A multi-step transcriptional and chromatin cascade underlies motor neuron programming (RNA-Seq)

Direct programming via the overexpression of transcription factors (TFs) aims to control cell fate at a precision that will be instrumental for clinical applications. However, direct programming of terminal fates remains an obscure process. Taking advantage of the rapid and uniquely efficient programming of spinal motor neurons by overexpression of Ngn2, Isl1 and Lhx3, we have characterized gene expression, chromatin and transcription factor binding time-course dynamics during complete motor neuron programming. Our studies point to a surprisingly dynamic programming process. Promoter chromatin and expression analysis reveals at least three distinct phases of gene activation, while programming factor binding shifts from one set of targets to another, controlling regulatory region activity and gene expression. Furthermore, our evidence suggest that the enhancers and genes activated in the final stage of motor neuron processing are dependent on the combined activities of Isl1 and Lhx3 factors with Ebf and Onecut TFs that are themselves activated midway through the programming process. Our results suggest an unexpected multi-stage model of motor neuron programming in which the programming TFs require activation of a set of intermediate regulators before they complete the programming process. Overall design: For bulk cell RNA-seq analysis, cells were collected at different time points after NIL induction and RNA isolated using TRIzol LS (Life Technologies) followed by purification using Qiagen RNAeasy kit

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Velasco S, Ibrahim MM, Kakumanu A, Garipler G, Aydin B, Al-Sayegh MA, Hirsekorn A, Abdul-Rahman F, Satija R, Ohler U, Mahony S, Mazzoni EO