Thyroid Transcription Factor-2 Research Articles

Nuclear localization domains in human thyroid transcription factor 2

Thyroid transcription factor-2 (TTF2) is a nuclear protein involved in morphogenesis and gene expression in thyroid gland, belonging to the family of the forkhead/winged-helix transcription factors. In the present study we have investigated the sequence determinants for transport and accumulation into the nucleus of the TTF2 protein. By transient expression of fusion proteins constructed by joining different parts of TTF2 to the reporter gene of the jellyfish green fluorescent protein (GFP) and, in a separate set of deleted constructs, the glutathione S-transferase (GST) coding sequence, we have demonstrated that a basic amino acid stretch present at both ends of the DNA-binding domain is a bona fide nuclear localization signal (NLS). We have analyzed the subcellular localization of deleted GFP-GST-TTF2 fusion proteins and have shown that residues inside the forkhead domain (FHD) contributed to the complete nuclear TTF2 protein accumulation. Furthermore, by means of GST binding assays we have shown that distinct TTF2 fragments, containing the NLS, were able to bind the nuclear import receptor importin α. Taken together, our results provide the first documentation about nuclear targeting of a forkhead protein containing two identical NLS signal flanking the DNA-binding domain.

Production and Application of Polyclonal Antibody to Human Thyroid Transcription Factor 2 Reveals Thyroid Transcription Factor 2 Protein Expression in Adult Thyroid and Hair Follicles and Prepubertal Testis

Germline mutations in thyroid transcription factor 2 (TTF2) cause thyroid agenesis, spiky hair, and cleft palate, indicating thyroidal and extrathyroidal expression. We sought to investigate this by producing and applying an antibody to human TTF2. The coding region of human TTF2 was cloned into a bacterial expression vector, production of the soluble TTF2 protein optimized, and pure TTF2 obtained by nickel chromatography. Rabbits were immunized and the resulting TTF2 polyclonal titrated on formalin-fixed, paraffin-embedded sections of thyroid. The optimized protocol was applied to a range of tissues. Nine milligrams of TTF2 protein was obtained per liter of culture and a high-titer antibody produced. This displayed specific staining of thyroid follicular cell nuclei/cytoplasm and not of the interstitium, connective tissue, smooth muscle, or endothelium. No staining was obtained with the preimmune serum in the same conditions, or with the majority of other tissues tested with the TTF2 polyclonal. The exceptions were testis and skin, in which nuclear TTF2 immunoreactivity was present in the seminiferous tubules and cells in the follicular outer root sheath, respectively. In conclusion, we have produced a polyclonal antibody for human TTF2 and demonstrated immunoreactivity for this transcription factor in adult human thyroid and hair follicles and prepubertal testis.

Distribution of the titf2/foxe1 gene product is consistent with an important role in the development of foregut endoderm, palate, and hair.

Titf2/foxe1 is a forkhead domain-containing gene expressed in the foregut, in the thyroid, and in the cranial ectoderm of the developing mouse. Titf2 null mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. In humans, mutations of the gene encoding for thyroid transcription factor-2 (TTF-2) result in the Bamforth syndrome, characterized by thyroid agenesis, cleft palate, spiky hair, and choanal atresia. Here, we report a detailed expression pattern of TTF-2 protein during mouse embryogenesis and show its presence in structures where it has not been described yet. At embryonic day (E) 10.5, TTF-2 is expressed in Rathke's pouch, in thyroid, and in the epithelium of the pharyngeal wall and arches, whereas it is absent in the epithelium of the pharyngeal pouches. According to this expression, at E13.5, TTF-2 is present in endoderm derivatives, such as tongue, palate, epiglottis, pharynx, and oesophagus. Later in embryogenesis, we detect TTF-2 in the choanae and whiskers. This pattern of expression helps to define the complex phenotype displayed by human patients. Finally, we show that TTF-2 is a phosphorylated protein. These results help to characterize the domains of TTF-2 expression, from early embryogenesis throughout organogenesis, providing more detail on the potential role of TTF-2 in the development of endoderm and ectoderm derived structures.

Polymorphism of the polyalanine tract of thyroid transcription factor-2 gene in patients with thyroid dysgenesis.

One of the thyroid-specific transcription factors, thyroid transcription factor-2 (TTF-2), performs a crucial role in the development of the thyroid gland. We performed genetic analysis of the TITF2 gene (encoding TTF-2) in patients with thyroid dysgenesis. By direct sequencing of the PCR products of TITF2, we screened the genomic DNA from 46 patients with thyroid dysgenesis (five had agenesis, six had hypoplasia, 15 had ectopy, and 20 were undetermined). We also studied the transcriptional activities of TITF2 by co-expressing the luciferase gene directed by the human thyroglobulin gene promoter. Human TITF2 consists of a forkhead domain, a polyalanine tract, and unique C-terminal residues. In one of the patients with an ectopic sublingual thyroid, we found a polyalanine tract of 11 alanine residues on one chromosome instead of the 14 alanine residues found in normal controls. In one patient with hypoplasia, the polyalanine tract consisted of 12 heterozygous alanine residues. The reduced polyalanine tracts were not detected in 101 normal individuals. However, the expression study showed that the transcriptional activities of TITF2 with reduced polyalanine-tract lengths were equal to that of TITF2 with an unreduced polyalanine tract. These results suggest that the polymorphism of the polyalanine tract of TITF2 is not a frequent cause of developmental defects of the human thyroid gland.

