Rfx5 acts as a master regulator of MHC class I and class II gene expression in zebrafish immunity

Regulatory Factor X5 (RFX5) is a well-established transcription activator essential for the expression of Major Histocompatibility Complex (MHC) class II genes. Consequently, its loss leads to severe immunodeficiency. While its function in mammals is well defined, the in vivo function of its ortholog in teleosts remains unclear. In this study, we investigated the role of Rfx5 in zebrafish. Bioinformatics analysis confirmed that Rfx5 is highly conserved across species. Rfx5 mRNA and protein exhibited ubiquitous expression profiles throughout embryogenesis and across adult tissues, though at varying levels. Subcellular localization studies revealed that Rfx5 is exclusively nuclear, and assembles into distinct foci. The AT-hook motif mediates this localization and the two low complexity regions (LCRs) are required for its foci formation. Functionally, rfx5 expression is upregulated in response to immune stimulation. Using both gain- and loss-of-function approaches, we demonstrated that Rfx5 regulates a comprehensive subset of MHC genes and immune-related signaling pathways. Notably, the Rfx5 targets include not only MHC class II genes but also a significant portion of the MHC class I repertoire. The L lineage, a non-canonical subgroup of MHC class I was identified as the predominant type affected by Rfx5 deficiency in zebrafish. This work uncovers the complex in vivo regulatory role of Rfx5 as a master modulator of both MHC class I and II genes expression, thereby participating in the immune response in zebrafish. These findings offer new insights into the mechanisms of immune homeostasis, and have significant implications for the evolutionary understanding of the vertebrate immune system.