Donato Civitareale - Academia.edu (original) (raw)

Papers by Donato Civitareale

Research paper thumbnail of Thyroid transcription factor 1 activates the promoter of the thyrotropin receptor gene

Molecular Endocrinology, Dec 1, 1993

The TSH receptor (TSHr) is one of the most important thyroid differentiation markers. The binding... more The TSH receptor (TSHr) is one of the most important thyroid differentiation markers. The binding of the TSH hormone to its receptor is an essential step in the modulation of thyroid function and differentiation. Here we report that the thyroid transcription factor 1 (TTF1), a transcription factor essential for thyroid-specific gene expression, binds to the TSHr minimal promoter. The promoter, when mutated at this binding site, shows a decreased activity in thyroid cells. In cotransfection experiments in nonthyroid cells, TTF1 is able to trans-activate the TSHr minimal promoter. This finding strengthens the importance of TTF1 in the maintenance of thyroid differentiation. The promoters of the main thyroid differentiation markers thyroglobulin, thyroperoxidase, and now TSHr, are regulated by TTF1.

Research paper thumbnail of PAX 8 activates the enhancer of the human thyroperoxidase gene

Biochemical Journal, Apr 1, 1998

In this study we report on a novel natural target of the paired domain transcription factor PAX 8... more In this study we report on a novel natural target of the paired domain transcription factor PAX 8 in the enhancer element of the human thyroperoxidase gene, one of the most important thyroid differentiation markers. It is the primary enzyme involved in thyroid hormone synthesis and PAX 8 has been previously identified as an activating factor of the rat thyroperoxidase gene promoter. In itro, PAX 8 binds a cis element of the human

Research paper thumbnail of Partial purification of a thyroid specific nuclear protein recognizing the thyroglobulin promoter

PubMed, 1987

We have used a gel retardation assay to follow the purification of a calf thyroid nuclear protein... more We have used a gel retardation assay to follow the purification of a calf thyroid nuclear protein that binds to the -70 region of the rat thyroglobulin promoter. The activity producing the observed band shift is thyroid specific. The same shift is in fact observed with extracts prepared from a differentiated rat thyroid cell line which synthesizes and secretes thyroglobulin, while no similar shift is detected when cell unable to express their endogenous thyroglobulin gene or tissues different from thyroid are used as a source of nuclear extract. Competition experiments suggest that the same protein may bind at two different sites within the promoter. The two sites display considerable sequence homology. Sequence comparisons between the rat, calf and human promoter suggest that more than the sequence is the geometry of the promoter which is conserved.

Research paper thumbnail of The Thyroid Hormone Inhibits the Thyrotropin Receptor Promoter Activity: Evidence for a Short Loop Regulation

Biochemical and Biophysical Research Communications, Nov 1, 1994

Research paper thumbnail of The Synergistic Activity of Thyroid Transcription Factor 1 and Pax 8 Relies on the Promoter/Enhancer Interplay

Molecular Endocrinology, Apr 1, 2002

The transcription factors, thyroid transcription factor 1 (TTF-1) and Pax 8, play a pivotal role ... more The transcription factors, thyroid transcription factor 1 (TTF-1) and Pax 8, play a pivotal role in the transcriptional regulation of the thyroid differentiation marker genes and in the differentiation of the thyroid follicular cells. They have a very restricted tissue distribution, and the thyrocyte is the only cell type with the simultaneous expression of these factors. Here we show that TTF-1 and Pax 8 cooperatively activate their target genes and that their synergistic activity requires the cross-talk between enhancer and gene promoter. We have characterized the cis and trans requirements of the TTF1/Pax 8 synergistic activity on the thyroperoxidase gene. We show that their synergy is also important for thyroglobulin gene transcription.

