Sequential regulatory loops as key gatekeepers for neuronal reprogramming in human cells [RNA-seq]

Direct conversion of somatic cells into neurons holds great promise for regenerative medicine. However, neuronal conversion is relatively inefficient in human cells compared to mouse cells. It has been unclear what might be the key barriers to reprogramming in human cells. We recently elucidated an RNA program mediated by the polypyrimidine tract binding protein PTB to convert mouse embryonic fibroblasts (MEFs) into functional neurons. In human adult fibroblasts (HAFs), however, we unexpectedly found that invoking the documented PTB–REST–miR-124 loop generates only immature neurons. We now report that the functionality requires sequential inactivation of PTB and the PTB paralog nPTB in HAFs. Inactivation of nPTB triggers another self-enforcing loop essential for neuronal maturation, which comprises nPTB, the transcription factor BRN2, and miR-9. These findings suggest that two separate gatekeepers control neuronal conversion and maturation and consecutively overcoming these gatekeepers enables deterministic reprogramming of HAFs into functional neurons. Overall design: Six RNA-seq libraries are generated by MAPS approach. Total RNA is extracted from induced neuronal cells derived from control shRNA or PTB shRNA treated human adult fibroblasts. Please note that the ''RNA-seq HAF hygro'' sample was on 6 days after switching to N3 media *without* nPTB depletion. The other two ''shPTB 6d'' and ''shPTB 3w'' were on 6 days and 3 weeks respectively after switching to N3 media *with* nPTB depletion.

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Xue Y, Qian H, Hu J, Zhou B, Zhou Y, Hu X, Karakhanyan A, Pang Z, Fu XD