FOXP3 recognizes microsatellites and bridges DNA through multimerization.

FOXP3 is a transcription factor that is essential for the development of regulatory T cells, a branch of T cells that suppress excessive inflammation and autoimmunity1-5. However, the molecular mechanisms of FOXP3 remain unclear. Here we here show that FOXP3 uses the forkhead domain-a DNA-binding domain that is commonly thought to function as a monomer or dimer-to form a higher-order multimer after binding to TnG repeat microsatellites. The cryo-electron microscopy structure of FOXP3 in a complex with T3G repeats reveals a ladder-like architecture, whereby two double-stranded DNA molecules form the two 'side rails' bridged by five pairs of FOXP3 molecules, with each pair forming a 'rung'. Each FOXP3 subunit occupies TGTTTGT within the repeats in a manner that is indistinguishable from that of FOXP3 bound to the forkhead consensus motif (TGTTTAC). Mutations in the intra-rung interface impair TnG repeat recognition, DNA bridging and the cellular functions of FOXP3, all without affecting binding to the forkhead consensus motif. FOXP3 can tolerate variable inter-rung spacings, explaining its broad specificity for TnG-repeat-like sequences in vivo and in vitro. Both FOXP3 orthologues and paralogues show similar TnG repeat recognition and DNA bridging. These findings therefore reveal a mode of DNA recognition that involves transcription factor homomultimerization and DNA bridging, and further implicates microsatellites in transcriptional regulation and diseases.

Domain tethering impacts dimerization and DNA-mediated allostery in the human transcription factor FoxP1.

Transcription factors are multidomain proteins with specific DNA binding and regulatory domains. In the human FoxP subfamily (FoxP1, FoxP2, FoxP3, and FoxP4) of transcription factors, a 90 residue-long disordered region links a Leucine Zipper (ZIP)-known to form coiled-coil dimers-and a Forkhead (FKH) domain-known to form domain swapping dimers. We used replica exchange discrete molecular dynamics simulations, single-molecule fluorescence experiments, and other biophysical tools to understand how domain tethering in FoxP1 impacts dimerization at ZIP and FKH domains and how DNA binding allosterically regulates their dimerization. We found that domain tethering promotes FoxP1 dimerization but inhibits a FKH domain-swapped structure. Furthermore, our findings indicate that the linker mediates the mutual organization and dynamics of ZIP and FKH domains, forming closed and open states with and without interdomain contacts, thus highlighting the role of the linkers in multidomain proteins. Finally, we found that DNA allosterically promotes structural changes that decrease the dimerization propensity of FoxP1. We postulate that, upon DNA binding, the interdomain linker plays a crucial role in the gene regulatory function of FoxP1.

[Research progress on the role of FOXOs family in cancer].

The Forkhead box class O proteins (FOXOs) family consists of highly conserved transcription factors, including FOXO1, FOXO3, FOXO4 and FOXO6. Each member of the FOXOs family is ubiquitously expressed and involved in regulating many biological activities such as tumor cell proliferation, apoptosis, migration and oxidative stress. The activity of FOXOs is mainly regulated by post-translational modification, and its inactivation is mainly mediated by the over-activation of its upstream modifying enzymes, which provides a possibility to use drugs to recover its activity. It is worth noting that FOXOs can not only inhibit, but also promote the occurrence and development of human tumors due to the complex effects of FOXOs. This review will summarize the structure and activity regulation of FOXOs, and discuss their tumor inhibiting effects by limiting cell proliferation and inducing apoptosis, as well as their tumor promoting effects by maintaining cell homeostasis, promoting metastasis and inducing drug resistance, so as to provide new ideas for the pathological research of related diseases and open up new ways to promote broader prevention and treatment strategies.

Reduced frequencies of Foxp3+GARP+ regulatory T cells in COPD patients are associated with multi-organ loss of tissue phenotype.

