Thyroid Transcription Factor-2 Research Articles

Forkhead box E1, frequently downregulted by promoter methylation, inhibits colorectal cancer cell growth and migration

Forkhead box E1 (FOXE1), also known as thyroid transcription factor 2 (TTF-2), belongs to a large family of forkhead transcription factors. It plays important roles in embryogenesis, cell growth, and differentiation. Cancer-specific FOXE1 hypermethylation events have been identified in several cancers. However, the expression and function of FOXE1 in the tumorigenesis of colorectal cancer remain still unknown. In this study, we examined FOXE1 expression and methylation in normal colon mucosa, colorectal cancer (CRC) cell lines, and primary tumors by immunohistochemistry, semi-quantitative RT-PCR, methylation-specific PCR, and bisulfite genomic sequencing. We found that FOXE1 was frequently methylated and silenced in CRC cell lines and was downregulated in CRC tissues compared with paired adjacent non-tumor tissues. Meanwhile, low FOXE1 expression was significantly correlated with lymph node metastasis and advanced TNM stages, indicating its potential as a tumor marker. Subsequently, we established colon cancer cell lines with stable FOXE1 expression to observe the biological effect on colorectal cancer, including cell growth, migration, actin cytoskeleton, and growth of human colorectal xenografts in nude mice. Ectopic expression of FOXE1 could suppress tumor cell growth and migration and affect the organization of the actin cytoskeleton together with suppressing tumorigenicity in vivo. FOXE1 methylation was frequently seen in association with a complete absence of or downregulated gene expression, and FOXE1 plays a suppressive role in the development and progression of colorectal cancer.

Drinking water disinfection byproduct iodoacetic acid affects thyroid hormone synthesis in Nthy-ori 3–1 cells

Iodoacetic acid (IAA) is an emerging and the most genotoxic iodinated disinfection byproduct to date. IAA can disrupt the thyroid endocrine function in vivo and in vitro, but the underlying mechanisms remain unclear. In this work, transcriptome sequencing was used to investigate the effect of IAA on the cellular pathways of human thyroid follicular epithelial cell line Nthy-ori 3–1 and determine the mechanism of IAA on the synthesis and secretion of thyroid hormone (TH) in Nthy-ori 3–1 cells. Results of transcriptome sequencing indicated that IAA affected the TH synthesis pathway in Nthy-ori 3–1 cells. IAA reduced the mRNA expression of thyroid stimulating hormone receptor, sodium iodide symporter, thyroid peroxidase, thyroglobulin, paired box 8 and thyroid transcription factor-2, inhibited the cAMP/PKA pathway and Na+-K+-ATPase, and decreased the iodine intake. The results were confirmed by our previous findings in vivo. Additionally, IAA downregulated glutathione and the mRNA expression of glutathione peroxidase 1, leading to increased reactive oxygen species production. This study is the first to elucidate the mechanisms of IAA on TH synthesis in vitro. The mechanisms are associated with down-regulating the expression of genes related to TH synthesis, inhibiting iodine uptake, and inducing oxidative stress. These findings may improve future health risk assessment of IAA on thyroid in human.

Resveratrol Alleviates the Inhibitory Effect of Tunicamycin-Induced Endoplasmic Reticulum Stress on Expression of Genes Involved in Thyroid Hormone Synthesis in FRTL-5 Thyrocytes.

Recently, ER stress induced by tunicamycin (TM) was reported to inhibit the expression of key genes involved in thyroid hormone synthesis, such as sodium/iodide symporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG), and their regulators such as thyrotropin receptor (TSHR), thyroid transcription factor-1 (TTF-1), thyroid transcription factor-2 (TTF-2) and paired box gene 8 (PAX-8), in FRTL-5 thyrocytes. The present study tested the hypothesis that resveratrol (RSV) alleviates this effect of TM in FRTL-5 cells. While treatment of FRTL-5 cells with TM alone (0.1 µg/mL) for 48 h strongly induced the ER stress-sensitive genes heat shock protein family A member 5 (HSPA5) and DNA damage inducible transcript 3 (DDIT3) and repressed NIS, TPO, TG, TSHR, TTF-1, TTF-2 and PAX-8, combined treatment with TM (0.1 µg/mL) and RSV (10 µM) for 48 h attenuated this effect of TM. In conclusion, RSV alleviates TM-induced ER stress and attenuates the strong impairment of expression of genes involved in thyroid hormone synthesis and their regulators in FRTL-5 thyrocytes exposed to TM-induced ER stress. Thus, RSV may be useful for the treatment of specific thyroid disorders, provided that strategies with improved oral bioavailability of RSV are applied.

