Deconstructing Stepwise Fate Conversion of Human Fibroblasts to Neurons by MicroRNAs: Subseries 5 [Figure 3, ATAC-seq]. Deconstructing Stepwise Fate Conversion of Human Fibroblasts to Neurons by MicroRNAs: Subseries 5 [Figure 3, ATAC-seq]

Cell-fate conversion generally presumes the activity of reprogramming effectors to both introduce fate programs of the target cell type and erase the identity of starting cell population. Here, we reveal novel insights into the activity of microRNAs, miR-9/9* and miR-124 (miR-9/9*-124) as reprogramming agents that orchestrate direct conversion of human fibroblasts by first eradicating fibroblast identity and promoting uniform transition to a neuronal state in sequence. Among the direct target genes of miR-9/9*-124, we identify KLF-family transcription factors whose repression is critical for erasing fibroblast fate. The subsequent upregulation of a small nuclear RNA, RN7SK induces accessibilities of chromatin regions and neuronal gene activation, pushing the reprogramming cells to a neuronal state. Our study defines deterministic components in the reprogramming cascade by microRNAs. Overall design: De-identified healthy human adult dermal fibroblasts were transduced with lentivirus to express the brain enriched microRNAs, miR-9/9* and miR-124 to directly convert fibroblasts into neurons as previously established (Yoo et al., Nature 2011; Richner et al., Nat. Prot. 2015; Abernathy, Kim, and McCoy et al., 2017) or motor neuron-enriched transcription factors ISL1 and LHX3 or pro-neural transcription factor MYT1L alone. Transposed (accessible) genomic DNA was collected from neurons and controls, then prepared for analysis as described in the Protocols.

细胞命运转换（Cell-fate conversion）通常依赖重编程效应因子（reprogramming effectors）的活性，以同时引入靶细胞类型的命运程序并消除起始细胞群的身份特征。本研究揭示了微小RNA（microRNAs）家族成员miR-9/9*与miR-124（合称为miR-9/9*-124）作为重编程因子的全新作用机制：它们通过时序性步骤协调人类成纤维细胞的直接重编程——首先清除成纤维细胞的身份特征，随后推动细胞均匀转变为神经元状态。在miR-9/9*-124的直接靶基因中，我们鉴定出KLF家族转录因子（KLF-family transcription factors），其表达抑制对于清除成纤维细胞命运至关重要。后续小核RNA RN7SK的上调可诱导染色质区域开放并激活神经元基因，推动重编程细胞向神经元状态转化。本研究明确了微小RNA介导的重编程级联反应中的决定性组分。

总体实验设计：将去标识化的健康人类成人真皮成纤维细胞通过慢病毒（lentivirus）转导，使其表达脑富集型微小RNA miR-9/9*与miR-124，以按照已建立的方法将成纤维细胞直接重编程为神经元（Yoo等，《Nature》，2011；Richner等，《Nature Protocols》，2015；Abernathy、Kim及McCoy等，2017）；同时设置对照组，仅转导运动神经元富集型转录因子ISL1与LHX3，或仅转导促神经元转录因子MYT1L。收集神经元样本与对照样本中的转座酶可及性基因组DNA（Transposed (accessible) genomic DNA），按照实验流程制备后进行分析。