Forkhead Box N1 Research Articles

Mechanism of pregnancy-induced thymus involution and regeneration and medication rules of postpartum prescriptions

In order to prevent the maternal immune defenses to the semi-allogeneic fetus, the maternal body will present a special adaptive immune system change represented by acute thymic involution(ATI) during pregnancy, which can be quickly regenerated after delivery. The ATI during pregnancy is related to the level of sex hormones, which is mainly caused by progesterone. Pregnancy-induced ATI is manifested as the continuous shrinkage of thymus volume, especially the cortex, and the wrinkle and phagocytosis of the subcapsular cortical thymic epithelial cells(cTECs), while other thymic epithelial cells(TECs) remain unchanged. The postpartum thymus is regenerated by the co-mediation of forkhead box N1(FOXN1) as well as its target genes chemokine(C-C motif) ligand 25(CCL25), chemokine(C-X-C motif) ligand 12(CXCL12), δ-like ligand 4(DLL4), cathepsin L(CTSL), and serine protease 16(PRSS16). Once the postpartum thymus is poorly repaired, immune dysfunction of the maternal body and several puerperal diseases will be induced, seriously endangering the survival of the mother and the newborn. In traditional Chinese medicine(TCM), Qi and blood are the cornerstone of pregnancy, and the thymus plays a key role in regulating Qi and blood. The deficiency of Qi and blood during pregnancy and childbirth is closely related to the abnormal ATI during pregnancy and the poor regeneration of the postpartum thymus. Based on this theory, TCM has profound academic ideas and rich clinical experience in postpartum recuperation. Based on the systematic description of the mechanism of ATI regeneration during pregnancy, as well as data mining and analysis of two classic gynecological works of TCM, Wan's Gynecology and Fu Qing-zhu's Treatise on Gynecology, this study found that the commonly used TCM for postpartum included Angelicae Sinensis Radix, Ginseng Radix et Rhizoma, Glycyrrhizae Radix et Rhizoma, and Chuanxiong Rhizoma. Among them, Ginseng Radix et Rhizoma, Angelicae Sinensis Radix, and Chuanxiong Rhizoma are high-frequency TCMs with positive effects on postpartum recovery.However, the mechanism of these TCMs in promoting postpartum thymus regeneration needs further investigation.

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

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.

Abstract 375: SR-B1-targeted nanoparticle delivery of novel anthracyclines to Ewing's Tumors