Functions of stem cells of thyroid glands in health and disease.

Thyroid gland has been implicated in the regulation of many functions using endocrine, paracrine and autocrine signals. Functional thyroid follicular cells derived from stem cells attracted a great interest from researchers as a strategy for thyroid's regenerative therapy. Thyroid has a very low rate of turnover; however, studies showed that the regenerative ability is enhanced following diseases or thyroidectomy, which promotes the role of stem cell. The objective of this review is to summarize the morphological characterization and the expression of stem cell genes/markers in the thyroid. Also, to highlight the mechanisms of tumor formation in thyroid via its stem cells. The most important thyroid stem cell's markers are: stem cell antigen 1 (SCA-1), octamer-binding transcription 4 (OCT-4), p63, CD34+ CD45-, paired box gene 8 (PAX-8), thyroid transcription factor 1 (TTF-1), thyroid transcription factor 2 (TTF-2), hematopoietically expressed homeobox protein HHEX, the transcription factor GATA-4, hepatocyte nuclear factor 4-α (HNF-4-α) and homeobox transcription factor Nanog (hNanog). This review highlights the functional characterization describing the mechanisms of stem cell's differentiation into functional thyroid follicle and proposing mechanisms involving in cancer formation through one of these cell types: fetal cell, thyroblasts, prothyrocytes, certain genetic mutation in the mature thyroid cells or presence of a special type of cells (cancer stem cell) which are responsible for different types of cancer formation. Understanding the mechanisms of thyroid's stem cell in cancer formation and the expression of the biomarkers in normal and abnormal thyroid status are promising physiological tools in promoting thyroid regeneration and in provision management for thyroid cancer.

Biological characteristics of cleft palate relevant gene thyroid transcription factor-2 transgenic mice

The aim of this study is to establish a transgenic mouse model for cleft palate relevant gene thyroid transcription factor-2 (TTF-2), which can be used to study palatal shelf development when the expression pattern and regular activation of TTF-2 is altered. The C57BL/6J mouse TTF-2 gene was cloned through polymerase chain reaction (PCR) from the mouse genomic DNA. The TTF-2 gene was inserted into the expression vector pBROAD3-mcs to construct the recombinant expression vector pBROAD3-TTF-2. This expression vector was then microinjected into the male pronuclei of the fertilized mouse ovum. Thus, the TTF-2 transgenic mice model was established. The genotype of the transgenic mice was identified by PCR and Southern blot analysis. Immunohistochemistry identified the consistent expression of TTF-2 gene during its palatal shelf development. TTF-2 genes were microinjected into 982 fertilized ova. A total of 580 two-cell-stage embryos cultured and transplanted into the oviducts of 48 pseudopregnant female mice. Overall, 68 embryos were obtained for analysis. The genotype of the mice was determined through PCR and Southern blot analysis using genomic DNA extracted from tail biopsies of the transgenic fetus. A total of 13 TTF-2 transgenic mice were detected. The expression of TTF-2 gene during the palatal shelf development of the transgenic mice was consistently detected by immunohistochemistry. The recombinant expression vector pBROAD3-TTF-2 was integrated into mouse genome through microinjection. The transgenic mouse in the palatal shelf that consistently expressed TTF-2 was successfully established and displayed a cleft palate phenotype.

