Disruption of higher order nuclear condensates by a dominant negative FOXN1 mutation causes immunodeficiency

The transcription factor FOXN1 acts in a gene-dosage sensitive way as a master regulator of thymic epithelial cell development and maintenance enabling effective thymopoiesis. Its autosomal recessive loss of function is the molecular cause of the “nude” phenotype, a severe combined immunodeficiency caused by athymia. Here we report on a spontaneously occurring, novel heterozygous FOXN1 mutation that initially permits regular thymus organogenesis but results in a failure of thymic maintenance beyond late stages of human gestation. Modeling the mutation in mice results in divergent disruptions of normal TEC subtype differentiation and function. Transcriptionally inactive, the mutant disrupts the formation of wild type FOXN1 homo-multimers and occupies canonical DNA binding sites, at which it operates in a dominant negative fashion to displace wild type FOXN1 from nuclear condensates. Comparing the interactome of the mutant and wild type FOXN1 identified binding partners that uniquely interact with wild type FOXN1 and are characteristic constituents of nuclear organelles required for transcription. Mutant FOXN1 moves in and out of condensates over a time-course indistinguishable from that of wild type FOXN1, suggesting that the mutation did not affect the molecular movement of FOXN1. Thus, we have identified a clinically relevant gain of function mutation of FOXN1 that actively prevents the transcriptional activity of wild type FOXN1 and provides critical insight into the mechanism by which FOXN1 operates in controlling sustained gene expression necessary for normal thymic epithelial cell differentiation, maintenance and function. Overall design: mTEC4D6 cell lines - RNA-seq: WT-FOXN1, d550-FOXN1, WT-FOXN1/d550-FOXN1 and empty control (n = 3 for each); ChIP-seq: WT-FOXN1, d550-FOXN1, WT-FOXN1-input and d550-FOXN1-input (n = 3 for each); mouse model - 10X gene expression: wt/wt-Foxn1 and d505/wt-Foxn1 (n = 3 for each at 5 weeks and 16 weeks of age)