Profiling of gene expression using RNA-Seq in fibroblasts, iPSCs, iPSC-derived neurons and cells overexpressing Onecut transcription factors

Remodeling of chromatin accessibility is necessary for successful reprogramming of fibroblasts to neurons. However, it is still not fully known which transcription factors can induce a neuronal chromatin accessibility profile when overexpressed in fibroblasts. To identify such transcription factors, we here used ATAC-sequencing to generate differential chromatin accessibility profiles between human fibroblasts and iNeurons, an in vitro neuronal model system obtained by overexpression of Neurog2 in induced pluripotent stem cells (iPSCs). We found that the ONECUT transcription factor sequence motif was strongly associated with differential chromatin accessibility between iNeurons and fibroblasts. All three ONECUT transcription factors associated with this motif (ONECUT1, ONECUT2 and ONECUT3) induced neuronal morphology and expression of neuronal genes within two days of overexpression in fibroblasts. We observed widespread remodeling of chromatin accessibility; in particular, we found that chromatin regions that contain the ONECUT motif were in- or lowly accessible in fibroblasts and became accessible after the overexpression of ONECUT1, ONECUT2 or ONECUT3. There was substantial overlap with iNeurons, still, many regions that gained accessibility following ONECUT overexpression were not accessible in iNeurons. Our study highlights the potential of ONECUT transcription factors for direct neuronal reprogramming.染色质可及性的重塑对于成纤维细胞向神经元的高效重编程必不可少。然而，目前仍未完全明确哪些转录因子在成纤维细胞中过表达时，能够诱导出神经元样的染色质可及性谱。为筛选此类转录因子，本研究通过ATAC测序（ATAC-sequencing）获取了人类成纤维细胞与诱导神经元（iNeurons）之间的差异染色质可及性谱；其中诱导神经元是一种通过在诱导多能干细胞（induced pluripotent stem cells, iPSCs）中过表达Neurog2所构建的体外神经元模型系统。我们发现，ONECUT转录因子的序列基序与诱导神经元和成纤维细胞之间的差异染色质可及性显著相关。与此基序相关的全部三种ONECUT转录因子（ONECUT1、ONECUT2与ONECUT3）在成纤维细胞中过表达后的两天内，即可诱导出神经元形态并激活神经元基因的表达。我们观察到了广泛的染色质可及性重塑；具体而言，成纤维细胞中原本处于不可及或低可及状态的、携带ONECUT基序的染色质区域，在过表达ONECUT1、ONECUT2或ONECUT3后可变为可及状态。尽管过表达ONECUT后获得可及性的染色质区域与诱导神经元存在大量重叠，但仍有许多区域在诱导神经元中并未处于可及状态。本研究凸显了ONECUT转录因子在直接神经元重编程中的应用潜力。