Transcriptional and epigenetic targets of MEF2C in human microglia contribute to cellular functions related to autism risk and age-related disease [ChIP-Seq]

MEF2C is a transcription factor with wide ranging roles and has specifically been implicated in restraining the microglial inflammatory response, participating in nervous system development, and contributing to aging and neurological disease. While MEF2C murine models have provided critical insights, the homeostatic and disease-associated functions of MEF2C in human microglia remain unclear. To examine this question, we profile microglia differentiated from isogenic, CRISPR-modified MEF2C haploinsufficient and knockout induced-pluripotent stem cell (iPSC) lines. A combination of transcriptomic, epigenetic and functional analyses revealed that loss of MEF2C leads to a hyperinflammatory microglial phenotype with broad impairment in phagocytosis, lipid accumulation, lysosomal dysfunction and elevated basal inflammatory cytokine and inflammasome activation. Transcriptomic and epigenetic approaches identified substantial overlap between reduced microglial MEF2C expression and primary brain idiopathic autism datasets, suggesting a broader role of microglial MEF2C dysregulation in idiopathic autism especially as relates to the transcription factors repressive functions. Reduced MEF2C expression has been associated with brain aging, and we find that reduction in MEF2C leads to a premature aging or senescence associated phenotype in microglia. These findings are extended to a murine xenotransplantation model wherein reductions in MEF2C expression in human microglia leads to an ameboid hyperinflammatory phenotype with lysosomal and lipid dysfunction. Taken together, these studies reveal novel aspects of microglial MEF2C function that contribute to homeostasis and development of neurological disease. To investigate the effect of the in MEF2C genotype, we generated a cohort of we profile microglia differentiated from isogenic, CRISPR-modified MEF2C haploinsufficient and knockout induced-pluripotent stem cell (iPSC) lines. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for transcription factor MEF2C as well as the histone modifications H3K27ac for acetylation and ATAC-seq in iPSC-derived microglia with different MEF2C genotypes--Control (+/+), haploinsufficient HET (+/-) and knockout (-/-).

MEF2C是一类功能广泛的转录因子（transcription factor），其已被证实可调控小胶质细胞（microglia）的炎症反应、参与神经系统发育，并与衰老及神经疾病密切相关。现有MEF2C小鼠模型虽已提供关键研究见解，但人类小胶质细胞中MEF2C的稳态维持功能及疾病相关功能仍尚不明确。
为探究这一科学问题，本研究对源自同基因、经CRISPR（成簇规律间隔短回文重复序列）修饰的MEF2C单倍体不足（haploinsufficient）及敲除（knockout）诱导多能干细胞（induced-pluripotent stem cell, iPSC）系分化得到的小胶质细胞开展了多组学表征分析。结合转录组学（transcriptomic）、表观遗传学（epigenetic）与功能实验分析结果显示，MEF2C缺失会导致小胶质细胞呈现过度炎症表型，并伴随吞噬作用广泛受损、脂质蓄积、溶酶体功能障碍，以及基础炎症细胞因子与炎症小体激活水平升高。
转录组学与表观组学分析发现，小胶质细胞MEF2C表达下调与原发性脑部特发性自闭症（idiopathic autism）数据集存在显著重叠，提示小胶质细胞MEF2C调控异常可能在特发性自闭症中发挥更为广泛的作用，尤其与其作为转录因子的抑制性功能密切相关。此前已有研究将MEF2C表达降低与脑衰老关联，本研究进一步发现，MEF2C水平下调会诱导小胶质细胞出现早衰或衰老相关表型。
上述研究发现已在小鼠异种移植模型中得到验证：人类小胶质细胞中MEF2C表达降低会引发阿米巴样过度炎症表型，并伴随溶酶体与脂质功能障碍。综上，本研究揭示了小胶质细胞MEF2C功能的全新维度，这些功能参与维持细胞稳态并参与神经疾病的发生发展。
为探究MEF2C基因型的影响，我们构建了一组源自同基因、经CRISPR修饰的MEF2C单倍体不足及敲除诱导多能干细胞系分化得到的小胶质细胞队列，并对其开展表征分析。我们对携带不同MEF2C基因型的iPSC来源小胶质细胞——对照(+/+)、单倍体不足杂合子(+/-)及敲除(-/-)——进行了转录因子MEF2C的染色质免疫沉淀测序（Chromatin immunoprecipitation DNA-sequencing, ChIP-seq）、乙酰化组蛋白修饰（histone modifications）H3K27ac的ChIP-seq以及ATAC测序（ATAC-seq）。