Abstract Purpose: Anthracyclines effectively treat many pediatric cancers, but their use is limited by cardiotoxicity and new approaches to mitigate anthracycline-induced cardiac damage are urgently needed. Nanoparticle drug delivery is one solution to maintain anticancer efficacy while mitigating both acute and long-term cardiac complications. Additionally, novel treatments are needed to improve cure rates in certain cancer diagnoses that have not seen advancements in decades, particularly Ewing Sarcoma (EWS). Experimental Design: We investigated the use of a self-assembling micelle structure composed of myristic acid conjugated to an Apo-A1 mimetic 5A peptide, termed Myr5A nanoparticles (Myr5A NPs). These nanoparticles are transported into cells via the cholesterol scavenger receptor class B type-1 (SR-B1). Doxorubicin (DOX) and the Myr5A NP cannot form a stable micelle due to the hydrophilic nature of DOX, which results in preferential binding to the peptide shell, as opposed to the hydrophobic core. To favor micelle stability, we opted to use the novel, highly hydrophobic anthracyclines valrubicin and AD198 to test the nanoparticle delivery system. The potency of these anthracyclines encapsulated in Myr5A NPs were evaluated in human EWS cell lines with high and low SR-B1 expression via MTT assay. Additionally, 5 mg/kg intraperitoneal injections were administered weekly to 4-week-old wild type C57BL6/J mice and immune compromised homozygous null forkhead box N1 mice (Foxn1null/null mice) over 5 weeks, to assess by echocardiography, acute and long-term cardiac dysfunction. Results: Treatment with Myr5A-encapsulated AD198 and Myr5A-encapsulated valrubicin significantly inhibited EWS cell proliferation at concentrations comparable to conventional anthracyclines. Moreover, compared to unencapsulated valrubicin and AD198, encapsulation resulted in a lower IC50 in EWS lines with higher SR-B1 surface expression. By 19 weeks of age, DOX-treated C57BL6/J mice exhibited significantly decreased ejection fraction (EF) compared to PBS control and Myr5A-encapsulated anthracyclines (p<0.05). Additionally, an increase in Left Ventricular Mass (LVM) was observed in all groups except DOX by 19 weeks (p<0.05). Lastly, DOX-treated Foxn1null/null mice showed diminished survival rate compared to all treatment groups, and similar results to C57BL6/J mice in EF and LVM. Conclusions: Our results indicate that AD198 and valrubicin, when encapsulated in Myr5A NPs, induce minimal cardiac dysfunction while maintaining antitumor efficacy in EWS tumors. Myr5A NP drug delivery may be a viable option for the treatment of high SR-B1 expressing tumors with a decreased risk of acute and long-term cardiac complications. Future studies will evaluate the drug potency in non-cancer cell lines, such as cardiac fibroblasts and myocytes, and the effects of Myr5A-encaspulated anthracyclines on EWS xenografts and other pediatric tumors. Citation Format: Kathryn Crouch, Logan Davis, Nirupama Sabnis, Alexander Pertsemlidis, Laura Rutledge, James Graham, Jason Yustein, Gregory J. Aune. SR-B1-targeted nanoparticle delivery of novel anthracyclines to Ewing's Tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 375.

Role of foxn1 in Xenopus laevis thymopoiesis.

Abstract The transcription factor forkhead box N1 (FOXN1) plays a pivotal role in differentiation of thymic epithelial cells (TECs) essential for intrathymic T cell education and keratinocyte development. Mutations in murine foxn1 are associated with T− B+ NK+ nude/severe combined immunodeficiency (SCID) phenotype. In humans FOXN1 mutations also lead to thymic aplasia or hypoplasia, hair loss and dysmorphic nails. In the amphibian Xenopus tropicalis, foxn1−/− line exhibited loss of thymi formation and absence of mature T-cells, similar with Foxn1−/− murine line. Due to expanded resources in immunobiology of X. laevis (a close relative of X. tropicalis), we generated a novel foxn1-deficient X. laevis model by using the CRISPR/Cas9. foxn1 crispants shows a high mutational efficiency (30–90%) and about 25% of tadpoles (stage 55–56), exhibit hypoplastic thymi. foxn1 disruption resulted in significant reduction of T-cell markers transcript levels, detected by RT-PCR. Furthermore, spleen of foxn1 crispants had markedly reduced CD3e staining compared to controls. Interestingly, Mycobacterium marinum (M. marinum) infection of tadpoles results in noninflammatory CD8 negative- and iT cell–mediated response, while foxn1 mutations in X. laevis appeared to affect tolerigenic innate immune response of infected tadpoles. This foxn1-deficient transgenic line will empower X. laevis as nonmammalian alternative experimental organism for studying host interaction with to non-tuberculosis mycobacteria. Currently, we are investigating mechanisms by which foxn1 mutation alter innate immunity of M. marinum inoculated tadpoles and evaluating tolerance/resistance by analysing the inflammatory/anti-inflammatory cytokine profile.

A family with forkhead box N1 (FOXN1) haploinsufficiency causing severe combined immunodeficiency (SCID)

Heterozygous loss-of-function variants in FOXN1 are associated with hypothymia, T lymphopenia and infection susceptibility. Herein, we present a mother and two children with FOXN1 haploinsufficiency causing SCID.

Development of the Nude Rabbit Model.