BACKGROUND: Expression of glycoprotein A dominant repeat (GARP) has been reported to occur only in activated human naturally occurring regulatory T cells (Tregs) and their clones, and not in activated effector T cells, indicating that GARP is a marker for bona fide Tregs. A different phenotype of chronic obstructive pulmonary disease (COPD) may have a different immunologic mechanism. OBJECTIVE: To investigate whether the distribution of Tregs defined by GARP is related to the multi-organ loss of tissue phenotype in COPD. METHODS: GARP expression on T cells from peripheral blood and bronchoalveolar lavage (BAL) collected from patients with COPD was examined by flow cytometry. The correlation of GARP expression to clinical outcomes and clinical phenotype, including the body mass index, lung function and quantitative computed tomography (CT) scoring of emphysema, was analyzed. RESULTS: Patients with more baseline emphysema had lower forced expiratory volume, body mass index (BMI), worse functional capacity, and more osteoporosis, thus, resembling the multiple organ loss of tissue (MOLT) phenotype. Peripheral Foxp3+GARP+ Tregs are reduced in COPD patients, and this reduction reversely correlates with quartiles of CT emphysema severity in COPD. Meanwhile, the frequencies of Foxp3+GARP- Tregs, which are characteristic of pro-inflammatory cytokine production, are significantly increased in COPD patients, and correlated with increasing quartiles of CT emphysema severity in COPD. Tregs in BAL show a similar pattern of variation in peripheral blood. CONCLUSION: Decreased GARP expression reflects more advanced disease in MOLT phenotype of COPD. Our results have potential implications for better understanding of the immunological nature of COPD and the pathogenic events leading to lung damage.

FOXO4 interacts with p53 TAD and CRD and inhibits its binding to DNA.

Transcription factor p53 protects cells against tumorigenesis when subjected to various cellular stresses. Under these conditions, p53 interacts with transcription factor Forkhead box O (FOXO) 4, thereby inducing cellular senescence by upregulating the transcription of senescence-associated protein p21. However, the structural details of this interaction remain unclear. Here, we characterize the interaction between p53 and FOXO4 by NMR, chemical cross-linking, and analytical ultracentrifugation. Our results reveal that the interaction between p53 TAD and the FOXO4 Forkhead domain is essential for the overall stability of the p53:FOXO4 complex. Furthermore, contacts involving the N-terminal segment of FOXO4, the C-terminal negative regulatory domain of p53 and the DNA-binding domains of both proteins stabilize the complex, whose formation blocks p53 binding to DNA but without affecting the DNA-binding properties of FOXO4. Therefore, our structural findings may help to understand the intertwined functions of p53 and FOXO4 in cellular homeostasis, longevity, and stress response.

Molecular modelling of the FOXO4-TP53 interaction to design senolytic peptides for the elimination of senescent cancer cells.

BACKGROUND: Senescent cells accumulate in tissues over time as part of the natural ageing process and the removal of senescent cells has shown promise for alleviating many different age-related diseases in mice. Cancer is an age-associated disease and there are numerous mechanisms driving cellular senescence in cancer that can be detrimental to recovery. Thus, it would be beneficial to develop a senolytic that acts not only on ageing cells but also senescent cancer cells to prevent cancer recurrence or progression. METHODS: We used molecular modelling to develop a series of rationally designed peptides to mimic and target FOXO4 disrupting the FOXO4-TP53 interaction and releasing TP53 to induce apoptosis. We then tested these peptides as senolytic agents for the elimination of senescent cells both in cell culture and in vivo. FINDINGS: Here we show that these peptides can act as senolytics for eliminating senescent human cancer cells both in cell culture and in orthotopic mouse models. We then further characterized one peptide, ES2, showing that it disrupts FOXO4-TP53 foci, activates TP53 mediated apoptosis and preferentially binds FOXO4 compared to TP53. Next, we show that intratumoural delivery of ES2 plus a BRAF inhibitor results in a significant increase in apoptosis and a survival advantage in mouse models of melanoma. Finally, we show that repeated systemic delivery of ES2 to older mice results in reduced senescent cell numbers in the liver with minimal toxicity. INTERPRETATION: Taken together, our results reveal that peptides can be generated to specifically target and eliminate FOXO4+ senescent cancer cells, which has implications for eradicating residual disease and as a combination therapy for frontline treatment of cancer. FUNDING: This work was supported by the Cancer Early Detection Advanced Research Center at Oregon Health & Science University.

Evaluation of urinary FOXP3 mRNA as a biomarker of lupus nephritis in Egyptian patients with systemic lupus erythematosus.

