Forkhead Box Gene Research Articles

FOXN1 in Organ Development and Human Diseases

FOXN1 gene belongs to the forkhead box gene family that comprises a diverse group of “winged-helix” transcription factors that have been implicated in a variety of biochemical and cellular processes, such as development, metabolism, aging and cancer. These transcription factors share the common property of being developmentally regulated and of directing tissue-specific transcription and cell-fate decisions. Foxn1 is selectively expressed in thymic and skin epithelial cells, where it acts through its molecular targets to regulate the balance between growth and differentiation. In particular, Foxn1 is required for thymic epithelial patterning and differentiation from the initial epithelial thymic anlage to a functional cortical and medullary thymic epithelial cells (TECs) meshwork necessary for the crosstalk with the lymphoid compartment. A mutation in FoxN1 generates alymphoid cystic thymic dysgenesis due to defective TECs, causing primary T-cell immunodeficiency, named Nude/SCID syndrome, and leads to a hairless “nude” phenotype in both mice and humans. This immune defect represents the first example of a Severe Combined Immunodeficiencies (SCID) phenotype not primarily related to an abnormality intrinsic of the hematopoietic cell, but rather to a peculiar alteration of the thymic epithelia cell. This review focuses on the key role of FOXN1 in cell development and its clinical implication in humans.

The Winged Helix Transcription Factor Foxa3 Regulates Adipocyte Differentiation and Depot-Selective Fat Tissue Expansion

Conversion of mesenchymal stem cells into terminally differentiated adipocytes progresses sequentially through regulated transcriptional steps. While it is clear that the late phases of adipocyte maturation are governed by the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ), less is known about the transcriptional control of the initial stages of differentiation. To identify early regulators, we performed a small interfering RNA (siRNA) screen of Forkhead-box genes in adipocytes and show here for the first time that the winged helix factor Foxa3 promotes adipocyte differentiation by cooperating with C/EBPβ and -δ to transcriptionally induce PPARγ expression. Furthermore, we demonstrate that mice with genetic ablation of Foxa3 have a selective decrease in epididymal fat depot and a cell-autonomous defect to induce PPARγ specifically in their visceral adipocytes. In obese subjects, FOXA3 is differentially expressed in visceral and subcutaneous adipose depots. Overall, our study implicates Foxa3 in the regulation of adipocyte differentiation and depot-selective adipose tissue expansion.

BRAF mutation-specific promoter methylation of FOX genes in colorectal cancer

BackgroundCancer-specific hypermethylation of (promoter) CpG islands is common during the tumorigenesis of colon cancer. Although associations between certain genetic aberrations, such as BRAF mutation and microsatellite instability, and the CpG island methylator phenotype (CIMP), have been found, the mechanisms by which these associations are established are still unclear. We studied genome-wide DNA methylation differences between colorectal tumors carrying a BRAF mutation and BRAF wildtype tumors.ResultsUsing differential methylation hybridization on oligonucleotide microarrays representing 32,171 CpG-rich regions, we identified 1,770 regions with differential methylation between colorectal tumor and paired normal colon. Next, we compared the tumor/normal methylation ratios between different groups of patients. Related to CIMP, we identified 749 differentially methylated regions, of which 86% had a higher tumor/normal methylation ratio in the CIMP-positive group. We identified 758 regions with a BRAF mutation-specific methylation change, of which 96% had a higher tumor/normal methylation ratio in the BRAF mutant group. Among the genes affected by BRAF mutation-specific methylation changes, we found enrichment of several cancer-related pathways, including the PI3 kinase and Wnt signaling pathways. To focus on genes that are silenced in a tumor-specific rather than a lineage-specific manner, we used information on the epigenetic silencing mark H3K27me3 in embryonic stem (ES) cells. Among the genes showing BRAF mutation-specific promoter methylation but no H3K27me3 mark in ES cells were forkhead box (FOX) transcription factors associated with the PI3 kinase pathway, as well as MLH1 and SMO. Repression of FOXD3 gene expression in tumors could be related to its promoter hypermethylation.ConclusionsWe identified new BRAF mutation-specific methylation changes in colorectal cancer. Epigenetic downregulation of these targets may contribute to mutationally active BRAF-driven tumorigenesis, explaining its association with aberrant DNA methylation.