SummaryLoss-of-function mutations in the forkhead box N1 (FOXN1) gene lead to nude severe combined immunodeficiency, a rare inherited syndrome characterized by athymia, severe T cell immunodeficiency, congenital alopecia, and nail dystrophy. We recently produced FOXN1 mutant nude rabbits (NuRabbits) by using CRISPR-Cas9. Here we report the establishment and maintenance of the NuRabbit colony. NuRabbits, like nude mice, are hairless, lack thymic development, and are immunodeficient. To demonstrate the functional applications of NuRabbits in biomedical research, we show that they can successfully serve as the recipient animals in xenotransplantation experiments using human induced pluripotent stem cells or tissue-engineered blood vessels. Our work presents the NuRabbit as a new member of the immunodeficient animal model family. The relatively large size and long lifespan of NuRabbits offer unique applications in regenerative medicine, cancer research, and the study of a variety of other human conditions, including immunodeficiency.

Alymphoid cystic thymic dysgenesis - FOXN1 gene mutation: a rare case report of two siblings

Alymphoid cystic thymic dysgenesis is a severe combined immunodeficiency (SCID) syndrome caused b y a mutation in fork head box N1 gene (FOXN1) on chromosome 17. It is a transcriptional factor regulating the development, differentiation and function of thymic epithelial cells; maintaining T-lineage progenitors in bone marrow; promoting terminal differentiation of epithelial cells of hair follicles. Mutation in FOXN1 is known to cause a rare disorder characterized by rudimentary thymus gland (primary lymphoid organ for T-cell differentiation), T-cell immunodeficiency, congenital alopecia totalis and nail dystrophy. Here we report two affected siblings from a non-consanguineous family with similar features of alopecia totalis, nail dystrophy and failure to thrive. The first child was a 7-month-old female baby, with history of two hospitalization in the past for lower respiratory tract infection, had left axillary lymphadenopathy (BCG adenitis), alopecia totalis, nail dystrophy and hepatosplenomegaly. Bronchoalveolar lavage secretion was positive for Mycobacterium tuberculosis and Pneumocystis carinii pneumonia by gene Xpert and polymerase chain reaction respectively. Immunodeficiency panel workup revealed combined T cell and B cell immunodeficiency, genetic analysis by whole exome sequencing revealed recessive missense mutation in exon 6 of FOXN1 gene on chromosome 17. Due to lack of sufficient literature it was reported as variant of unknown significance and to establish its clinical significance the carrier status of both the parents was established. Second child presented to us at 3 months of age, also had similar phenotypic features and on evaluation had very low lymphocyte subset count however mutational analysis could not be done in this child due to parent’s denial. Hence, we conclude this child also was affected.

Foxn1 Control of Skin Function

The forkhead box N1 (Foxn1) transcription factor regulates biological processes of the thymus and skin. Loss-of-function mutations in Foxn1 cause the nude phenotype in humans, mice, and rats, which is characterized by hairless skin and a lack of thymus. This review focuses on the role of Foxn1 in skin biology, including epidermal, dermal, and dermal white adipose tissue (dWAT) skin components. In particular, the role of Foxn1 in the scar-forming skin wound healing process is discussed, underscoring that Foxn1 inactivity in nude mice is permissive for scar-less cutaneous wound resolution.

The crystal structure of human forkhead box N1 in complex with DNA reveals the structural basis for forkhead box family specificity

Forkhead box N1 (FOXN1) is a member of the forkhead box family of transcription factors and plays an important role in thymic epithelial cell differentiation and development. FOXN1 mutations in humans and mice give rise to the “nude” phenotype, which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognizes an alternative forkhead-like (FHL) consensus sequence (GACGC) that is different from the more widely recognized forkhead (FKH) sequence RYAAAYA (where R is purine, and Y is pyrimidine). Here, we present the FOXN1 structure in complex with DNA containing an FHL motif at 1.6 Å resolution, in which the DNA sequence is recognized by a mixture of direct and water-mediated contacts provided by residues in an α-helix inserted in the DNA major groove (the recognition helix). Comparisons with the structure of other FOX family members revealed that the FKH and FHL DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identified a single alternative rotamer within the recognition helix itself as an important determinant of DNA specificity and found protein sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally, we demonstrate that the C-terminal region of FOXN1 is required for high-affinity DNA binding and that FOXN1 has a significantly reduced affinity for DNA that contains 5′-methylcytosine, which may have implications for the role of FOXN1 in thymic involution.