AIM: Lupus nephritis (LN) is one of the most serious complications of SLE. Tregs (Regulatory T lymphocytes) are thought to play a part in the pathogenesis of SLE. According to recent research, Foxp3, a Treg identification marker, plays a significant role in the pathogenesis of SLE. This study aimed to compare the urinary Foxp3 mRNA levels of patients with active and inactive forms of LN and healthy control subjects to see whether it played a role in disease activity. METHODS: We measured FOXP3 messenger RNA (mRNA) expression in the urine of 50 people with active LN, 50 people with inactive lupus, and 50 healthy people. RESULTS: We found that the expression level of FOXP3 was significantly higher in urine from patients with active LN than from subjects with inactive lupus and healthy controls (22.93 ± 4.13 vs 5.66 ± 0.47 vs 0.57 ± 0.15copy; P < 0.001).Urinary FOXP3 mRNA level significantly correlated with SLEDAI (0.000057) In the active group, urinary FOXP3 mRNA level also significantly correlated with histological activity index (< 0.00001). CONCLUSION: We concluded that urinary FOXP3 mRNA is elevated in patients with active LN and that it is linked to the SLEDAI and the severity of the disease. FOXP3 mRNA in urine sediment may be used as a non-invasive biomarker for evaluating the severity of LN and risk stratification.

Multiple regulatory intrinsically disordered motifs control FOXO4 transcription factor binding and function.

Transcription factors harbor defined regulatory intrinsically disordered regions (IDRs), which raises the question of how they mediate binding to structured co-regulators and modulate their activity. Here, we present a detailed molecular regulatory mechanism of Forkhead box O4 (FOXO4) by the structured transcriptional co-regulator ß-catenin. We find that the disordered FOXO4 C-terminal region, which contains its transactivation domain, binds ß-catenin through two defined interaction sites, and this is regulated by combined PKB/AKT- and CK1-mediated phosphorylation. Binding of ß-catenin competes with the autoinhibitory interaction of the FOXO4 disordered region with its DNA-binding Forkhead domain, and thereby enhances FOXO4 transcriptional activity. Furthermore, we show that binding of the ß-catenin inhibitor protein ICAT is compatible with FOXO4 binding to ß-catenin, suggesting that ICAT acts as a molecular switch between anti-proliferative FOXO and pro-proliferative Wnt/TCF/LEF signaling. These data illustrate how the interplay of IDRs, post-translational modifications, and co-factor binding contribute to transcription factor function.

The SARS-CoV-2 SSHHPS Recognized by the Papain-like Protease.

Viral proteases are highly specific and recognize conserved cleavage site sequences of ∼6-8 amino acids. Short stretches of homologous host-pathogen sequences (SSHHPS) can be found spanning the viral protease cleavage sites. We hypothesized that these sequences corresponded to specific host protein targets since >40 host proteins have been shown to be cleaved by Group IV viral proteases and one Group VI viral protease. Using PHI-BLAST and the viral protease cleavage site sequences, we searched the human proteome for host targets and analyzed the hit results. Although the polyprotein and host proteins related to the suppression of the innate immune responses may be the primary targets of these viral proteases, we identified other cleavable host proteins. These proteins appear to be related to the virus-induced phenotype associated with Group IV viruses, suggesting that information about viral pathogenesis may be extractable directly from the viral genome sequence. Here we identify sequences cleaved by the SARS-CoV-2 papain-like protease (PLpro) in vitro within human MYH7 and MYH6 (two cardiac myosins linked to several cardiomyopathies), FOXP3 (an X-linked Treg cell transcription factor), ErbB4 (HER4), and vitamin-K-dependent plasma protein S (PROS1), an anticoagulation protein that prevents blood clots. Zinc inhibited the cleavage of these host sequences in vitro. Other patterns emerged from multispecies sequence alignments of the cleavage sites, which may have implications for the selection of animal models and zoonosis. SSHHPS/nsP is an example of a sequence-specific post-translational silencing mechanism.

E3 ubiquitin ligase PJA1 regulates lung adenocarcinoma apoptosis and invasion through promoting FOXR2 degradation.

Among all lung cancer cases, lung adenocarcinoma (LAC) represents nearly 40% and remains the leading cause of cancer deaths worldwide. Although the combination therapy of surgical treatment with radiotherapy, chemotherapy, and immunotherapy, has been used to treat LAC, unfortunately, high recurrence rates and poor survival remain. Therefore, novel prognostic markers and new targets for molecular targeted therapy in LAC is urgently needed. Fork-head box R2 (FOXR2) plays a key role in a wide range of cellular processes, including cellular proliferation, invasion, differentiation, and apoptosis, and it has been reported to be implicated in progression of LAC, thus inhibition of FOXR2 may be a novel targeting therapy for lung cancer. This current study found that E3 ligase PJA1 regulates ubiquitin-mediated degradation of FOXR2 and predicts good outcome of patients with LAC. In addition, it was showed force expression of PJA1 significantly inhibited LAC cells invasion and induced apoptosis in vitro through inactivating Wnt/ß-catenin signaling pathway. In short, our findings reveal that PJA1 could be a potential diagnostic and prognostic biomarkers and the PJA1- FOXR2 axis could be served as a promising target for LAC therapy.