Genome Wide DNA Methylation Analysis of Patients with Myelodysplastic Syndrome and Isolated Deletion (5q) Reveals Characteristic Methylation Profiles in Low and Intermediate-1 Risk Groups

AbstractAbstract 3801 BackgroundAberrant DNA methylation at promoter CpG islands is recognized as one of the hallmarks driving the pathogenesis of myeloid malignancies, especially myelodysplastic syndromes (MDS). Shen et al. recently characterized an algorithm of 10 aberrantly methylated gene loci which was predictive for overall survival and progression-free survival in a large cohort of MDS patients. A particular cytogenetic subgroup of MDS patients with a deletion on the long arm of chromosome 5 (5q-syndrome) has been shown to benefit from treatment with lenalidomide. However, the exact underlying molecular mechanism of MDS with isolated deletion (5q) is still not understood. To further elucidate on the role of deregulated DNA methylation we analyzed DNA-methylation profiles of bone marrow cells from patients with MDS with an isolated deletion (5q). MethodsAll patients were diagnosed and treated within a German multicenter trial investigating the safety of Lenalidomide in patients with low risk myelodysplastic syndromes and an isolated deletion (5q) after informed consent and according to the declaration of Helsinki. Bone marrow cells of 47 MDS patients with deletion (5q) at initial diagnosis were analyzed (median age 70 years, range 41 – 88 years, IPSS score: low n= 22; intermediate-1 n = 25).DNA was extracted using the QIAGEN Allprep Kit® (Qiagen, Hilden, Germany). Genome wide DNA methylation analysis was performed using the HumanMethylation450 BeadChip (Illumina, San Diego, USA). Differential methylation of CpGs was defined by a minimum mean methylation difference of 15% as expressed by the beta-value of the array data and statistical significance set at q ≤ 0.01 according to the Benjamini-Hochberg-method for multiple significance testing. Analysis of array data was performed using Genome-Studio Software® (Illumina, San Diego, USA), Qlucore Omics explorer 2.3 (Qlucore software. Lund, Sweden) and Microsoft Excel 10.1® (Microsoft Software, Redmond, USA). Gene ontology analysis was performed using GATHER (http://http://gather.genome.duke.edu/). ResultsUsing a q-value of ≤ 0.05 for the beta-value of the array and excluding gender-specific chromosomal CpGs, 473,929 CpGs were evaluable for analysis. Gene Ontology analysis using the GATHER Tool showed a significant enrichment of genes mapped to 5q31 (p<0.0001).Highly significant differential methylation profiles between MDS patients with isolated (5q) were found between patients with low and intermediate-1 IPSS score.CpGs differentially hypermethylated in intermediate-1 risk versus lows risk patients affected the coding regions of interesting candidate genes such as platelet-derived growth factor receptor, beta polypeptide (PDGFRB), clathrin interactor 1 (CLINT1), both located at 5q33 and suspected to be involved in the pathogenesis of 5q deleted MDS. Furthermore, transcriptional regulators such as proline, glutamate and leucine rich protein 1 (PELP1), v-myb myeloblastosis viral oncogene homolog (avian) (MYB), genes known to be involved in cancer like trichorhinophalangeal 1(TRPS1), and tumor suppressors like forkhead box P1 (FOXP1) and genes thought to be involved in the pathogenesis of MDS like Minichromosome maintenance protein2(MCM2) did show differentially DNA-methylation according to our selection criteria. ConclusionsWe present a comprehensive genome wide methylation analysis of MDS patients with an isolated deletion (5q) with low and intermediate-1 risk according to IPSS. Thereby we detected sets of significantly differentially methylated CpGs between both risk groups. Correlation of these data to clinical parameters might help to further elucidate the contribution of aberrant methylation to the phenotype of MDS with isolated deletion (5q) and could possibly help establishing novel prognostic markers based on differential methylation. Moreover, unraveling the role of aberrant methylation patterns might result in new therapeutic treatment approaches at least in a subset of patients. Disclosures:Nowak:Celgene: Research Funding. Platzbecker:Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Giagounidis:Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Götze:Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Ottmann:Celgene: Clinical trial participation Other. Haase:Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Schlenk:Celgene: Research Funding. Ganser:Celgene: Research Funding. Germing:Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Hofmann:Celgene: Honoraria, Research Funding. Nolte:Celgene: Research Funding.