Recurrent microdeletions at chromosome 2p11.2 are associated with thymic hypoplasia and features resembling DiGeorge syndrome

Thymic hypoplasia/aplasia occurs as a part of DiGeorge syndrome, which has several known genetic causes, and with loss-of-function mutations in forkhead box N1 (FOXN1). We sought to determine the cause of selective T-cell lymphopenia with inverted kappa/lambda ratio in several kindreds. Patients were identified through newborn screening for severe combined immunodeficiency using the T-cell receptor excision circle assay. Those found to have selective T-cell lymphopenia underwent testing with chromosomal microarray analysis. Three-week-old mice heterozygous for a loss-of-function mutation in forkhead box I3 (FOXI3), a candidate gene within the common deleted region found in patients, were compared with wild-type littermates. Assessments included body and organ weights, flow cytometric analysis of thymocytes and splenocytes, and histologic/transcriptomic analyses of thymic tissue. Five kindreds with similar immunophenotypes that included selective T-cell lymphopenia had overlapping microdeletions at chromosome 2p11.2 that spanned FOXI3 and, in most cases, the immunoglobulin kappa light chain locus. Studies in a mouse knockout strain for FOXI3 revealed smaller body weights and relatively lower thymus weights in heterozygous compared with wild-type animals. Histology and flow cytometry on spleens and thymi from 3-week-old pups for T- and B-cell subsets and epithelial cells did not show any significant qualitative or quantitative differences. Transcriptomic analysis of thymic RNA revealed divergence in global transcriptomic signatures, and Ingenuity Pathway Analysis revealed predicted dysfunction in epithelial adherens junctions. Microdeletions at chromosome 2p11.2 are associated with T-cell lymphopenia and probable thymic hypoplasia in human subjects, and haploinsufficiency for FOXI3, a candidate gene within the deleted region, is the likely underlying cause.

22q11.2 Deletion Syndrome (DiGeorge) and Mutations in Forkhead Box N1 (FOXN1) cause a Thymic Hypoplasia through distinct Developmental Processes

Abstract Patients with 22q11.2 deletion syndrome and those with mutations in the Forkhead Box N1(FOXN1) transcription factor (Nude/SCID) can both present with a thymic hypoplasia that results in a severe T cell lymphopenia. In both clinical conditions, the thymic anlage fails to develop properly within the 3rdpharyngeal pouch during embryogenesis. We characterized the development of the thymus in mouse models of 22q11.2 deletion syndrome (22q11.2del) and a new set of mice with mutations in Foxn1that genocopied a SCID patient with novel compound heterozygous mutations in FOXN1. Both sets of mice develop hypoplastic thymic lobes. An analysis of thymopoiesis in embryos revealed distinct development problems. The hypoplastic thymii from the 22q11.2del mice were primarily sized restricted, with normal percentages of all thymocyte subsets apparent. This contrasted a severe deficiency of thymocytes due to the Foxn1mutations, which primarily affected the development and expansion of thymic epithelial cells. Comparative gene expression analyses of e13.5 fetal thymii revealed differentially regulated transcripts that define the basis of the hypoplasia. A dysregulated mesenchymal cell signature was apparent in the 22q11.2del model, which contrasted the epithelial transcript disruption due to the Foxn1mutations. These results suggest different strategies are necessary to correct the thymic tissue abnormalities in patients who present with thymic hypoplasias due to their congenital disorders.

