体（AAV9-hSYN-Egr1-shRNA-eGFP-WPRE）、eGFP对照载体（AAV9-hSYN-eGFP-WPRE）或乱序shRNA对照载体（AAV9-hSYN-scramble-eGFP-WPRE），以此探究Egr1过表达或敲低对该脑区神经元的性别依赖性效应。

经3周孵育后，于10-11周龄处死小鼠，取双侧腹侧海马并置于液氮中快速冷冻。随后从冷冻组织中提取细胞核，通过靶向神经元特异性核蛋白NeuN的抗体，利用荧光激活细胞分选（FACS）分离得到神经元细胞核。从NeuN阳性细胞核中提取核RNA，使用KAPA RNA HyperPrep试剂盒构建RNA测序文库。在NovaSeq 6000测序仪的S1流动槽单通道中开展150 bp双端测序，每个样本可获得6000万至9000万条读段。