Forkhead Transcription Factor Fd3F Cooperates with Rfx to Regulate a Gene Expression Program for Mechanosensory Cilia Specialization

SummaryCilia have evolved hugely diverse structures and functions to participate in a wide variety of developmental and physiological processes. Ciliary specialization requires differences in gene expression, but few transcription factors are known to regulate this, and their molecular function is unclear. Here, we show that the Drosophila Forkhead box (Fox) gene, fd3F, is required for specialization of the mechanosensory cilium of chordotonal (Ch) neurons. fd3F regulates genes for Ch-specific axonemal dyneins and TRPV ion channels, which are required for sensory transduction, and retrograde transport genes, which are required to differentiate their distinct motile and sensory ciliary zones. fd3F is reminiscent of vertebrate Foxj1, a motile cilia regulator, but fd3F regulates motility genes as part of a broader sensory regulation program. Fd3F cooperates with the pan-ciliary transcription factor, Rfx, to regulate its targets directly. This illuminates pathways involved in ciliary specialization and the molecular mechanism of transcription factors that regulate them.

APCI1307K Mutations and Forkhead Box Gene (FOXO1A): Another Piece of an Interesting Correlation

Germline nonsense and frameshift mutations in the adenomatous polyposis coli (APC) gene are found in approximately 90% of individuals affected by familial adenomatous polyposis (FAP) and a genotype-phenotype relationship has been observed. Missense mutations have also been found in a few cases, even if their role in FAP is still unknown. An association between a missense mutation, APC I1307K, and the risk of sporadic colorectal cancer (CRC) has been reported. In order to improve the knowledge about the genetic effect of APC I1307K on the phenotype, we tried a new approach using matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS). An APC mutation (I1307K) was found in an index case of a non-Jewish woman and her son with attenuated familial adenomatous polyposis (A-FAP) and no family history of cancer. In order to evaluate whether the presence and abundance of the ionic species are related to the presence of cancer or the presence of mutation, comparative analyses of 11 healthy clean-colon subjects, 59 patients with CRC (stage II n=19, stage III n=23, stage IV n=17) without polyps, and 9 FAP patients, carriers of a nonsense mutation in the APC gene, were evaluated. Comparative analysis of serum protein profiles of the index patient and her healthy son, FAP and sporadic CRC patients, and subjects with preneoplastic lesions showed a characteristic abundance of ionic species at m/z 905, which was not present in healthy controls. Two peptides were identified from MALDI/MS/MS spectra of m/z 905 belonging to the kininogen-1 precursor and the human forkhead box protein 01A (FOXO1A). FOXO1A was present in only two subjects carrying I1307K, but not in other patients. Our findings seem to suggest a relationship between m/z 905, FOXO1A and the development and growth of colorectal cancer. FOXO1A fragment determination in serum with MALDI/MS might be a promising approach for early detection of colon carcinoma or for the development of targeted therapies.

16q24.1 microdeletion in a premature newborn: Usefulness of array-based comparative genomic hybridization in persistent pulmonary hypertension of the newborn

