A multi-step transcriptional and chromatin cascade underlies motor neuron programming (Histone modification ChIP-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: Histone modifications were characterized during the direct programming of ES cells into motor neurons using over-expression of Ngn2-Isl1-Lhx3 programming factors. ChIP-seq experiments were performed targeting H3K4me3, H3K4me2, H3K4me1, H3K27ac and H3K27me3 at various time-points during the programming process. Input sequencing at each time-point was performed as a control. Input experiments are provided under GEO accession number: GSE80321