Profiling of chromatin accessibility in fibroblasts, iPSCs, iPSC-derived neurons and cells overexpressing Onecut transcription factors (OC1, OC2, OC3)

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，目前仍未完全明确在成纤维细胞中过表达何种转录因子，可诱导出神经元样的染色质可及性特征。为筛选此类转录因子，本研究采用转座酶可及性染色质测序（ATAC-sequencing）技术，绘制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图谱。其中，诱导神经元是通过在诱导多能干细胞（iPSCs）中过表达神经素2（Neurog2）构建的体外神经元模型系统。研究发现，ONECUT转录因子家族的序列基序与诱导神经元和成纤维细胞间的差异染色质可及性显著相关。该基序对应的全部三种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封闭或低可及状态的、携带ONECUT基序的染色质区域，在过表达ONECUT1、ONECUT2或ONECUT3后均可变为开放可及状态。尽管该重塑与诱导神经元的染色质开放状态存在大量重合，但仍有诸多在ONECUT过表达后获得可及性的染色质区域，在诱导神经元中并未开放。本研究凸显了ONECUT转录因子家族在直接神经元重编程领域的应用潜力。