Report of a 16q24.1 deletion in a premature newborn, demonstrating the usefulness of array-based comparative genomic hybridization in persistent pulmonary hypertension of the newborn and multiple congenital malformations. Descriptive case report. Genetic department and neonatal intensive care unit of a tertiary care children's hospital. None. We report the case of a preterm male infant, born at 26 wks of gestation. A cardiac malformation and bilateral hydronephrosis were diagnosed at 19 wks of gestation. Karyotype analysis was normal, and a 22q11.2 microdeletion was excluded by fluorescence in situ hybridization analysis. A cesarean section was performed due to fetal distress. The patient developed persistent pulmonary hypertension unresponsive to mechanical ventilation and nitric oxide treatment and expired at 16 hrs of life. An autopsy revealed partial atrioventricular canal malformation and showed bilateral dilation of the renal pelvocaliceal system with bilateral ureteral stenosis and annular pancreas. Array-based comparative genomic hybridization analysis (Agilent oligoNT 44K, Agilent Technologies, Santa Clara, CA) showed an interstitial microdeletion encompassing the forkhead box gene cluster in 16q24.1. Review of the pulmonary microscopic examination showed the characteristic features of alveolar capillary dysplasia with misalignment of pulmonary veins. Some features were less prominent due to the gestational age. Our review of the literature shows that alveolar capillary dysplasia with misalignment of pulmonary veins is rare but probably underreported. Prematurity is not a usual presentation, and histologic features are difficult to interpret. In our case, array-based comparative genomic hybridization revealed a 16q24.1 deletion, leading to the final diagnosis of alveolar capillary dysplasia with misalignment of pulmonary veins. It emphasizes the usefulness of array-based comparative genomic hybridization analysis as a diagnostic tool with implications for both prognosis and management decisions in newborns with refractory persistent pulmonary hypertension and multiple congenital malformations.

Expression of FOXL2 in human normal pituitaries and pituitary adenomas

Many transcription factors have important roles in the function and differentiation of the human pituitary adenomas. Forkhead box gene transcription factor L2, Foxl2, is expressed during mouse pituitary development and co-localizes with the expression of α-glycoprotein hormone subunit (αGSU). In addition, Foxl2 regulates expression of the αGSU gene (Cga) in cell culture. To elucidate the functional role of FOXL2 in the human pituitary, we examined the expression and localization of FOXL2 in normal human pituitaries and various types of pituitary adenomas. Human pituitary adenomas were obtained by trans-sphenoidal surgery from 67 patients. Three normal adult pituitaries were obtained from autopsies of non-endocrine cases. The localization of FOXL2 and pituitary hormones in these pituitary patients was examined by immunohistochemical staining and RT–PCR. Quantitative analysis of FOXL2 protein was performed by immunoblotting. FOXL2 was localized in the nuclei of ∼20% of normal pituitary cells that also co-expressed gonadotropins including follicule-stimulating hormone β (FSHβ), luteinizing hormone β (LHβ), and αGSU, whereas it was observed in minor proportion of thyroid-stimulating hormone (TSH)-producing cells, prolactin (PRL)-producing cells, and precursor of adrenocorticotropic hormone (ACTH)-producing cells. FOXL2 immunoreactivity was not detected in growth hormone (GH)-producing cells or S100-positive folliculo-stellate cells. In human pituitary adenomas, FOXL2 was expressed in the nuclei of the adenoma cells. FOXL2 was detected in 13 of 15 gonadotropin-subunit-producing adenoma (Gn-oma) cases and 8 of 11 null cell adenoma cases, but its incidence was reduced or not detected in the other types of adenomas. The results of this study suggest that FOXL2 contributes to the human-specific functional expression and the differentiation of gonadotroph cells and adenomas.

Abstract 4828: Genome-wide DNA methylation analysis identifies tumor-specifically methylated genes in non-small cell lung cancer patients