Inducible thymic epithelial cell-based rejuvenation in thymic aging-associated inflammaging (Supported by NIH/NIAID R01AI121147)

Abstract Age-related thymic involution primarily results from a decline in the transcription factor forkhead box N1 (FOXN1), while input of exogenous FOXN1 is able to significantly rescue thymic aging. Given that FOXN1 over-expressing fibroblasts (inducible thymic epithelial cells, iTECs) can generate an ectopic de novo thymus and engrafted young TECs can lead to middle-aged thymus regrowth, we asked whether engrafting iTECs directly into the aged thymus and perithymus can be a strategy to rescue aged thymic function. The re-establishment of central tolerance is crucial to attenuate age-associated chronic inflammation in the elderly (termed inflammaging). This will reduce increased self(auto)-reactivity, an important contributor to inflammaging. Intrathymic transplantation of iTECs into the aged murine thymus revealed that iTECs drove partial regrowth of the aged thymus, equally well in both male and female mice. The iTECs also rejuvenated aged thymic architecture, including regeneration of well-organized thymic medulla, and improvement of aged thymic function, including re-enforcement of thymocyte negative selection-related Aire gene expression. This led to reduction of self-reactivity-resulted inflammaging, including pro-inflammatory cytokines (IL-6 and IL-1b) and lymphoinfiltration to non-lymphoid organs, in old mice. Our findings support this potential novel strategy for enforcing the declined thymocyte negative selection to partially restore central tolerance in the elderly and reduce inflammaging.

Compound Heterozygous Mutations in Forkhead Box N1 (FOXN1) Lead to a Severe Immunodeficiency but Normal Hair and Nail Development in Patients

Abstract Patients with mutations in the Forkhead Box N1 (FOXN1) transcription factor are born with a severe T-cell lymphopenia in conjunction with alopecia and nail dystrophy (OMIM # 600838). The T-cell lymphopenia results from the impaired development and/or function of the thymic epithelial cells (TECs). TECs are essential regulators of positive and negative selection of developing thymocytes. We report on 3 independent patients with compound heterozygous mutations in FOXN1. All 3 patients had a severe T cell lymphopenia. However, each had normal hair growth and nail bed formation, suggesting that the compound heterozygous mutations result in clinical presentations distinct from classic cases. To determine how the mutations impact murine Foxn1 function, transcriptional reporter assays and protein expression studies were done. Only one of the mutations affected the transcriptional activity of murine Foxn1, with Western blot analyses indicating that this mutation caused production of a truncated protein. CRISPR/Cas9 technologies were used to create mouse lines with compound heterozygous mutations in Foxn1. The mice are currently being intercrossed. The impact of the compound heterozygous Foxn1 mutations on T cell development in the thymus will be presented. Findings from this study may suggest novel therapeutic strategies at restoring thymopoiesis in different clinical settings.

MIR205HG is a Long Noncoding RNA with Distinct Functions in the Thymus versus the Anterior Pituitary

Abstract MicroRNAs (miRNAs) are processed from primary RNA transcripts (pri-miRNAs) encoded in host genes. Several miRNAs including miR-205 are present within long noncoding RNAs (lncRNAs). MiR-205 is an epithelial-specific miRNA that supports thymopoiesis by positively regulating Forkhead Box N1 (Foxn1) expression. We assessed whether the host gene for miR-205, MIR205HG, has independent functions as a lncRNA. The more severe stress-induced thymic atrophy reported in miR-205-deficient mice is also evident in MIR205HG knockout lines. However, MIR205HG knockouts have a small stature phenotype. A new set of miR-205KI mice did not have this phenotype. The smaller mouse size of the MIR205HG null animals is partly a consequence of reduced levels of endocrine hormones produced by the anterior pituitary. In addition, the MIR205HG null animals had abnormal development of the lacrimal and harderian glands that produce eye secretions. Transcriptome analyses revealed that MIR205HG regulates gene expression in the anterior pituitary unlike miR-205. In contrast, transcripts regulated by miR-205 and MIR205HG in the epithelial cells of the skin and thymus overlap significantly. These data indicate that MIR205HG has a specific function as a lncRNA in the anterior pituitary in addition to its primary role as the host gene for miR-205 in thymic epithelial cells.

Taking advantage from phenotype variability in a local animal genetic resource: identification of genomic regions associated with the hairless phenotype in Casertana pigs.