Functions of Forkhead Box O on Glucose Metabolism in Abalone Haliotis discus hannai and Its Responses to High Levels of Dietary Lipid.

The forkhead box O (FoxO) subfamily is a member of the forkhead transcription factor family. It has regulation functions in glucose metabolism in mammals and fish. In the present study, a gene of the foxo homolog in abalone Haliotis discus hannai was cloned. A conservative forkhead (FH) domain and a transactivation (FoxO-TAD) domain were identified. Abalone foxo-specific siRNA (small interfering RNA) was injected to investigate the functions of foxo on glucose metabolism. Knockdown of foxo inhibited expression of phosphoenolpyruvate carboxykinase (pepck) and significantly increased expressions of hexokinase (hk) and pyruvate kinase (pk), but it failed to inhibit the relative mRNA level of glucose-6-phosphatase (g6pase). Then, a 100-day feeding trial was conducted to investigate the response of foxo and glucose metabolism in abalone fed with 1.57% (LFD, low-fat diet), 3.82% (MFD, middle-fat diet) and 6.72% (HFD, high-fat diet) of dietary lipid, respectively. The insulin-signaling pathway (AKT) was depressed and FoxO was activated by the HFD, but it did not inhibit glycolysis (hk) or improved gluconeogenesis significantly (pepck and g6pase). At the same time, impaired hepatopancreas glycogen storage raised hemolymph glucose levels. In conclusion, abalone foxo can be regulated by dietary lipid and can regulate gluconeogenesis or glycolysis in response to changes of dietary lipid levels, in which glycogen metabolism plays an important role.

Mechanism of forkhead transcription factors binding to a novel palindromic DNA site.

Forkhead transcription factors bind a canonical consensus DNA motif, RYAAAYA (R = A/G, Y = C/T), as a monomer. However, the molecular mechanisms by which forkhead transcription factors bind DNA as a dimer are not well understood. In this study, we show that FOXO1 recognizes a palindromic DNA element DIV2, and mediates transcriptional regulation. The crystal structure of FOXO1/DIV2 reveals that the FOXO1 DNA binding domain (DBD) binds the DIV2 site as a homodimer. The wing1 region of FOXO1 mediates the dimerization, which enhances FOXO1 DNA binding affinity and complex stability. Further biochemical assays show that FOXO3, FOXM1 and FOXI1 also bind the DIV2 site as homodimer, while FOXC2 can only bind this site as a monomer. Our structural, biochemical and bioinformatics analyses not only provide a novel mechanism by which FOXO1 binds DNA as a homodimer, but also shed light on the target selection of forkhead transcription factors.

FOXO4 Transactivation Domain Interaction with Forkhead DNA Binding Domain and Effect on Selective DNA Recognition for Transcription Initiation.

Forkhead box O4 (FOXO4) is a human transcription factor (TF) that participates in cell homeostasis. While the structure and DNA binding properties of the conserved forkhead domain (FHD) have been thoroughly investigated, how the transactivation domain (TAD) regulates the DNA binding properties of the protein remains elusive. Here, we investigated the role of TAD in modulating the DNA binding properties of FOXO4 using solution NMR. We found that TAD and FHD form an intramolecular complex mainly governed by hydrophobic interaction. Remarkably, TAD and DNA share the same surface of FHD for binding. While FHD did not differentiate binding to target and non-target DNA, the FHD-TAD complex showed different behaviors depending on the DNA sequence. In the presence of TAD, free and DNA-bound FHD exhibited a slow exchange with target DNA and a fast exchange with non-target DNA. The interaction of the two domains affected the kinetic function of FHD depending on the type of DNA. Based on these findings, we suggest a transcription initiation model by which TAD modulates FOXO4 recognition of its target promoter DNA sequences. This study describes the function of TAD in FOXO4 and provides a new kinetic perspective on target sequence selection by TFs.