Abstract Epigenetic changes largely contribute to the regulation of gene expression in cancer cells. DNA methylation is part of this epigenetic gene regulation complex and it has been shown that methylation of certain genes occurs frequently in non-small cell lung cancers (NSCLC). So far, in primary NSCLCs mainly studies investigating methylation of small numbers of genes have been performed. Thus, we performed a genome-wide search for methylated CpG islands in primary tumors and corresponding non-malignant lung tissue samples of 100 stage I-III NSCLC patients by combining methylated DNA immunoprecipitation (MeDIP) and microarray analysis (MeDIP-chip). 385K Human CpG Island plus Promoter arrays from NimbleGen which cover ∼28,000 CpG islands were used. To test for differences in methylation between tumors and corresponding non-malignant lung tissues we calculated paired t-statistics and performed a multiplicity adjusted permutation test. By this approach we identified 303 tumor-specifically methylated genes. These genes include 263 well characterized protein encoding genes and 40 predicted genes. Tumor-specifically methylated genes were classified according to Gene Ontology (GO) categories and over-representation of certain GO terms was calculated. This analysis demonstrated that a large number of tumor-specifically methylated genes act as regulators of gene expression (including members of homeobox, forkhead box and paired box gene clusters) or mediate homophilic cell adhesion (including members of protocadherin alpha and gamma gene clusters). To confirm our results obtained by MeDIP-chip analysis we additionally performed methylation-sensitive high-resolution melting (MS-HRM) analysis of selected genes (e.g. HOXA2, SHOX2 and TAL1). These results corresponded with the results obtained by MeDIP-chip analysis. A comparison of our methylation results obtained by MeDIP-chip analysis and clinico-pathological characteristics of the patients will be performed. In conclusion, using a genome-wide approach we identified a large number of tumor-specifically methylated genes in NSCLC patients. From many of these genes epigenetic regulation was unknown so far. Moreover, our data suggest that transcriptional regulation and cell adhesion are frequently affected by DNA methylation in NSCLC. Overall, our results stress the importance of DNA methylation for the pathogenesis of NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4828. doi:10.1158/1538-7445.AM2011-4828

Update of human and mouse forkhead box (FOX) gene families

The forkhead box (FOX) proteins are transcription factors that play complex and important roles in processes from development and organogenesis to regulation of metabolism and the immune system. There are 50 FOX genes in the human genome and 44 in the mouse, divided into 19 subfamilies. All human FOX genes have close mouse orthologues, with one exception: the mouse has a single Foxd4, whereas the human gene has undergone a recent duplication to a total of seven (FOXD4 and FOXD4L1 → FOXD4L6). Evolutionarily ancient family members can be found as far back as the fungi and metazoans. The DNA-binding domain, the forkhead domain, is an example of the winged-helix domain, and is very well conserved across the FOX family and across species, with a few notable exceptions in which divergence has created new functionality. Mutations in FOX genes have been implicated in at least four familial human diseases, and differential expression may play a role in a number of other pathologies -- ranging from metabolic disorders to autoimmunity. Furthermore, FOX genes are differentially expressed in a large number of cancers; their role can be either as an oncogene or tumour suppressor, depending on the family member and cell type. Although some drugs that target FOX gene expression or activity, notably proteasome inhibitors, appear to work well, much more basic research is needed to unlock the complex interplay of upstream and downstream interactions with FOX family transcription factors.

Expression of Ol-foxi3 and Na +/K +-ATPase in ionocytes during the development of euryhaline medaka ( Oryzias latipes) embryos

Osmoregulation is a vital function that is essential to all vertebrates. Ionocytes are epithelial cells responsible for this function and have been extensively studied in adult teleost fish gills. The euryhaline medaka ( Oryzias latipes) has recently emerged as an investigative model because of its ability to acclimatize easily to water presenting various salinities. However, no studies to date have focused on the development of ionocytes in medaka embryos. We first analyzed the distribution of ionocytes in the skin and gills during development, using a specific marker of differentiated ionocytes (the Na +/K +-ATPase pump, or NKA). Strikingly, we were able to identify two ionocyte domains on the yolk surface ectoderm, that we named the Vitellin Zone (VZ) and the Lateral Zone (LZ). In zebrafish, ionocyte differentiation has been shown to be controlled by two forkhead-box genes, foxi3a and foxi3b. We cloned the medaka foxi3 ortholog which appeared to be highly similar to foxi3b. Whole-mount in situ hybridizations performed on medaka embryos revealed that Ol-foxi3 is expressed in differentiated ionocytes of the pharyngeal endoderm, the branchial arches and the yolk epidermis, as well as in epibranchial placode territories. We further focused on the expression patterns of the yolk epidermis and compared the expression of Ol-foxi3 with that of the non-neural progenitor marker p63. We evidenced that Ol-foxi3 is expressed in progenitor cells which are first of all located uniformly in the VZ and then transitorily clustered in the LZ. Taken together, these data contribute to a clearer understanding of osmoregulatory tissue ontogenesis in euryhaline fish.