The Thyroid Transcription Factor 2 (TTF-2) Is a Promoter-Specific DNA-Binding Independent Transcriptional Repressor

The thyroid transcription factor TTF-2 is a forkhead-containing protein involved in thyroid-specific gene expression and necessary for thyroid morphogenesis. In this paper, we demonstrate that TTF-2 is able to inhibit the activity of the thyroid-specific transcription factors TTF-1 and Pax-8 only on certain promoters. We identified the minimal protein domain responsible for repressor activity, which behaves as an independent functional domain, and we show that repression by TTF-2 is DNA-binding independent. We suggest that TTF-2 is able to interfere with a specific cofactor required for TTF-1 and Pax-8 activity.

Sequence Analysis of Thyroid Transcription Factor-2 (TTF-2) Gene in Ten Patients with Congenital Hypothyroidism due to Thyroid Dysgenesis.

The etiology of thyroid dysgenesis is still unknown. One of the thyroid specific transcription factors, thyroid transcription factor-2 (TTF-2), performs a crucial role in the development of thyroid gland as shown by TTF-2 knock out mice. In the present study, we analyzed the TTF-2 gene in patients with congenital hypothyroidism due to thyroid malformation: three patients with thyroid agenesis, two with hypoplasia, and five with ectopy. Genomic DNA was isolated from peripheral leukocytes, and the TTF-2 gene, consisting of a single exon with extremely high GC content, was amplified by polymerase chain reaction (PCR) with 2 pairs of primers. The PCR products were directly sequenced. One of the patients with ectopic thyroid showed the deletion of 9 bases (GCCGCCGCC), which corresponded to 3 alanine repeats, at the region of 1, 226-1, 234 bp in one chromosome. Also in the patient’s mother the same 9 bases deletion was noted at the same position. However, the mother had normal thyroid function and 99mTc thyroid scintigraphy revealed no abnormalities of thyroid morphology. Whether this heterozygous deletion of 9 bases corresponding 3 alanine repeats from TTF-2 gene observed in the patient with ectopy was involved in the development of thyroid abnormalities is unclear in the present study and further studies are required.

Tumor necrosis factor-alpha and interferon-gamma suppress both gene expression and deoxyribonucleic acid-binding of TTF-2 in FRTL-5 cells.

Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) are cytokines that can individually or additively suppress thyroid cell function and the expression of thyroid-specific genes, such as thyroglobulin (TG) and thyroperoxidase (TPO). Thyroid transcription factor-2 (TTF-2) is a DNA-binding protein that modulates the expression of TG and TPO genes. In the present study, we examine the effects of TNF-alpha and IFN-gamma on TTF-2 gene expression, as well as the DNA-binding activity of TTF-2. FRTL-5 cells were maintained in 5H medium containing 0.2% calf serum for 7 days, then incubated with TNF-alpha, IFN-gamma, or TNF-alpha plus IFN-gamma. Total RNA was isolated and Northern blotted. TNF-alpha (50 ng/ml) only slightly suppressed (61+/-2% compared with control), whereas IFN-gamma (100 U/ml) modestly decreased TTF-2 messenger RNA (mRNA) levels (34+/-4%). TNF-alpha and IFN-gamma simultaneously caused a marked decrease in TTF-2 mRNA levels (13+/-2%). The suppressive effects of TNF-alpha and IFN-gamma on TTF-2 mRNA levels were concentration dependent and maximal at 50 ng/ml TNF-alpha with 100 U/ml IFN-gamma. The suppressive effect was also time dependent, reaching a maximum 12 h after exposure. Moreover, the suppressive effects of TNF-alpha and IFN-gamma upon rat TG and TTF-2 mRNA levels were similar. To test whether TNF-alpha and IFN-gamma alter TTF-2-binding to DNA, we performed electrophoretic mobility shift assays using a TTF-2-binding element in the rat TG gene as a probe. Formation of the TTF-2/DNA complex was decreased by TNF-alpha and/or IFN-gamma. Our results demonstrate that TNF-alpha and IFN-gamma additively reduce the gene expression and DNA-binding of TTF-2. These data suggest that TTF-2 is involved in the TNF-alpha and IFN-gamma-induced suppression of thyroid-specific gene expression.

Cloning, chromosomal localization and identification of polymorphisms in the human thyroid transcription factor 2 gene (TITF2)

The human gene encoding the thyroid transcription factor 2 (TTF-2) was cloned and mapped to human chromosome 9q22. Three polymorphisms were identified in the gene by SSCP and direct sequencing: two consist of a third base substitution in the triplet encoding Leu129 and Ser273, and the third is an alanine stretch that varies from 12 to 17 residues. TTF-2 plays a critical role during thyroid morphogenesis in mice, and in man the TITF2 gene is associated with congenital hypothyroidism and cleft palate with thyroid dysgenesis. The polymorphisms identified in this study can be used as markers to study the role of the TITF2 gene in other cases of thyroid dysgenesis, especially in familial cases.