Expanding the Nude SCID/CID Phenotype Associated with FOXN1 Homozygous, Compound Heterozygous, or Heterozygous Mutations.

Human nude SCID is a rare autosomal recessive inborn error of immunity (IEI) characterized by congenital athymia, alopecia, and nail dystrophy. Few cases have been reported to date. However, the recent introduction of newborn screening for IEIs and high-throughput sequencing has led to the identification of novel and atypical cases. Moreover, immunological alterations have been recently described in patients carrying heterozygous mutations. The aim of this paper is to describe the extended phenotype associated with FOXN1 homozygous, compound heterozygous, or heterozygous mutations. We collected clinical and laboratory information of a cohort of 11 homozygous, 2 compound heterozygous, and 5 heterozygous patients with recurrent severe infections. All, except one heterozygous patient, had signs of CID or SCID. Nail dystrophy and alopecia, that represent the hallmarks of the syndrome, were not always present, while almost 50% of the patients developed Omenn syndrome. One patient with hypomorphic compound heterozygous mutations had a late-onset atypical phenotype. A SCID-like phenotype was observed in 4 heterozygous patients coming from the same family. A spectrum of clinical manifestations may be associated with different mutations. The severity of the clinical phenotype likely depends on the amount of residual activity of the gene product, as previously observed for other SCID-related genes. The severity of the manifestations in this heterozygous family may suggest a mechanism of negative dominance of the specific mutation or the presence of additional mutations in noncoding regions.

Forkhead Transcription Factors in Health and Disease.

Forkhead box (FOX) proteins belong to an evolutionarily conserved family of transcription factors that has evolved by gene/genome duplication. FOX family members have undergone sequence and regulatory diversification. However, they have retained some degree of functional redundancy, in addition to playing specific roles, both during development and in the adult. Genetic alterations or misregulation of FOX genes underlie human genetic diseases, cancer, and/or aging. In this review, we provide an updated overview of the main characteristics of the members of this family, in terms of breadth of expression, protein domain composition, evolution, and function.

The influence of various regions of the FOXP2 sequence on its structure and DNA-binding function.

FOX proteins are a superfamily of transcription factors which share a DNA-binding domain referred to as the forkhead domain. Our focus is on the FOXP subfamily members, which are involved in language and cognition amongst other things. The FOXP proteins contain a conserved zinc finger and a leucine zipper motif in addition to the forkhead domain. The remainder of the sequence is predicted to be unstructured and includes an acidic C-terminal tail. In the present study, we aim to investigate how both the structured and unstructured regions of the sequence cooperate so as to enable FOXP proteins to perform their function. We do this by studying the effect of these regions on both oligomerisation and DNA binding. Structurally, the FOXP proteins appear to be comparatively globular with a high proportion of helical structure. The proteins multimerise via the leucine zipper, and the stability of the multimers is controlled by the unstructured interlinking sequence including the acid rich tail. FOXP2 is more compact than FOXP1, has a greater propensity to form higher order oligomers, and binds DNA with stronger affinity. We conclude that while the forkhead domain is necessary for DNA binding, the affinity of the binding event is attributable to the leucine zipper, and the unstructured regions play a significant role in the specificity of binding. The acid rich tail forms specific contacts with the forkhead domain which may influence oligomerisation and DNA binding, and therefore the acid rich tail may play an important regulatory role in FOXP transcription.

ß-catenin regulates FOXP2 transcriptional activity via multiple binding sites.

The transcription factor forkhead box protein P2 (FOXP2) is a highly conserved key regulator of embryonal development. The molecular mechanisms of how FOXP2 regulates embryonal development, however, remain elusive. Using RNA sequencing, we identified the Wnt signaling pathway as key target of FOXP2-dependent transcriptional regulation. Using cell-based assays, we show that FOXP2 transcriptional activity is regulated by the Wnt coregulator ß-catenin and that ß-catenin contacts multiple regions within FOXP2. Using nuclear magnetic resonance spectroscopy, we uncovered the molecular details of these interactions. ß-catenin contacts a disordered FOXP2 region with α-helical propensity via its folded armadillo domain, whereas the intrinsically disordered ß-catenin N terminus and C terminus bind to the conserved FOXP2 DNA-binding domain. Using RNA sequencing, we confirmed that ß-catenin indeed regulates transcriptional activity of FOXP2 and that the FOXP2 α-helical motif acts as a key regulatory element of FOXP2 transcriptional activity. Taken together, our findings provide first insight into novel regulatory interactions and help to understand the intricate mechanisms of FOXP2 function and (mis)-regulation in embryonal development and human diseases. DATABASE: Expression data are available in the GEO database under the accession number GSE138938.