HoxA-11 and FOXO1A Cooperate to Regulate Decidual Prolactin Expression: Towards Inferring the Core Transcriptional Regulators of Decidual Genes

BackgroundDuring the menstrual cycle, the ovarian steroid hormones estrogen and progesterone control a dramatic transcriptional reprogramming of endometrial stromal cells (ESCs) leading to a receptive state for blastocyst implantation and the establishment of pregnancy. A key marker gene of this decidualization process is the prolactin gene. Several transcriptional regulators have been identified that are essential for decidualization of ESCs, including the Hox genes HoxA-10 and HoxA-11, and the forkhead box gene FOXO1A. While previous studies have identified downstream target genes for HoxA-10 and FOXO1A, the role of HoxA-11 in decidualization has not been investigated. Here, we show that HoxA-11 is required for prolactin expression in decidualized ESC. While HoxA-11 alone is a repressor on the decidual prolactin promoter, it turns into an activator when combined with FOXO1A. Conversely, HoxA-10, which has been previously shown to associate with FOXO1A to upregulate decidual IGFBP-1 expression, is unable to upregulate PRL expression when co-expressed with FOXO1A. By co-immunoprecipitation and chromatin immunoprecipitation, we demonstrate physical association of HoxA-11 and FOXO1A, and binding of both factors to an enhancer region (−395 to −148 relative to the PRL transcriptional start site) of the decidual prolactin promoter. Because FOXO1A is induced upon decidualization, it serves to assemble a decidual-specific transcriptional complex including HoxA-11. These data highlight cooperativity between numerous transcription factors to upregulate PRL in differentiating ESC, and suggest that this core set of transcription factors physically and functionally interact to drive the expression of a gene battery upregulated in differentiated ESC. In addition, the functional non-equivalence of HoxA-11 and HoxA-10 with respect to PRL regulation suggests that these transcription factors regulate distinct sets of target genes during decidualization.

Involvement of a Forkhead Transcription Factor, FOXO1a, in UV-Induced Changes of Collagen Metabolism

Transcription factors belonging to the forkhead box gene, group O (FOXOs) family have been found to be crucial in downstream suppression of life-shortening effects of the insulin/insulin-like growth factor-1 (IGF-1) signaling pathway, which accelerates aging by suppressing FOXOs. Thus, FOXOs could hold the key for counteracting aging. Although FOXOs may play a critical role in aging, the effects of FOXOs on UV-induced changes of collagen metabolism by dermal fibroblasts are unknown. In this study, UV-induced changes in FOXO1a expression and the roles of FOXO1a in the regulation of collagen synthesis and matrix metalloproteinase (MMPs) expression in human dermal fibroblasts were investigated. In UVA- or UVB-irradiated fibroblasts, the expression of FOXO1A mRNA decreased significantly. The expression of type I collagen (COLIAI) also decreased. On the other hand, MMP-1 and MMP-2 mRNA levels increased. FOXO1A-small interfering RNA transfection induced the downregulation of FOXO1A expression, it also induced a decrease in COLIAI expression, and it increased MMP-1 and MMP-2 expression. These changes are similar to those observed in UV-irradiated fibroblasts. Furthermore, FOXO1a-peptide induced opposite changes in COLIAI, MMP-1, and MMP-2 expression. Therefore, FOXO1a is involved in the UV-induced changes of type I collagen and MMPs expression.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 60-62; doi:10.1038/jidsymp.2009.2.