Casertana is an endangered autochthonous pig breed (raised in south-central Italy) that is considered to be the descendant of the influential Neapolitan pig population that was used to improve British breeds in the 19th century. Casertana pigs are characterized by a typical, almost complete, hairless phenotype, even though a few Casertana pigs are normal haired. In this work, using Illumina PorcineSNP60 BeadChip data, we carried out a genome-wide association study and an FST analysis with this breed by comparing animals showing the classical hairless phenotype (n=81) versus pigs classified as haired (n=15). Combining the results obtained with the two approaches, we identified two significant regions: one on porcine chromosome (SSC) 7 and one on SSC15. The SSC7 region contains the forkhead box N3 (FOXN3) gene, the most plausible candidate gene of this region, considering that mutations in another gene of the same family (forkhead box N1; Foxn1 or FOXN1) are responsible for the nude locus in rodents and alopecia in humans. Another potential candidate gene, rho guanine nucleotide exchange factor 10 (ARHGEF10), is located in the SSC15 region. FOXN3 and ARHGEF10 have been detected as differentially expressed in androgenetic and senescent alopecia respectively. This study on an autochthonous pig breed contributes to shed some light on novel genes potentially involved in hair development and growth and demonstrates that local animal breeds can be valuable genetic resources for disclosing genetic factors affecting unique traits, taking advantage of phenotype variability segregating in small populations.

Mechanisms in hypertension and target organ damage: Is the role of the thymus key? (Review).

A variety of cells and cytokines have been shown to be involved in the whole process of hypertension. Data from experimental and clinical studies on hypertension have confirmed the key roles of immune cells and inflammation in the process. Dysfunction of the thymus, which modulates the development and maturation of lymphocytes, has been shown to be associated with the severity of hypertension. Furthermore, gradual atrophy, functional decline or loss of the thymus has been revealed to be associated with aging. The restoration or enhancement of thymus function via upregulation in the expression of thymus transcription factors forkhead box N1 or thymus transplantation may provide an option to halt or reverse the pathological process of hypertension. Therefore, the thymus may be key in hypertension and associated target organ damage, and may provide a novel treatment strategy for the clinical management of patients with hypertension in addition to different commercial drugs. The purpose of this review is to summarize and discuss the advances in our understanding of the impact of thymus function on hypertension from data from animal and human studies, and the potential mechanisms.

Forkhead box N1 inhibits the progression of non-small cell lung cancer and serves as a tumor suppressor.

Forkhead box N1 (FOXN1) belongs to the FOX family of transcription factors, which comprises a diverse group of winged-helix proteins. FOXN1 is a ubiquitously expressed member that has been implicated in the embryo development, metabolism, aging and cancer. However, little is known regarding the role of FOXN1 in non-small cell lung cancer (NSCLC). The aim of the study was to investigate the function of FOXN1 in NSCLC and examine the relevant mechanism. In the present study, using reverse transcription-quantitative polymerase chain reaction, western blotting, transwell assay, MTT assay, luciferase report assy, it was identified that knockdown of FOXN1 increased the proliferation of A549 and H1299 cells, while overexpression of FOXN1 evidently suppressed the cell growth. A Transwell assay was used to determine the relative cell invasion ability, and it was observed that the invading cells were markedly decreased in the FOXN1 overexpression groups; by contrast, reduced expression of FOXN1 demonstrated the potential to promote cell invasion. Furthermore, lower expression of FOXN1 was observed in NSCLC tissues and cell lines as compared with the adjacent non-tumor tissues or human bronchial epithelial cells, respectively. A higher level of FOXN1 was associated with a better prognosis of NSCLC patients. Quantitative chromatin immunoprecipitation analysis and luciferase reporter gene assays revealed that enhancer of zeste homolog 2 (EZH2) and β-catenin were two target genes of FOXN1. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis indicated that FOXN1 suppressed the expression levels of these target genes at the transcriptional level. In conclusion, the present study demonstrated that FOXN1 served major roles in NSCLC proliferation and invasion by directly repressing EZH2 and β-catenin, which suggested that FOXN1 may function as a tumor suppressor in NSCLC.