Genetic analysis of TTF2 gene in congenital hypothyroid infants with thyroid dysgenesis

Thyroid transcription factor 2 (TTF2) also known as FOXE1 is one of the candidate genes thought to has been involved in thyroid development. Impairment in this gene has been reported in a few cases of patients with congenital hypothyroidism resulting from thyroid dysgenesis (TD). In this study we analyzed the entire coding-region of TTF2 genes in 50 infants who were referred to the Endocrine and Metabolism Research Center of Isfahan University of Medical Sciences TD patients by direct sequencing. The analysis revealed a known polymorphism in ser 273 (TCC.TCT) in 74% unrelated patients. Furthermore, we found that the length of the alanine tract of TTF2 was 14 in some of our TD patients. This data may point to a role of the TTF2 polyA tract length in modulating genetic susceptibility to TD. doi: http://dx.doi.org/10.12669/pjms.291(Suppl).3526 How to cite this:Mahjoubi F, Hashemipour M, Moshiri F, Iranpour R, Amini M, Hovsepian S, et al. Genetic analysis of TTF2 gene in congenital hypothyroid infants with thyroid dysgenesis. Pak J Med Sci 2013;29(1)Suppl:325-328. doi: http://dx.doi.org/10.12669/pjms.291(Suppl).3526 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Overexpression of mouse TTF‐2 gene causes cleft palate

In humans, mutations of the gene encoding for thyroid transcription factor-2 (TTF-2 or FOXE1) result in Bamforth syndrome. Bamforth syndrome is characterized by agenesis, cleft palate, spiky hair and choanal atresia. TTF-2 null mice (TTF-2−/−) also exhibit cleft palate, suggesting its involvement in the palatogenesis. However, the molecular pathology and genetic regulation by TTF2 remain largely unknown. In the present study, the recombinant expression vector pBROAD3-TTF-2 containing the promoter of the mouse ROSA26 gene was created to form the structural gene of mouse TTF-2 and was microinjected into the male pronuclei of fertilized ova. Sequence analysis confirmed that the TTF-2 transgenic mouse model was established successfully. The transgenic mice displayed a phenotype of cleft palate. In addition, we found that TTF-2 was highly expressed in the medial edge epithelium (MEE) from the embryonic day 12.5 (E12.5) to E14.5 in TTF-2 transgenic mice. These observations suggest that overexpression of TTF-2 during palatogenesis may contribute to formation of cleft palate.

Suppression of estrogen receptor-alpha transactivation by thyroid transcription factor-2 in breast cancer cells

Estrogen receptors (ERs), which mediate estrogen actions, regulate cell growth and differentiation of a variety of normal tissues and hormone-responsive tumors through interaction with cellular factors. In this study, we show that thyroid transcription factor-2 (TTF-2) is expressed in mammary gland and acts as ERα co-repressor. TTF-2 inhibited ERα transactivation in a dose-dependent manner in MCF-7 breast cancer cells. In addition, TTF-2 directly bound to and formed a complex with ERα, colocalizing with ERα in the nucleus. In MCF-7/TTF-2 stable cell lines, TTF-2 repressed the expression of endogenous ERα target genes such as pS2 and cyclin D1 by interrupting ERα binding to target promoters and also significantly decreased cell proliferation. Taken together, these data suggest that TTF-2 may modulate the function of ERα as a corepressor and play a role in ER-dependent proliferation of mammary cells.

Hesperetin Activates The Notch1 Signaling Cascade, Induces Cellular Differentiation, And Causes Apoptosis In Anaplastic Thyroid Cancer