FOXA1 Is a Potential Oncogene in Anaplastic Thyroid Carcinoma

FOXA1 is a mammalian endodermal transcription factor belonging to the human forkhead box gene family that plays a role in certain tumor types. Here, we investigated the potential role of FOXA1 in human thyroid carcinomas. We examined the level of FOXA1 expression and gene copy number by immunohistochemistry and fluorescence in situ hybridization, respectively, in a cohort of benign and malignant thyroid tumors. In addition, we examined the role of FOXA1 in the proliferation of an undifferentiated thyroid carcinoma cell line by short hairpin RNA-mediated silencing. We show that FOXA1 is overexpressed in human anaplastic thyroid carcinomas (ATC). In addition, we identify FOXA1 DNA copy number gain within the 14q21.1 locus in both an ATC cell line and human ATC cases. Silencing of FOXA1 in an ATC cell line causes G(1) growth arrest and reduction of cell proliferation. Moreover, we observe a potential link between FOXA1 and the cell cycle machinery by identifying p27(kip1) up-regulation on FOXA1 silencing. FOXA1 is overexpressed in aggressive thyroid cancers and involved in cell cycle progression in an ATC cell line. Therefore, FOXA1 may be an important oncogene in thyroid tumorigenesis and a potential new therapeutic target for the treatment of anaplastic thyroid cancers.

Evolutionary selection pressure of forkhead domain and functional divergence

Forkhead-box ( Fox) genes encode a family of transcription factors defined by a “winged helix” DNA-binding domain which have been identified in many metazoans, and play important roles in diverse biological processes. Here we aimed to extend previous evolutionary selection analysis to fungi, using available sequences from E. cuniculi (Ec), Eremothecium gossypii (Eg), Saccharomyces cerevisiae (Sc), etc. The phylogeny of 335 Fox protein sequences was reconstructed, revealing the existence of 26 orthologous groups that were well supported by gene phylogeny which arose following a series of gene duplication events. Gene conversion events may also play important roles in the evolution of Fox genes. The nonsynonymous to synonymous substitution ratios (d N/d S) for orthologous groups suggested that after gene duplication and/or speciation of forkhead clusters, rapid differentiation and the negative selection have occurred, prompting the formation of distinct Fox subclasses and new functions. SDPpred was used to produce a set of the alignment positions (specificity determining positions) which is involved in conferring differential functional specificity. These findings explained the functional divergence of Fox gene family.

The Medaka FoxP2, a Homologue of Human Language Gene FOXP2, has a Diverged Structure and Function

Forkhead box (Fox) genes are involved in organogenesis and cell differentiation. A mutation of FOXP2 was discovered in patients with severe defects in speech and language. The medaka FoxP2 was cloned in order to clarify the molecular evolution and difference in the protein structure and function by comparing human/mouse and medaka genes. The result showed that medaka FoxP2 had a 73.7% homology to the human and mouse counterparts, and its zinc finger, leucine zipper and forkhead domain structures were conserved. However, medaka FoxP2 lacked a long polyglutamine repeat and had two insertions of unique amino acid sequences. FoxP2 expression was found in the epiphysis and retina, in addition to the midbrain and cerebellum. The transcriptional assay revealed that medaka FoxP2 showed a very weak repressive activity to the CC10 promoter while mouse Foxp2 exhibited a strong repressive activity. Mutational analyses of medaka FoxP2 showed that the three amino acids of forkhead domain were responsible for the weak repressive activity. These results suggest that medaka FoxP2 may play a different function in the development of the medaka fish.

High-Throughput Analysis of Promoter Occupancy Reveals Direct Neural Targets of FOXP2, a Gene Mutated in Speech and Language Disorders

We previously discovered that mutations of the human FOXP2 gene cause a monogenic communication disorder, primarily characterized by difficulties in learning to make coordinated sequences of articulatory gestures that underlie speech. Affected people have deficits in expressive and receptive linguistic processing and display structural and/or functional abnormalities in cortical and subcortical brain regions. FOXP2 provides a unique window into neural processes involved in speech and language. In particular, its role as a transcription factor gene offers powerful functional genomic routes for dissecting critical neurogenetic mechanisms. Here, we employ chromatin immunoprecipitation coupled with promoter microarrays (ChIP-chip) to successfully identify genomic sites that are directly bound by FOXP2 protein in native chromatin of human neuron-like cells. We focus on a subset of downstream targets identified by this approach, showing that altered FOXP2 levels yield significant changes in expression in our cell-based models and that FOXP2 binds in a specific manner to consensus sites within the relevant promoters. Moreover, we demonstrate significant quantitative differences in target expression in embryonic brains of mutant mice, mediated by specific in vivo Foxp2-chromatin interactions. This work represents the first identification and in vivo verification of neural targets regulated by FOXP2. Our data indicate that FOXP2 has dual functionality, acting to either repress or activate gene expression at occupied promoters. The identified targets suggest roles in modulating synaptic plasticity, neurodevelopment, neurotransmission, and axon guidance and represent novel entry points into in vivo pathways that may be disturbed in speech and language disorders.

