Defining the impact of FOXN1 variants with functional assays and reaggregate thymus organ cultures reveals those with loss- and gain- of function and dominant negative consequences

Abstract

Thymus hypoplasia can occur in individuals with autosomal recessive, compound heterozygous and single allelic Forkhead Box N1 (FOXN1) mutations. FOXN1 regulates thymus epithelial cell (TEC) development and function, which is essential for T cell development. The impact of an ever-increasing number of FOXN1 mutations, identified in patients, on TECs and consequently, thymopoiesis, remains unclear for most variants. We developed a systematic approach to determine the consequence of different FOXN1 variants on protein activity (luciferase reporter assays), localization (cell imaging), and dominant negative capabilities (co-expression studies). With these assays, we mapped the nuclear localization signal in FOXN1, identified de novo sequences in human FOXN1 that mediate dominant negative functions and uncovered two domains that modulate transcriptional activities. Such strategies reveal both loss- and gain- of function variants. To determine the impact of the variants on thymopoiesis, we modified reaggregate thymus organ cultures (RTOC) by incorporating flow sorting to isolate and recombine different cell subsets. In this system, only FOXN1 sufficient TECs can support thymopoiesis when combined with hematopoietic, mesenchymal, and endothelial cell subsets. Murine TECs from “nude” embryos (lacking FOXN1) were unable to support RTOC growth. The flow sorted “nude” TECs were reconstituted with purified TAT-Foxn1 fusion proteins prior to reaggregation. The transduction of wildtype TAT-Foxn1 re-established RTOC growth, thymopoiesis and Tcell development within a 10-day culture period. This RTOC strategy can enable one to define the FOXN1 variants of unknown significance (VUS) as benign, partially, or fully attenuated in their capacity to support thymopoiesis. Taken together, our findings establish FOXN1 genotype-phenotype relationships and reveal a thymopoietic screening strategy that obviates the need for generating mouse knock-in lines.