Research paper thumbnail of Absence of Mutations in the Gene EncodingThyroid Transcription Factor-1 (TTF-1) in Patients with Thyroid Dysgenesis

Thyroid, Jun 1, 1997

Thyroid transription factor-1 (TTF-1) is a homeodomain-containing nuclear transcription factor, i... more Thyroid transription factor-1 (TTF-1) is a homeodomain-containing nuclear transcription factor, important in reg¬ ulation of the thyroid-specific genes thyroglobulin (Tg), thyroperoxidase (TPO), and thyrotropin receptor (TSHR). TTF-1 is an early biochemical marker of thyroid differentiation, essential for thyroid development and mainte¬ nance of the thyroid differentiated state. It is possible that mutations in titfl gene encoding TTF-1 could result in failure of the thyroid gland to develop. Single strand conformation polymorphism (SSCP) was used to detect the presence of titfl gene mutation in a group of 15 patients with congenital hypothyroidism. The etiology of the con¬ genital hypothyroidism included thyroid agenesis (9), sublingual ectopie thyroid (4), and severe hypoplasia (2). The analysis did not identify any titfl gene mutation, among these patients. These results rule out the presence of titfl mutations, at least in the coding region, in our thyroid dysgenesis patients. Mutations in titfl coding region may be an extremely rare event, and was not detected in our small sample size or, alternatively, such a mutant might even be viable since TTF-1 plays an important role in lung, brain, and pituitary development.

Research paper thumbnail of A thyroid-specific nuclear protein essential for tissue-specific expression of the thyroglobulin promoter

The EMBO Journal, Sep 1, 1989

A rat thyroglobulin promoter fragment, capable of directing thyroid-specific transcription, binds... more A rat thyroglobulin promoter fragment, capable of directing thyroid-specific transcription, binds at least three different factors, TTF-1, TTF-2 and UFA, which are all present in nuclear extracts of the differentiated rat thyroid cell line FRTL-5. TTF-1 and TTF-2 are FRTL-5 specific, as demonstrated by their absence in nuclear extracts prepared from cell lines that do not express any thyroid-differentiated function, while UFA is present in all cell lines tested. TTF-1 has been extensively purifled. It binds to the rat throyglobulin promoter at three diffrerent sites which share sequence homology. Mutations in two of the three sites decrease both binding of TTF-1 in vitro and promoter function in vivo. This suggests that the tissue-specific expression of the thyroglobulin genes is mediated, at least in part, by the presence of a transcription factor exclusively in thyroid cells.

Research paper thumbnail of Role for p300 in Pax 8 Induction of Thyroperoxidase Gene Expression

Journal of Biological Chemistry, Nov 1, 2000

Research paper thumbnail of Large scale analysis of transcription factor TTF-1/NKX2.1 target genes in GnRH secreting cell line GT1-7

Molecular and Cellular Endocrinology, Jul 1, 2010

TTF-1/Nkx2.1 is a homeodomain-containing transcription factor required for the proper development... more TTF-1/Nkx2.1 is a homeodomain-containing transcription factor required for the proper development of ventral forebrain, including some structures of the hypothalamus. TTF-1/Nkx2.1 remains expressed in the hypothalamus after birth and it plays a crucial role during sexual development. To identify putative TTF-1/Nkx2.1 target genes in GnRH neurons, we have studied the gene expression profile of the GT1-7 cells exogenously expressing TTF-1/Nkx2.1 coding gene. Our transcriptome analysis confirms that TTF-1/Nkx2.1 is involved in neuron morphogenesis and differentiation. Many of the newly identified TTF-1/Nkx2.1 target genes have a direct involvement with the central regulation of sexual maturity. In particular, we have identified Sparc as a gene directly regulated by TTF-1/Nkx2.1 at the promoter level. To further support the role of TTF-1 in GnRH neurons, we show that Sparc is involved in the regulation of the GnRH secretion in GT1-7 cells.

