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. 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)转录因子FOXN1（transcription factor FOXN1）以基因剂量敏感的方式，作为胸腺上皮细胞（thymic epithelial cell, TEC）发育与维持的核心调控因子，保障高效的胸腺造血生成（thymopoiesis）。其常染色体隐性功能缺失突变是“裸表型”（nude phenotype）的分子病因——该表型表现为无胸腺症（athymia）引发的重度联合免疫缺陷。本研究报道一例自发产生的新型杂合FOXN1突变体：该突变初始可支持正常胸腺器官发生，但会导致人类妊娠晚期之后的胸腺维持失败。在小鼠体内模拟该突变，会引发胸腺上皮细胞亚型分化与功能的异常紊乱。该突变体转录活性缺失，可破坏野生型FOXN1同源多聚体的形成，并结合经典DNA结合位点，以显性负调控的方式将野生型FOXN1从核凝聚体中置换出来。对比突变型与野生型FOXN1的互作组（interactome），我们鉴定出仅与野生型FOXN1结合的互作蛋白，这些蛋白是转录所需核细胞器的特征性组成成分。突变型FOXN1在核凝聚体中的进出动态与野生型FOXN1无显著差异，提示该突变并未影响FOXN1的分子运动特性。综上，我们鉴定出一例具有临床相关性的FOXN1功能获得性突变，该突变可主动抑制野生型FOXN1的转录活性，并为阐明FOXN1调控维持正常胸腺上皮细胞分化、维持与功能所需的持续基因表达的分子机制提供了关键见解。本研究数据集包含：1. mTEC4D6细胞系RNA测序（RNA-seq）：设置野生型FOXN1（WT-FOXN1）、d550-FOXN1、野生型FOXN1/d550-FOXN1及空载体对照组，每组生物学重复数n=3；2. 染色质免疫共沉淀测序（ChIP-seq）：设置野生型FOXN1、d550-FOXN1、野生型FOXN1-input及d550-FOXN1-input组，每组n=3；3. 小鼠模型10X基因表达分析：设置wt/wt-Foxn1与d505/wt-Foxn1组，分别在小鼠5周龄与16周龄时取样，每组n=3。