Introduction: Anaplastic thyroid cancer (ATC) is characterized by aggressive growth and extremely poor prognosis, for which there is no standardized therapy. In addition, the undifferentiated feature of ATC in which sodium iodide symporter (NIS) expression is reduced makes treatment such as radioactive iodine ineffective. Therefore, there is an urgent need for strategies that sensitize ATC cells to iodine treatment. It has been reported that the Notch1 signaling pathway, which affects cell proliferation and differentiation, is inactivated in ATC. However, it is unknown whether activation of Notch1 could yield therapeutic effects in ATC. Therefore, in this study we evaluated whether Hesperetin, a naturally occurring flavanone and potential Notch activator, effects ATC cell proliferation and possibly up-regulates thyroid-specific differentiation markers such as NIS. Methods: In-vitro studies were carried out to evaluate the drug effects using a human derived ATC cell line (HTH7). To determine whether Hesperetin activates Notch1 in ATC, expression of Notch1 mRNA and protein was evaluated upon treatment. In addition, mRNA levels of Notch1 response genes (Hes1 and Hey1) were measured, and the Notch1 activity was assessed using CBF-1-luc binding assay. Cellular proliferation was measured by viable cell counts every 24 hours post-treatment (0-200mM) for 72 hours, and the underlying mechanism was investigated by western blot. Expression of thyroid-specific differentiation markers including Thyroid Transcription Factor 1 (TTF-1), Thyroid Transcription Factor 2 (TTF-2), Paired Box Gene 8 (PAX-8), Thyroid Stimulating Hormone Receptor (TSHR), and NIS were also measured. Results: Hesperetin caused activation of the Notch1 signaling pathway. Treated ATC cells displayed elevated Notch1 protein and increased mRNA levels of Hes1 and Hey1, important Notch1 response genes. Treatment induced a dose-dependent increase in Notch1 activity as evidenced by increased luciferase activity. Hesperetin treatment also resulted in a time and dose-dependent decrease in cell proliferation: at 72 hours, 50mM and 100mM Hesperetin resulted in a 27% and 47% reduction respectively compared to control. Protein expression of apoptotic markers cleaved PARP, cleaved Caspase-3, and BAD increased with treatment. Importantly, Hesperetin up-regulated NIS mRNA levels, a thyroid-specific marker responsible for sensitivity to radioactive iodine. Other thyrocyte markers (TTF-1, TTF-2, PAX-8 and TSHR) were also increased, further evidence that Hesperetin induces differentiation in ATC. Conclusions: Hesperetin activates the Notch1 signaling cascade and has a significant antiproliferative effect on ATC that can be largely attributed to apoptosis. Hesperetin also induces differentiation in ATC, and by up-regulating markers such as NIS, could be useful in combination with radioactive iodine for treating ATC. Further investigation to elucidate the in-vivo effects of Hesperetin on ATC is warranted.

Hesperetin Activates the Notch1 Signaling Cascade, Induces Cellular Differentiation, and Causes Apoptosis in Anaplastic Thyroid Cancer

Introduction: Anaplastic thyroid cancer (ATC) is characterized by aggressive growth and extremely poor prognosis, for which there is no standardized therapy. in addition, the undifferentiated feature of ATC in which sodium iodide symporter (NIS) expression is reduced makes treatment such as radioactive iodine ineffective. Therefore, there is an urgent need for strategies that sensitize ATC cells to iodine treatment. It has been reported that the Notch1 signaling pathway, which affects cell proliferation and differentiation, is inactivated in ATC. However, it is unknown whether activation of Notch1 could yield therapeutic effects in ATC. Therefore, in this study we evaluated whether Hesperetin, a naturally occurring flavanone and potential Notch activator, effects ATC cell proliferation and possibly up-regulates thyroid-specific differentiation markers such as NIS. Methods: In-vitro studies were carried out to evaluate the drug effects using a human derived ATC cell line (HTH7). to determine whether Hesperetin activates Notch1 in ATC, expression of Notch1 mRNA and protein was evaluated upon treatment. in addition, mRNA levels of Notch1 response genes (Hes1 and Hey1) were measured, and the Notch1 activity was assessed using CBF-1-luc binding assay. Cellular proliferation was measured by viable cell counts every 24 hours post-treatment (0-200mM) for 72 hours, and the underlying mechanism was investigated by western blot. Expression of thyroid-specific differentiation markers including Thyroid Transcription Factor 1 (TTF-1), Thyroid Transcription Factor 2 (TTF-2), Paired Box Gene 8 (PAX-8), Thyroid Stimulating Hormone Receptor (TSHR), and NIS were also measured. Results: Hesperetin caused activation of the Notch1 signaling pathway. Treated ATC cells displayed elevated Notch1 protein and increased mRNA levels of Hes1 and Hey1, important Notch1 response genes. Treatment induced a dose-dependent increase in Notch1 activity as evidenced by increased luciferase activity. Hesperetin treatment also resulted in a time and dose-dependent decrease in cell proliferation: at 72 hours, 50mM and 100mM Hesperetin resulted in a 27% and 47% reduction respectively compared to control. Protein expression of apoptotic markers cleaved PARP, cleaved Caspase-3, and BAD increased with treatment. Importantly, Hesperetin up-regulated NIS mRNA levels, a thyroid-specific marker responsible for sensitivity to radioactive iodine. Other thyrocyte markers (TTF-1, TTF-2, PAX-8 and TSHR) were also increased, further evidence that Hesperetin induces differentiation in ATC. Conclusions: Hesperetin activates the Notch1 signaling cascade and has a significant antiproliferative effect on ATC that can be largely attributed to apoptosis. Hesperetin also induces differentiation in ATC, and by up-regulating markers such as NIS, could be useful in combination with radioactive iodine for treating ATC. Further investigation to elucidate the in-vivo effects of Hesperetin on ATC is warranted.

