Expression of Terminal Effector Genes in Mammalian Neurons Is Maintained by a Dynamic Relay of Transient Enhancers

Generic spinal motor neuron identity is established by cooperative binding of programming transcription factors (TFs), Isl1 and Lhx3, to motor-neuron-specific enhancers. How expression of effector genes is maintained following downregulation of programming TFs in maturing neurons remains unknown. High-resolution exonuclease (ChIP-exo) mapping revealed that the majority of enhancers established by programming TFs are rapidly deactivated following Lhx3 downregulation in stem-cell-derived hypaxial motor neurons. Isl1 is released from nascent motor neuron enhancers and recruited to new enhancers bound by clusters of Onecut1 in maturing neurons. Synthetic enhancer reporter assays revealed that Isl1 operates as an integrator factor, translating the density of Lhx3 or Onecut1 binding sites into transient enhancer activity. Importantly, independent Isl1/Lhx3- and Isl1/Onecut1-bound enhancers contribute to sustained expression of motor neuron effector genes, demonstrating that outwardly stable expression of terminal effector genes in postmitotic neurons is controlled by a dynamic relay of stage-specific enhancers.