Genome-wide analysis of enhancers repressed by Nkx2.2 in spinal motor neuron progenitors

Unambiguous consolidation of cell identity relies on transcription factors (TFs) acting to suppress alternative lineages, yet few general principles have emerged explaining how repressors manipulate chromatin landscapes to achieve stable gene silencing, especially in instances when these TFs are only temporarily activated. The TF Nkx2.2 is transiently expressed in CNS progenitors yet permanently abrogates somatic motor neuron (sMN) differentiation via interactions with Groucho (Grg) co-repressors. We show that Nkx2.2 inhibits regulatory elements of progenitor and postmitotic sMN transcripts by utilizing Grg to evict co-activators. Surprisingly, co-activator eviction was accomplished using Grg proteins already present at enhancers, rather than through the additional recruitment of Grg or other co-repressors. Nkx2.2-mediated enhancer silencing also led to the loss of H3K27ac modifications from H3K27me3+H3K27ac+ proximal promoters of key sMN genes, facilitating persistent repression of the sMN progenitor program after Nkx2.2 downregulation. These results imply that active loci are poised for suppression, and repressor binding mediates the transition through co-activator displacement. Examination of ChIP-seq profiles of 2 transcription factors (TFs), 3 histone modifications, 4 TF cofactors, and total RNA profiles during the differentiation of mouse ES cell-derived spinal motor neuron progenitors expressing a wildtype copy of Nkx2.2 tagged by FLAG under the control of a doxycycline (DOX) inducible promotor (iNkx2.2), a TN-domain mutant copy of Nkx2.2 tagged by FLAG under the control of a doxycycline (DOX) inducible promotor (iTNmut), or no Nkx2.2 (pMN). Replicates are provided for all ChIP-seq samples except Sox2, Med1, and H3K27me3; an input sample is also provided. RNA samples are provided in triplicates.