Increasing Incidence of Congenital Hypothyroidism: Some Answers, More Questions

In 2007, Harris and Pass (1) reported that the incidence (birth prevalence) of congenital hypothyroidism (CH) detected by newborn screening programs in the United States had nearly doubled over the previous two decades, increasing from 1:3985 (in 1987) to 1:2273 (in 2002). In an analysis of data from their state of New York, Harris and Pass (1) found an even higher incidence, increasing from 1:3373 (in 1978) to 1:1415 (in 2005). The report of an apparent doubling was not limited to the United States. Newborn screening programs in Western Australia (1: 5747 to 1:2825) (2), Italy (1:2654 to 1:1154) (3), the northern region of the United Kingdom (1:2702 to 1:1078) (4), and Greece (1:3384 to 1:1749) (5) also reported an increasing incidence of CH. After these reports, questions naturally arose. Was this increasing incidence real, signaling an increase in the underlying etiology(ies) of CH? Or was the increasing incidence an artifact, explained by modifications of screening programs such as a change in test cutoffs? Furthermore, did the additional babies benefit from detection and early treatment? What do we know about the etiology of CH? CH is a heterogeneous disorder, with a spectrum of underlying etiologies. The most common cause is thyroid dysgenesis (ectopy, aplasia, hypoplasia, or hemiagenesis); historically, dysgenesis accounts for approximately 85% of cases. The next most common cause is thyroid dyshormonogenesis (defects in thyroid hormone biosynthesis), accounting for 10–15% of cases. Defects in thyroid hormone biosynthesis are familial, generally inherited in an autosomal recessive manner. Determination of an underlying defect is generally made by an imaging study, either ultrasonography or thyroid scintigraphy. Although imaging can determine thyroid anatomy and, if radionuclide uptake is done, some information on function, it does not elucidate the exact etiology. In fact, the underlying etiology of thyroid dysgenesis remains unknown for the vast majority of cases. Mutations in transcription factors that regulate thyroid gland development [thyroid transcription factor 2 (TTF-2), NKX2.1 (also termed TTF-1), or PAX-8] are found in only 2% of cases shown to have thyroid dysgenesis by imaging (6). Most cases of thyroid dysgenesis appear to be sporadic. It has long been known that there is a 2:1 female-to-male ratio in thyroid dysgenesis. Although this would seem to be a clue to the underlying etiology, its significance remains unknown. Previous studies from the screening program in Quebec report that the incidence of thyroid dysgenesis has remained relatively stable over the last few decades (7). Of note, with reports of increasing incidence, imaging studies now find a thyroid problem not reported in older studies—a normal-appearing thyroid gland in a eutopic location, so-called “thyroid in situ” (3, 5). Because this is associated with hypothyroidism, thyroid in situ is thought to be the result of a “functional abnormality.” Some cases of thyroid in situ have been shown to be the result of resistance to TSH binding (TSH receptor mutation), or signaling (G protein defect) (8), or to mild forms of dyshormonogenesis (hydrogen peroxide generation or maturation defects) (9). However, the exact etiology remains unknown for most cases. Hypothyroidism tends to be milder with thyroid in situ, and some cases are transient, with recovery to euthyroidism (9). Other cases of transient CH have a known etiology. Examples include babies born to mothers with Graves’ disease on antithyroid drugs, babies exposed to excess iodine (either maternal transmission or postnatal, usually from topical iodine antiseptics), and babies born to mothers with autoimmune thyroid disease

