Comparative analysis of salmonid non-classical MHC class I U lineage genes: Promoter architecture and transcriptional responses with a focus on Atlantic salmon

In addition to highly polymorphic classical MHC class I genes, teleost fish also possess variable numbers of non-classical MHC class I genes, the functions of which are largely unknown. This work focuses on genes belonging to the non-classical MHC class I U lineage revealing unique evolutionary and regulatory patterns across salmonids, indicative of sub-clade specific and diversified functional roles. Comparative analysis of a total of 175 salmonid U lineage genes supported a classification into seven subclades following one of two main evolutionary patterns indicative of different selective pressures. Genes belonging to the ULA, UC/DA, UFA, and UZA sub-clades exhibited variable degrees of species-specific diversification, characterized by gene duplications, gene loss and pseudogenization. In contrast, the UGA, UEA, and UHA sub-clades have remained conserved, with minimal sequence divergence across divergent species, suggesting functional constraints. Notably, the UGA subclade represents a striking example of conservation across divergent salmonid species. Promoter analyses of the U lineage subclades revealed two main regulatory architectures: i) overall conserved promoter elements similar to the UBA gene in ULA, UFA, and UZA, implying shared transcriptional regulation; and ii) divergent promoters in UGA, UEA, UC/DA and UHA suggesting diversified regulatory mechanisms. Expression profiling further supported functional diversity, revealing non-classical U lineage subclade specific expression patterns and variable expression levels. While non-classical U lineage genes, as a group, showed limited response to stimulation with viral mimics, subclade specific responses were observed following infection with salmon alpha virus 3 (SAV3) and in response to interferon stimulation. Similar to the classical UBA gene, UGA and, to a lesser extent, UHA transcript expression levels were upregulated in response to stimulation with either type I IFNa1 or type II IFNγ, as well as in response to SAV3 infection but not to IFNc stimulation. In contrast, UC/DA and UEA genes showed minimal response to interferon stimulation or SAV3 challenge. Combined, these findings underscore the adaptability and complexity of the salmonid MHC class I system and provide a comprehensive framework for understanding the diversification of salmonid MHC class I genes, offering valuable insights into the variation, regulation, and potential roles of these genes in salmonid immunity.