The homeobox transcription factor MNX1 regulates the expression of many non-MN-specific neuronal genes in motor neurons.

Motor neurons (MNs) control muscle movement and are essential for breathing, walking and fine motor skills. MN and Pancreas Homeobox 1 (MNX1) has long been recognized as a key marker of the MN lineage, and its deficiency in mice results in early postnatal lethality-likely by causing abnormal MN development and respiratory malfunction. However, its genome-wide targets and regulatory mechanism remain unresolved. Using an in vitro model for efficient MN induction, we identified ∼6000 MNX1-bound loci, of which half are conserved enhancers frequently overlap peaks for core MN-inducing factors ISL1 and LHX3. Despite its widespread binding, Mnx1 knockout affects only a few dozen bound loci and causes mis-regulation of ∼100 genes, the majority are up-regulated neuronal genes which routinely have comparable or even higher expression in brain relative to MNs. Integrative analysis predicts Pbx3 and Pou6f2 as two putative direct targets-both are homeobox genes highly expressed in the central nervous system, and adjacent to MNX1-repressed enhancers according to luciferase reporter assay. Collectively, our results suggest MNX1 restrains the expression of non-MN-specific neuronal genes, likely mainly through an indirect fashion. Further, the rarity of direct targets in contrast to widespread binding reflects a distinctive mode of transcriptional regulation.