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. Overall design: Each RNA-Seq experiment was performed in duplicate (library constructed from different wells of the same cell line in the same cell culture experiment). Bclxl controls were generated for the overexpression. experiments.

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van der Raadt J, van Gestel SHC, Nadif Kasri N, Albers CA