Sp1 is an important transcriptional regulation factor for forkhead box N1 in pig

The transcription factor forkhead box N1 (Foxn1) plays an important role in the development and function of thymic epithelial cells (TECs) in vertebrates. However, the transcriptional regulation of Foxn1 is still unknown. A series of dual luciferase report vectors were constructed and their relative activities were also detected. The 5'-untranslated regions contains two cis-acting elements, Sp1 and GATA-1, as well as trans-acting elements between positions -332 to -438. Nevertheless, the relative activities of Foxn1 promoter were significantly increased in PK15 cells only when the Sp1 was overexpressed, suggesting that the Sp1 was the most important sequence for Foxn1 transcription activation in the pig. Regrettably, the exact trans-acting elements were not found.

Genome-wide association analysis identifies a GLUL haplotype for familial hepatitis B virus-related hepatocellular carcinoma.

A family history of liver cancer increases the risk of developing hepatocellular carcinoma (HCC) by 2-fold to 10-fold among patients with chronic hepatitis B virus (HBV). Previous genome-wide association studies have identified many possible susceptible loci associated with sporadic HBV-related HCC. However, despite family history being a well-known risk factor for HBV-related HCC, to the authors' knowledge its genetic mechanisms and associating loci remain largely unknown or unexplored, most likely due to the relative rarity of familial HCC and the difficulty of sample collection. The authors conducted a genome-wide association study with 139 male cases with familial HBV-related HCC and 139 non-HCC male controls with chronic HBV. The results were corroborated further with an independent cohort of 101 patients with familial HBV-related HCC and comparison with both the 1000 Genomes Project and the Taiwan Biobank. A total of 51 risk single-nucleotide polymorphisms (P≤1E-04) were identified in the association analyses, which included 2 clusters of associated single-nucleotide polymorphisms and haplotypes at 1q25.3 (glutamate-ammonia ligase [GLUL]/transmembrane epididymal protein 1 [TEDDM1]/long intergenic non-protein-coding RNA 272 [LINC00272]/regulator of G-protein signaling-like 1 [RGSL1]) and 17q11.2 (solute carrier family 13 member 2 [SLC13A2]/forkhead box N1 [FOXN1]). Both the GLUL and SLC13A2/FOXN1 haplotypes have large effect sizes and were found to be different from those found from genome-wide association studies of sporadic HCCs. To the authors' knowledge, the current study is the first genome-wide association study to identify genetic factors for familial HBV-related HCC. The results identified 2 large effect susceptible haplotypes located at GLUL and SLC13A2/FOXN1. The current study findings also suggest different genetic susceptibility between familial and sporadic HBV-related HCC. Cancer 2017;123:3966-76. © 2017 American Cancer Society.

FOXN1 Italian founder mutation in Indian family: Implications in prenatal diagnosis

The Forkhead box N1 (FOXN1) is a transcriptional factor regulating the development, differentiation and function of thymic epithelial cells; maintaining T-lineage progenitors in bone marrow; promoting terminal differentiation of epithelial cells of hair follicles. Mutation in FOXN1 was reported to cause a rare disorder characterized by rudimentary thymus gland, T-cell immunodeficiency, congenital alopecia and nail dystrophy within an Italian community. This is the first report of FOXN1 p.R255X mutation from India, outside this endogamous Italian community. Out of the two affected children, only one was alive during the genetic evaluation and had all the clinical manifestations such as alopecia totalis and nail dystrophy. The proband was homozygous for FOXN1 p.R255X Italian founder mutation. The carrier status of both the parents was established. Immunological study of the proband revealed total absence of T-cells confirming T-cell immunodeficiency. Prenatal diagnosis during third pregnancy revealed absence of FOXN1 mutation. To conclude, this is the first report of FOXN1 mutation from India highlighting that diseases once confined to certain geographical areas are spreading across the globe probably due to human migrations.