Expression of the forkhead transcription factor FOXP1 is associated with that of estrogen receptorβ in primary invasive breast carcinomas

We previously identified a correlation between estrogen receptor alpha (ERalpha) and the candidate tumour suppressor gene Forkhead Box P1 (FOXP1), whose nuclear protein expression in breast tumours was associated with improved patient survival. However, the expression pattern of FOXP1 in normal breast tissue is more reminiscent of the second receptor, ERbeta, which has an emerging role as a tumour suppressor in breast cancer and critically may underlie the ability of some ERalpha-negative tumours to respond to tamoxifen. In a series of 283 breast cancers, in which ERalpha-positive tumours were treated with tamoxifen, the nuclear expression of ERbeta correlated significantly with ERalpha (p = 0.004), low-tumour grade (p = 0.008) and nuclear FOXP1 (p = 0.01). High-grade tumours exhibited significantly more cytoplasmic ERbeta than the low-grade tumours (p = 0.006). Regression analysis demonstrated that FOXP1 expression was most closely related to nuclear ERbeta (p = 0.021). Neither, nuclear or cytoplasmic ERbeta expression demonstrated prognostic significance. FOXP1 is not estrogen regulated and silencing FOXP1 expression, using siRNA, did not affect ERalpha, ERbeta or progesterone receptor expression, suggesting ER and FOXP1 co-expression may reflect a common regulatory mechanism.

Variation in numbers of CD4+CD25highFOXP3+T cells with normal immuno-regulatory properties in long-term graft outcome

Chronic rejection (CR) is a major cause of long-term graft loss that would be avoided by the induction of tolerance. We previously showed that renal transplant patients with CR have lower numbers of peripheral CD4(+)CD25(high) T cells than operationally tolerant patients, patients with stable graft function and healthy volunteers (HV). We explored here the profile of CD4(+)CD25(high) blood T cells in these patients focusing on their expression of the regulatory T cells (Treg) gene Forkhead Box P3 (FOXP3) and their suppressive function. We show that CR is associated with a decreased number of CD4(+)CD25(high)FOXP3(+)T cells with normal regulatory profile, whereas graft acceptance is associated with CD4(+)CD25(high)FOXP3(+)T cell numbers similar to HVs. These data suggest that Treg numbers, rather than their intrinsic suppressive capacity, may contribute to determining the long-term fate of renal transplants.

The developmental expression of foxl2 in the dogfish Scyliorhinus canicula

The FoxL2 genes are a subfamily of the Fox (forkhead box) gene family. FOXL2 is mutated in the disorder Blepharophimosis, Ptosis, and Epicanthus Inversus Syndrome (BPES), which is characterized by eyelid malformations, and Premature Ovarian Failure (POF). In the mouse expression is seen in the perioptic mesenchyme, developing eyelids, ovary and pituitary. We have isolated a foxl2 cDNA from the dogfish Scyliorhinus canicula (also known as the lesser spotted catshark), allowing the characterisation of this gene’s sequence and expression from a lineage that diverged early in the evolution of gnathostomes. Molecular phylogenetic analysis strongly grouped this sequence with the gnathostomes within the FoxL2 subfamily. We demonstrate the early expression of Scyliorhinus canicula foxl2 in the mandibular head mesoderm and later in continuous populations of mandibular arch cells and mandibular head mesenchyme cells around the developing pituitary. As development proceeds expression decreases in the mesenchyme of the head but is seen in the mesenchyme around the eye and later in the developing eyelids. Additionally expression is seen in regions of pharyngeal arch mesoderm and in ectoderm from which gill buds will form. This expression is maintained in the developing and elongating gill buds. Thus, S. canicula foxl2 is a marker for the mandibular mesoderm and gill buds and its expression is conserved in the perioptic mesenchyme, developing eyelids and pituitary.