Intrinsically Disordered Regions of the DNA-Binding Domain of Human FoxP1 Facilitate Domain Swapping.

Forkhead box P (FoxP) proteins are unique transcription factors that spatiotemporally regulate gene expression by tethering two chromosome loci together via functional domain-swapped dimers formed through their DNA-binding domains. Further, the differential kinetics on this dimerization mechanism underlie an intricate gene regulation network at physiological conditions. Nonetheless, poor understanding of the structural dynamics and steps of the association process impedes to link the functional domain swapping to human-associated diseases. Here, we have characterized the DNA-binding domain of human FoxP1 by integrating single-molecule Förster resonance energy transfer and hydrogen-deuterium exchange mass spectrometry data with molecular dynamics simulations. Our results confirm the formation of a previously postulated domain-swapped (DS) FoxP1 dimer in solution and reveal the presence of highly populated, heterogeneous, and locally disordered dimeric intermediates along the dimer dissociation pathway. The unique features of FoxP1 provide a glimpse of how intrinsically disordered regions can facilitate domain swapping oligomerization and other tightly regulated association mechanisms relevant in biological processes.

Mouse Double Minute 2 Homolog-Mediated Ubiquitination Facilitates Forkhead Box P3 Stability and Positively Modulates Human Regulatory T Cell Function.

Regulatory T cells (Treg cells) are essential for maintaining immune tolerance, and the dysfunction of Treg cells may cause autoimmune diseases and tumors. Forkhead box P3 (FOXP3) is the key transcription factor controlling Treg cell development and suppressive function. Mouse double minute 2 homolog (MDM2), an E3 ubiquitin ligase, has been identified as an oncoprotein that mediates the ubiquitination and degradation of tumor suppressor p53; however, whether it has functions in Treg cells remains unknown. Here, we demonstrate that MDM2 positively regulates human Treg cell suppressive function via its mediated ubiquitination and stabilization of FOXP3. Knockdown of MDM2 with shRNA in human primary Treg cells leads to the impaired ability of FOXP3 to regulate the expression levels of downstream genes and the attenuated suppressive capacity of Treg cells, due to FOXP3 instability. Consistently, MDM2 overexpression in human Treg cells enhances FOXP3 stability and Treg cell suppressive capacity. Mechanistically, MDM2 interacts with FOXP3, and mainly mediates monoubiquitination and polyubiquitination of FOXP3, thus stabilizing the protein level of FOXP3. We have also found lysine residues in FOXP3 required for MDM2-mediated ubiquitination. In addition, TCR/CD28 signaling upregulates the expression level of MDM2 and its mediated FOXP3 ubiquitination in human Treg cells. Therefore, our findings reveal that MDM2 in Treg cells could be a potential therapeutic target for treating autoimmune diseases and tumors.

FOXC2 Disease Mutations Identified in Lymphedema Distichiasis Patients Impair Transcriptional Activity and Cell Proliferation.

FOXC2 is a member of the human forkhead-box gene family and encodes a regulatory transcription factor. Mutations in FOXC2 have been associated with lymphedema distichiasis (LD), an autosomal dominant disorder that primarily affects the limbs. Most patients also show extra eyelashes, a condition known as distichiasis. We previously reported genetic and clinical findings in six unrelated families with LD. Half the patients showed missense mutations, two carried frameshift mutations and a stop mutation was identified in a last patient. Here we analyzed the subcellular localization and transactivation activity of the mutant proteins, showing that all but one (p.Y109*) localized to the nucleus. A significant reduction of transactivation activity was observed in four mutants (p.L80F, p.H199Pfs*264, p.I213Tfs*18, p.Y109*) compared with wild type FOXC2 protein, while only a partial loss of function was associated with p.V228M. The mutant p.I213V showed a very slight increase of transactivation activity. Finally, immunofluorescence analysis revealed that some mutants were sequestered into nuclear aggregates and caused a reduction of cell viability. This study offers new insights into the effect of FOXC2 mutations on protein function and shows the involvement of aberrant aggregation of FOXC2 proteins in cell death.