Research paper thumbnail of Cloning and sequence analysis of human thyroid transcription factor 1

Biochimica et biophysica acta (N), Apr 1, 1995

The thyroid transcription factor 1 (Iq'F-1) is a homeodomain-containing transcription factor that... more The thyroid transcription factor 1 (Iq'F-1) is a homeodomain-containing transcription factor that activates the transcriptional activity of thyroid-specific gene promoters by binding to them. Hence, qTF-1 is crucial in the maintenance of the thyroid differentiation phenotype. The authors isolated and analysed the human TIT-1 gene, which shows a striking homology with the rat T177-1 gene.

Research paper thumbnail of Synergistic transcriptional activation of the thyrotropin receptor promoter by cyclic AMP-responsive-element-binding protein and thyroid transcription factor 1

Biochemical Journal, Sep 1, 1995

In this study we have investigated the molecular mechanisms involved in hormonal induction of thy... more In this study we have investigated the molecular mechanisms involved in hormonal induction of thyroid-specific transcription of the thyrotropin receptor (TSHr). A cyclic AMP-responsive element (CRE) has been characterized in the minimal TSHr promoter, and promoter activity shown to be also induced by thyroid transcription factor 1 (TTF-1). We here describe a cooperative effect between TTF-l and CRE-binding protein on the

Research paper thumbnail of Retinoblastoma protein acts as Pax 8 transcriptional coactivator

Oncogene, Jun 27, 2005

Control of cell proliferation and differentiation by the retinoblastoma protein (pRb) depends on ... more Control of cell proliferation and differentiation by the retinoblastoma protein (pRb) depends on its interactions with key cellular substrates. Available data indicate that pRb and the transcription factor Pax 8 play a crucial role in the differentiation of thyroid follicular cells. In this study, we show that pRb takes part in the complex assembled on the thyroperoxidase gene promoter acting as a transcriptional coactivator of Pax 8. Accordingly, pRb interacts with and potentiates Pax 8 transcriptional activity. In addition, we show that the downregulation of pRb gene expression, in thyrocytes, through RNA interference results in a reduction of the thyroperoxidase gene promoter activity mediated by the Pax 8-binding site. In agreement with these results and with the ability of the adenoviral protein E1A to bind pRb, we show that E1A downregulates Pax 8 activity and that such inhibition requires the E1A-Rb interaction. Furthermore, we show that the Pax 8/pRb synergy plays a role on the sodium/ iodide symporter gene expression as well.

Research paper thumbnail of The tissue-specific expression of the thyroglobulin gene requires interaction between thyroid-specific and ubiquitous factors

European journal of biochemistry, Oct 1, 1990

Thyroid-specific expression of the rat thyroglobulin gene is mediated by transcriptional control.... more Thyroid-specific expression of the rat thyroglobulin gene is mediated by transcriptional control. Sufficient DNA sequence information to confer thyroid-specific expression to a heterologous gene is contained between positions-168 and + 39. DNA-binding studies have demonstrated that this region interacts with two thyroidspecific factors (TTF-1 and TTF-2), and a ubiquitous factor (UFA). Here we have characterized three elements within the promoter, A, K, and C, which are important for promoter activity in thyroid cells. We have shown by mutational analysis that the interaction of TTF-1 with the A and C regions, UFA with the A region, and TTF-2 with the K region are required for full promoter activity. The complex interactions in the A region can be replaced by the substitution of the UFA/TTF-1-binding site with a high-affinity TTF-1 binding site. There is a correlation between the presence of TTF-1 and TTF-2 DNA-binding activities and the expression of thyroglobulin, which implies that the mechanism restricting thyroglobulin expression to thyroid cells is mediated through the control of the expression, or the activity, of TTF-1 and TTF-2.