An upstream regulatory element confers orientation-independent enhancement of the TSG101 promoter activity in transformed cells

Tumor susceptibility gene 101 (TSG101), a mammalian homologue of yeast vps23, is involved in protein sorting, vesicular trafficking and maintenance of genomic integrity. Upregulation of the TSG101 gene was found in human thyroid papillary and breast tumors. Here, we define the proximal promoter of human TSG101 at -1 to -436 by reporter assay. Intact Sp1 and MAZ binding sequences within this region are essential, and mutation of both sites eliminates proximal promoter activity implying cooperation between these two cis-elements. Chromatin immunoprecipitation and DNA affinity precipitation assay confirmed in vivo Sp1 binding on the GGGGCGGGTT sequence. MAZ protein was essential for TSG101 promoter activity because its knockdown using siRNA decreased reporter activity. An upstream regulatory element (URE) at the -1280 to -1757 region was identified to confer the orientation-independent enhancement of the promoter activity in transformed COS-1, ARO and WRO cell lines but not in a normal thyroid FRTL cell line. The sequence of this URE region contains putative binding sites for thyroid transcription factor 2 (TTF-2) and thyroid hormone receptor (T3R), which might be relevant to differential regulation of TSG101 promoter activity in transformed and primary cells.

Human sodium–iodide symporter (hNIS) gene expression is inhibited by a trans-active transcriptional repressor, NIS-repressor, containing PARP-1 in thyroid cancer cells

Radioiodine remains the only tumoricidal therapy for disseminated thyroid carcinomas; however, dedifferentiated tumors lose the expression of human sodium-iodide symporter (hNIS) gene, and cannot respond to this treatment. Previous studies suggested that a trans-active protein factor (NIS-repressor) represses endogenous hNIS transcription, likely contributing to the loss of radioiodine uptake, and defined the NIS-repressor binding site (NRBS) in the proximal hNIS promoter. Using electrophoretic mobility shift assay (EMSA), we found evidence of NIS-repressor in the nuclear extract from KAK-1 cells, and confirmed this result using nuclear extracts prepared from multiple verified thyroid cell lines. Luciferase reporter assays of hNIS promoter constructs and EMSA were used to define two core sequences, NRBS-P and NRBS-D, in the hNIS promoter as the binding sites for NIS-repressor. Electrophoretic analysis of KAK-1 nuclear extract proteins cross-linked with NRBS-P suggests that NIS-repressor is a protein complex. Analysis of KAK-1 nuclear extract proteins bound to NRBS-P, via liquid chromatography coupled with tandem mass spectroscopy, demonstrated poly(ADP-ribose) polymerase-1 (PARP-1) as a NIS-repressor component. Pharmacological inhibition of PARP-1 enzymatic activity using PJ34 stimulated both the luciferase reporter activity driven by hNIS promoter and the endogenous hNIS mRNA level. Supershift studies suggest that thyroid transcription factor 2 (TTF-2) is also associated with the NIS-repressor complex. NIS-repressor, including its PARP-1 component, presents a potential therapeutic target to restore radioiodine uptake in dedifferentiated thyroid carcinomas.

Thyroid Development—Revisiting a Foxeing Case

Migration and organogenesis of the embryonic thyroid gland is dependent on transcription factors regulating expression of target genes involved in its differentiation and function. Thyroid transcription factor 2 (TTF-2), first cloned in the rat, contains a central forkhead domain and regulates both thyroglobulin and thyroperoxidase gene transcription (1). Independently, the FOXE1 gene (formerly FKHL15), which also contains a central forkhead domain, was identified in humans (2). We observed that the predicted amino acid sequence of FOXE1 was very similar to rodent Ttf-2, suggesting that this protein is its human homologue.

