Summary Defects in motor neuron (MN) function or survival result in severe human pathologies, such as amyotrophic lateral sclerosis and spinal muscular atrophy, with distinct MN subtypes differing in susceptibility to disease. There is currently no effective treatment for MN disorders in part due to a lack of understanding of the molecular mechanisms that allow distinct MN subtypes to acquire and maintain their function-defining properties. Thus, basic research in model organisms such as nematodes, flies, and mice is needed to reveal such mechanisms. MN subtype function is endowed by the differential expression of terminal identity genes. Such genes encode proteins (e.g., ion channels, neurotransmitter receptors, neuropeptides, trans-membrane receptors, adhesion molecules) that are expressed continuously, from the last steps of development through adulthood, and thereby define the unique functional features of a given MN subtype. Hence, revealing the molecular mechanisms that induce (during development) and maintain (throughout life) expression of terminal identity genes will help us understand how MNs become and remain functional, a key goal in the fields of MN development and disease. A remarkable wealth of terminal identity markers is available for all cholinergic MN subtypes of the C. elegans nerve cord that control locomotion, providing a unique model system to elucidate how MNs acquire and maintain their functional features. Leveraging these tools, we discovered that the conserved Collier/Olf/Ebf-type transcription factor (TF) UNC-3 is required for the continuous function of all these cholinergic MN subtypes, and that this outcome arises from UNC-3-dependent induction and maintenance of MN subtype-specific terminal identity genes. Through unbiased genetic screens, we recently identified several conserved regulatory factors (6 TFs, 2 chromatin factors) that control the terminal identity of individual MN subtypes. Intriguingly, our preliminary results suggest that while UNC-3 activates expression of all MN subtype-specific terminal identity genes, these regulatory factors counteract the activator function of UNC-3 by repressing UNC-3 targets in specific MN subtypes. These observations suggest a general principle for the control of MN terminal identity, in which the transcriptional targets of a broadly acting activator (UNC-3) are repressed in a MN subtype-specific fashion by distinct TFs and chromatin factors. To test this hypothesis within the 5-year R01 timeframe, this proposal will focus on one TF (BNC-1/mammalian BNC1-2) and one chromatin factor (PBRM-1/mammalian BAF180) that counteract UNC-3 in different MN subtypes. Specifically, we seek to: (a) determine whether these two factors are required throughout life to secure subtype identity (Aim 1), (b) define the mechanism underlying the repressor activity of BNC-1 (Aim 2), and (c) decipher the function of PBRM-1 by identifying its downstream targets (Aim 3). The proposed experiments will e...