A novel thyroid transcription factor is essential for thyrotropin-induced up-regulation of Na+/I- symporter gene expression.

The stimulation of iodide (I-) transport by TSH in FRTL-5 thyroid cells is partly due to an increase in Na+/I- symporter (NIS) gene expression. The identification of a TSH-responsive element (TRE) in the NIS promoter and its relationship to the action of thyroid transcription factor-1 (TTF-1) on the promoter are the subjects of this report. By transfecting NIS promoter-luciferase chimeric plasmids into FRTL-5 cells in the presence or absence of TSH, we identify a TRE between -420 and -370 bp of the NIS 5'-flanking region. Nuclear extracts from FRTL-5 cells cultured in the absence of TSH form two groups of protein-DNA complexes, A and B, in gel mobility shift assays using an oligonucleotide having the sequence from -420 to -385 bp. Only the A complex is increased by exposure of FRTL-5 cells to TSH or forskolin. The addition of TSH to FRTL-5 cells can increase the A complex at 3-6 h, reaching a maximum at 12 h. FRTL-5, but not nonfunctioning FRT thyroid or Buffalo rat liver (BRL) cell nuclear extracts, form the A complex. The TSH-increased nuclear factor in FRTL-5 cells interacting with the NIS TRE is distinct from TTF-1, thyroid transcription factor-2, or Pax-8, as evidenced by the absence of competition using oligonucleotides specific for these factors in gel shift assays. Neither is it the nuclear protein interacting with cAMP response element. The TRE is in the upstream of a TTF-1-binding site, -245 to -230 bp. Mutation of the TRE causing a loss of TSH responsiveness also decreases TTF-1-induced promoter activity in a transfection experiment. The formation of the A complex between FRTL-5 nuclear extracts and the NIS TRE is redox-regulated. In sum, TSH/cAMP-induced up-regulation of the NIS requires a novel thyroid transcription factor, which also appears to be involved in TTF-1-mediated thyroid-specific NIS gene expression.

Hepatocyte Nuclear Factor 3β Participates in the Transcriptional Regulation of the Thyroperoxidase Promoter

The promoters of the thyroglobulin (Tg) and thyroperoxidase (TPO) genes both contain sequences that match the consensus recognized by the fork-head family of transcription factors. The fork-head recognition element in the TPO promoter is recognized by two proteins, one of which is the transcription factor HNF-3β, a member of the hepatocyte nuclear factor 3 (HNF-3) family, and the other is a thyroid-specific binding activity previously described as thyroid transcription factor 2 (TTF-2). Differently, only TTF-2 is capable of recognizing the fork-head recognition element in the thyroglobulin promoter. HNF-3β protein is present in cultured thyroid cells and in the adult thyroid gland, suggesting that it does indeed have a role in modulating thyroid-specific gene expression. The transcriptional activities of TPO promoter mutants impaired in either TTF-2 or HNF-3β binding suggest that both factors participate in regulating transcription from the TPO promoter.

Identification of a novel insulin-responsive element in the rat thyrotropin receptor promoter.

By transfecting TSH receptor (TSHR)-chloramphenicol acetyltransferase (CAT) chimeras into FRTL-5 thyroid cells in the presence or absence of insulin, we identify an insulin-responsive element (IRE) between -220 and -190 bp of the TSHR 5'-flanking region. The region between -220 and -192 bp is footprinted by nuclear extracts from FRTL-5 cells and, coupled to a heterologous SV40-CAT chimera, an oligonucleotide containing the protected region induces insulin responsiveness in FRTL-5 cells. FRTL-5 cell nuclear extracts form two groups of protein-DNA complexes, A and B, in gel shift assays using an oligonucleotide having the protected sequence; mutation data indicate only the A complexes are increased by exposure of FRTL-5 cells to insulin; TSH can also increase A complex formation, but the TSH action is insulin-dependent. The nuclear factor(s) in FRTL-5 cells that interact with the TSHR IRE are distinct from thyroid transcription factor-2 (TTF-2), the insulin regulatory factor of the thyroglobulin promoter, as evidenced by the absence of competition in gel shift assays; there is no apparent sequence similarity of this region with other known IREs. The IRE is immediately upstream of a thyroid transcription factor-1 (TTF-1) binding site, -189 to -175 bp; mutation of the TTF-1 site causing a loss of TTF-1 activity also causes a loss of insulin responsiveness when the TSHR-CAT chimera at -220 bp is transfected into FRTL-5 cells and an altered IRE footprint by nuclear extracts. The TSHR appears, therefore, to contain a novel IRE whose activity depends at least in part on TTF-1, a thyroid-specific, homeodomain-containing transcription factor important both for thyroid-specific TSHR gene expression and TSH/cAMP autoregulation of the TSHR.