Research paper thumbnail of Sequence-specific DNA recognition by the thyroid transcription factor-1 homeodomain

Nucleic Acids Research, 1994

The molecular basis for the DNA binding specificity of the thyroid transcription factor 1 homeodo... more The molecular basis for the DNA binding specificity of the thyroid transcription factor 1 homeodomain (TTF-1 HD) has been investigated. Methylation and ethylation interference experiments show that the TTF-1 HD alone recapitulates the DNA binding properties of the entire protein. Studies carried out with mutant derivatives of TTF-1 HD indicate a precise correspondence of some of its amino acid residues with specific bases in its binding site, allowing a crude orientation of the TTF-1 HD within the protein-DNA complex. TTF-1 HD shows an overall geometry of interaction with DNA similar to that previously observed for Antennapedia class HDs, even though the binding specificities of these two types of HDs are distinct. We demonstrate that the crucial difference between the binding sites of Antennapedia class and TTF-1 HDs is in the motifs 5'-TAAT-3', recognized by Antennapedia, and 5'-CAAG-3', preferentially bound by TTF-1. Furthermore, the binding of wild type and mutants TTF-1 HD to oligonucleotides containing either 5'-TAAT-3' or 5'-CAAG-3' indicate that only in the presence of the latter motif the GIn50 in TTF-1 HD is utilized for DNA recognition. Since the Gin at position 50 is an essential determinant for DNA binding specificity for several other HDs that bind to 5'-TAAT-3' containing sequences, we suggest that utilization by different HDs of key residues may depend on the sequence context and probably follows a precise hierarchy of contacts.

Research paper thumbnail of TTF-1/Nkx2.1 functional connection with mutated EGFR relies on LRIG1 and β-catenin pathways in lung cancer cells

Biochemical and Biophysical Research Communications, Nov 1, 2018

In non-small lung cancer, the expression of the transcription factor TTF-1/Nkx2.1 correlates with... more In non-small lung cancer, the expression of the transcription factor TTF-1/Nkx2.1 correlates with the presence of EGFR mutations, therefore TTF-1/Nkx2.1 expression is used to optimize an EGFR testing strategy and to guide clinical treatment. We investigate the molecular mechanisms underlying the functional connection between EGFR and TTF-1/Nkx2.1 gene expression in lung adenocarcinoma. Using the H1975 cell line as a non-small cell lung cancer model system and short hairpin RNA, we have selected clones with TTF-1/Nkx2.1 silenced expression. We have found that Leucine-rich immunoglobulin repeats-1 (LRIG1) gene is a direct target of TTF-1/Nkx2.1 and the transcription factor binding to the LRIG1 genomic sequence inhibits its gene expression. In TTF-1/Nkx2.1 depleted clones, we have found high levels of LRIG1 and decreased presence of EGFR protein. Furthermore, in TTF-1/Nkx2.1 depleted clones we detected a reduced β-catenin level and we provide experimental evidence indicating that TTF-1/Nkx2.1 gene expression is regulated by β-catenin. Published studies indicate that LRIG1 triggers EGFR degradation and that mutated EGFR induces β-catenin activity. Hence, with the present study we show that mutated EGFR, enhancing β-catenin, stimulates TTF-1/Nkx2.1 gene expression and, at the same time, TTF-1/Nkx2.1, down-regulating LRIG1, sustains EGFR pathway. Therefore, LRIG1 and β-catenin mediate the functional connection between TTF-1/Nkx2.1 and mutated EGFR.

Research paper thumbnail of Functional characterization of two novel mutations in TTF-1/NKX2.1 homeodomain in patients with benign hereditary chorea

Journal of the Neurological Sciences, 2016

The thyroid transcription factor 1 (TTF-1) is encoded, on chromosome 14q13, by the gene termed TI... more The thyroid transcription factor 1 (TTF-1) is encoded, on chromosome 14q13, by the gene termed TITF-1/NKX2.1. Mutations in this gene have been associated with chorea, hypothyroidism, and lung disease, all included in the "brain-thyroid-lung syndrome." We here describe two cases of novel missense mutations [NM_003317.3:c.516GNT and c.623GNC resulting in p.(Gln172His) and p.(Trp208Ser), respectively] in TITF-1/ NKX2-1 in non-consanguineous patients. We provide a functional study of the role of the two mutations on the TTF-1 ability to bind DNA and to trans-activate both thyroid and lung specific gene promoters. Our results confirm the difficulty to correlate the TTF-1 activity with the clinical phenotype of affected patients and highlight the need to increase the limited knowledge we have on the activity of TTF-1 in neuronal cells.

