Functional characterisation of a novel heterozygous mutation in the transcriptional activation domain of the human FOXN1 gene

The competence of the human body to fight infections relies on lymphocytes, which differentiate in the thymus, a primary organ of the immune system. There, thymic epithelial cells (TECs) have a unique role to instruct developing T lymphocytes to discriminate self-proteins from foreign pathogens and react only against the latter. The development, function and maintenance of TECs are primarily dependent on the transcription factor FOXN1, which acts as a master regulator of the thymus organogenesis and maintenance. Its loss of function results in athymia followed by severe immunodeficiency and is the molecular cause of the “nude” phenotype in humans and mice. Despite its crucial role in thymic development, very little is known about the molecular mechanism of its function. We have recently identified 3 individuals that presented, from as early as a few months of life, with significant lymphopenia, reduced frequency of naïve T cells, absent T cell receptor excision circles (TRECs) and a lack of radiographic signs of thymus tissue. Surprisingly, these individuals were otherwise clinically healthy. Each was heterozygous for a deletion in the genomic sequence encoding the C-terminus of FOXN1 thus causing a frameshift and consequently a premature stop at amino acid 550, leading to a significantly shortened protein (designated Δ550). Using both in-vitro and in-vivo models that mimicked the heterozygous patients we explored how a co-expression of the wild-type and mutant FOXN1 proteins compromise thymus development and function. Using RNA-seq coupled with ChIPSeq we explored differences in gene expression regulated by the two proteins. We also employed mass-spectrometry proteomics analysis followed by CRISPR arrayed screening, to reveal, to our best of knowledge for the first time, the molecular interactome of FOXN1 and how the Δ550 mutation may interfere with the binding of these interaction partners. With advanced microscopy we showed that FOXN1 participates in the formation of nuclear bodies, essential for its activity as a transcriptional regulator. Finally, we studied the impact of the Δ550 mutation on TEC differentiation and function in a mouse model heterozygous for a mutation equivalent to that of human Δ550. Taken together our studies elucidate aspects of the molecular mechanism by which FOXN1 operates and provide a molecular understanding on how heterozygous FOXN1 mutations can lead to pathologies.