Identification of the Transcriptional Targets of FOXP2, a Gene Linked to Speech and Language, in Developing Human Brain

Mutations in FOXP2, a member of the forkhead family of transcription factor genes, are the only known cause of developmental speech and language disorders in humans. To date, there are no known targets of human FOXP2 in the nervous system. The identification of FOXP2 targets in the developing human brain, therefore, provides a unique tool with which to explore the development of human language and speech. Here, we define FOXP2 targets in human basal ganglia (BG) and inferior frontal cortex (IFC) by use of chromatin immunoprecipitation followed by microarray analysis (ChIP-chip) and validate the functional regulation of targets in vitro. ChIP-chip identified 285 FOXP2 targets in fetal human brain; statistically significant overlap of targets in BG and IFC indicates a core set of 34 transcriptional targets of FOXP2. We identified targets specific to IFC or BG that were not observed in lung, suggesting important regional and tissue differences in FOXP2 activity. Many target genes are known to play critical roles in specific aspects of central nervous system patterning or development, such as neurite outgrowth, as well as plasticity. Subsets of the FOXP2 transcriptional targets are either under positive selection in humans or differentially expressed between human and chimpanzee brain. This is the first ChIP-chip study to use human brain tissue, making the FOXP2-target genes identified in these studies important to understanding the pathways regulating speech and language in the developing human brain. These data provide the first insight into the functional network of genes directly regulated by FOXP2 in human brain and by evolutionary comparisons, highlighting genes likely to be involved in the development of human higher-order cognitive processes.

TTF-2/FOXE1 gene polymorphisms in Sicilian patients with permanent primary congenital hypothyroidism

Thyroid transcription factor-2 (TTF-2/FOXE1) is a polyalanine domain protein that regulates thyroid embryogenesis, but very few patients with permanent primary congenital hypothyroidism (pCH) harbor germline mutations of this or other transcription factors that are involved in thyroid development that might explain the etiology of pCH. Variations within the polyalanine tract are found in a variety of genes and are often reported in association with malformation syndromes; pCH is frequently associated with thyroid malformations and extra-thyroidal malformations. Therefore, in this study we investigated whether alanine (Ala) length polymorphisms and non-polymorphic mutations of the TTF-2 gene in pCH patients might be involved in the pathogenesis of pCH. The entire coding region of the TTF-2 gene was analyzed in 57 Sicilian patients and 142 healthy controls. We found that the homozygous Ala14 polymorphism (Ala14/14) was less frequent in the pCH group than in the controls. In contrast, significantly more pCH patients than controls harbored the Ala16 polymorphism (Ala16/16 and Ala14/16). However, neither the Ala14/14 nor the Ala16 polymorphism was related to extra-thyroidal malformations. Two of the 57 patients carried Ala11/14 and Ala12/14, and one Ala14/14 patient also had the silent polymorphism 387 C/T (Leu129Leu). Other than known polymorphic variants we found no mutation in the TTF-2 gene. Therefore, this study demonstrates that mutations in the TTF-2 gene are rare in pCH patients and suggests that variations in the length of the Ala-tract could at least partially explain the etiology of pCH but not that of extra-thyroidal malformations.

A Novel Missense Mutation in Human TTF-2 (FKHL15) Gene Associated with Congenital Hypothyroidism But Not Athyreosis

Thyroid dysgenesis is the most frequent cause of congenital hypothyroidism (CH), and its genetic basis is largely unknown. Hitherto, two mutations in the human thyroid transcription factor 2 (TTF-2) gene have been described in unrelated cases of CH with cleft palate, spiky hair, variable choanal atresia, and complete thyroid agenesis. Here, we describe a novel TTF-2 mutation in a female child resulting in syndromic CH in the absence of thyroid agenesis. The index case is homozygous for an arginine to cysteine mutation (R102C) of a highly conserved residue within the forkhead, DNA binding domain of TTF-2. Her consanguineous, heterozygous parents are unaffected, and the mutation was not detected in 100 control chromosomes. Consonant with its location, the R102C mutant TTF-2 protein showed loss of DNA binding and was transcriptionally inactive. CH in the proposita was associated with cleft palate, spiky hair, and bilateral choanal atresia. However, radiological studies showed the presence of thyroid tissue in a eutopic location. Our findings indicate that human thyroid development can occur despite loss of TTF-2 function and suggest that TTF-2 gene defects should also be considered in cases of syndromic CH without total athyreosis.

