Nuclear microRNA-mediated transcriptional control determines adult microglial homeostasis and brain function [ATAC-seq]

Microglia are versatile regulators in brain development and disorders. Emerging evidence links miRNA-mediated regulation to microglial function; however, the exact underlying mechanism remains largely unknown. Here, we discover that miR-137, a neuropsychiatric disorders-associated miRNA, is abundant in the microglial nucleus and exhibits unexpected epigenetic functions in maintaining the global transcriptomic state, core properties, and functions of microglia. Mechanistically, microglial Mir137 deletion increases the accessibility of chromatin, which harbors binding motifs of microglial master transcription factor Pu.1. Our biochemical and bioinformatics analyses suggest that miR-137 can interact with Pu.1, thus influencing Pu.1-mediated gene expression. Importantly, we find that increased Pu.1 binding upregulates the target gene Jdp2, and Jdp2 knockdown markedly suppresses the impaired cellular phenotypes in Mir137 knockout microglia. Collectively, our study provides evidence supporting a notion that miR-137 acts as an epigenetic regulator, and this microglia-specific function is essential to maintain normal adult brain function. We generated microglia-specific Mir137 knockout mice to investigate the role of miR-137 in microglia in adult mice. To do so, we crossed Mir137loxP/loxP mice with transgenic mice bearing a tamoxifen-inducible Tmem119-CreERT2 allele . After 7 consecutive days injection of tamoxifen at 7–8 weeks of age, we obtained the mice with correct genotypes, including Mir137+/+;Tmem119-CreERT2 (cWT) and Mir137loxP/loxP;Tmem119-CreERT2 (cKO). The primary microglia of adult mice (n = 4) were gently isolated and purified. To investigate the most active transcription factors upon the loss of miR-137, we performed transposase-accessible chromatin sequencing (ATAC-seq) for Mir137 cWT and cKO microglia. Three replicates were included for each genotype.

小胶质细胞（Microglia）是脑发育与疾病进程中兼具多种功能的调控因子。日益增多的研究证据显示，微小RNA（microRNA, miRNA）介导的基因调控与小胶质细胞功能紧密相关，但其背后的确切分子机制仍未完全明晰。本研究发现，与神经精神疾病相关的miR-137在小胶质细胞核内富集度较高，并在维持小胶质细胞的全局转录组状态、核心生物学特性及生理功能方面，发挥了此前未被认知的表观遗传调控作用。机制层面，小胶质细胞Mir137基因敲除可提升染色质开放程度，此类开放染色质区域携带有小胶质细胞核心转录因子Pu.1的结合基序。生化与生物信息学分析结果表明，miR-137可与Pu.1发生相互作用，进而影响Pu.1介导的基因表达程序。值得关注的是，我们发现Pu.1结合位点的增加会上调其靶基因Jdp2的转录，而敲低Jdp2可显著逆转Mir137基因敲除小胶质细胞中出现的细胞功能受损表型。综上，本研究为miR-137作为表观遗传调控因子的功能提供了直接实验证据，且这一小胶质细胞特异性的调控功能对于维持成年大脑的正常生理功能不可或缺。为探究miR-137在成年小鼠小胶质细胞中的作用，我们构建了小胶质细胞特异性Mir137基因敲除小鼠：将Mir137loxP/loxP小鼠与携带他莫昔芬诱导型Tmem119-CreERT2等位基因的转基因小鼠进行杂交。待小鼠生长至7~8周龄时，连续7天注射他莫昔芬，最终获得基因型正确的小鼠，包括Mir137+/+;Tmem119-CreERT2（对照野生型，cWT）与Mir137loxP/loxP;Tmem119-CreERT2（条件性敲除型，cKO）。我们温和分离并纯化了成年小鼠的原代小胶质细胞（样本量n=4）。为明确miR-137缺失后活性发生显著变化的转录因子，我们对Mir137 cWT与cKO小胶质细胞开展了转座酶可及性测序（ATAC-seq），每种基因型设置3个生物学重复。