Restricted expression of NADPH oxidase/peroxidase gene (Duox) in zone VII of the ascidian endostyle

The ascidian Ciona intestinalis, a marine invertebrate chordate, is an emerging model system for developmental and evolutionary studies. The endostyle, one of the characteristic organs of ascidians, is a pharyngeal structure with iodine-concentrating and peroxidase activities and is therefore considered to be homologous to the follicular thyroid of higher vertebrates. We have previously reported that a limited part of the endostyle (zone VII) is marked by the expression of orthologs of the thyroid peroxidase (TPO) and thyroid transcription factor-2 (TTF-2/FoxE) genes. In this study, we have identified the Ciona homolog of NADPH oxidase/peroxidase (Duox), which provides hydrogen peroxide (H(2)O(2)) for iodine metabolism by TPO in the vertebrate thyroid. Expression patterns assessed by in situ hybridization have revealed that Ciona Duox (Ci-Duox) is predominantly expressed in the dorsal part of zone VII of the endostyle. Furthermore, two-color fluorescent in situ hybridization with Ci-Duox and Ciona TPO (CiTPO) has revealed that the ventral boundary of the Ci-Duox domain of expression is more dorsal than that of CiTPO. We have also characterized several genes, such as Ci-Fgf8/17/18, 5HT7, and Ci-NK4, which are predominantly expressed in the ventral part of zone VII, in a region complementary to the Ci-Duox expression domain. These observations suggest that, at the molecular level, zone VII has a complex organization that might have some impact on the specification of cell types and functions in this thyroid-equivalent element of the ascidian endostyle.

Immunohistochemical analysis of thyroid‐specific transcription factors in thyroid tumors

Thyroid transcription factor 1 (TTF1), thyroid transcription factor 2 (TTF2) and paired box gene 8 (Pax8) are demonstrated to play a crucial role for the differentiation and organogenesis of thyroid follicular cells. Their roles in thyroid carcinogenesis are not very clear. Because dedifferentiation is a common process in thyroid carcinogenesis, thyroid-specific transcription factors seem also to be involving in thyroid carcinogenesis. The purpose of the present paper was to investigate their expression in a broad spectrum of follicular cell tumors in different degrees of differentiation, from well-differentiated benign follicular adenoma to anaplastic carcinoma. Medullary (C cell) carcinoma was also included in the investigation. Results of immunohistochemical staining showed that nuclear localization of these transcription factors was gradually decreased corresponding to the progressive dedifferentiation of thyroid tumors. Also, abnormal cytoplasmic accumulation of TTF2 and Pax8 was detected in many tumors samples, which may indicate a subtle regulation mechanism on the function of these transcription factors. In conclusion, abnormal expression of TTF1, TTF2 and Pax8 was closely related to thyroid tumorigenesis.

TTF-2 stimulates expression of 17 genes, including one novel thyroid-specific gene which might be involved in thyroid development

Thyroid dysgenesis is the most frequent cause of congenital hypothyroidism, but its molecular pathophysiology is largely unknown. Our hypothesis that some genes downstream to thyroid transcription factor-2 (TTF-2) might be responsible for development of the thyroid prompted us to identify genes whose expression is stimulated by TTF-2. PCR products of cDNA clones obtained by a subtraction PCR method in TTF-2 expressing cell lines were screened with labeled cDNA by microarray analysis. We isolated 17 genes up-regulated by TTF-2, which were subsequently confirmed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). One of them is a novel gene designated T1560 that showed a highly thyroid-specific expression pattern. Luciferase reporter assays showed that expression of all of the 14 genes tested was stimulated by both TTF-2 and TTF-1, another thyroid-specific transcription factor. Our results have important implications for understanding normal thyroid development as well as the molecular defects underlying thyroid dysgenesis.