Research paper thumbnail of Functional characterization of a novel mutation in TITF-1 in a patient with benign hereditary chorea

Journal of the Neurological Sciences, 2008

Benign hereditary chorea (BHC) is an autosomal dominant disorder of early onset characterised by ... more Benign hereditary chorea (BHC) is an autosomal dominant disorder of early onset characterised by non progressive choreic movements with normal cognitive function occasionally associated with hypothyroidism and respiratory problems. Numerous pieces of evidence link BHC with TITF-1/NKX2.1 gene mutations. We studied a patient with a familial benign hereditary chorea and normal thyroid and respiratory function. Sequence analysis of TITF-1 revealed the presence of a heterozygous C>T substitution at nucleotide 532, predicted to change an arginine (CGA) with a stop codon (TGA) at position 178 (R178X). A functional analysis shows that the mutated TTF-1 is not binding DNA, nor activating the canonical thyroid target gene promoter or interfering with the ability of wild type TTF-1 to activate transcription. In addition, the mutated protein is predominantly cytoplasmic, rather than nuclear as in the case of the wild type TTF-1. Thus, we have identified a new mutation in the TTF-1 coding gene in a patient with benign hereditary chorea. The results show that the mutation leads to a haploinsufficiency of TITF-1 and opens the question of genotype/phenotype correlation.

Research paper thumbnail of Adipocyte signaling affects thyroid-specific gene expression via down-regulation of TTF-2/FOXE1

Journal of Molecular Endocrinology, 2023

Obesity affects thyroid gland function. Hypothyroidism, thyroid nodules, goiter, and thyroid canc... more Obesity affects thyroid gland function. Hypothyroidism, thyroid nodules, goiter, and thyroid cancer are more frequent in patients with higher body mass index values. Although these data are supported by many clinical and epidemiological studies, very scarce is our knowledge at the molecular level. In this study, we present the first experimental evidence that adipocyte signaling down-regulates the expression of thyroid-specific transcription factor 2 (TTF-2/FOXE1). It plays a crucial role in thyroid development and thyroid homeostasis and it is strictly connected to thyroid cancer as well. We provide in vivo and in vitro evidence that inhibition of TTF-2/FOXE1 gene expression is mediated by adipocyte signaling.

Research paper thumbnail of Thyroid transcription factor 1 activates the promoter of the thyrotropin receptor gene

Molecular Endocrinology, Dec 1, 1993

The TSH receptor (TSHr) is one of the most important thyroid differentiation markers. The binding... more The TSH receptor (TSHr) is one of the most important thyroid differentiation markers. The binding of the TSH hormone to its receptor is an essential step in the modulation of thyroid function and differentiation. Here we report that the thyroid transcription factor 1 (TTF1), a transcription factor essential for thyroid-specific gene expression, binds to the TSHr minimal promoter. The promoter, when mutated at this binding site, shows a decreased activity in thyroid cells. In cotransfection experiments in nonthyroid cells, TTF1 is able to trans-activate the TSHr minimal promoter. This finding strengthens the importance of TTF1 in the maintenance of thyroid differentiation. The promoters of the main thyroid differentiation markers thyroglobulin, thyroperoxidase, and now TSHr, are regulated by TTF1.

Research paper thumbnail of A thyroid-specific nuclear protein essential for tissue-specific expression of the thyroglobulin promoter

The EMBO Journal, 1989

A rat thyroglobulin promoter fragment, capable of directing thyroid-specific transcription, binds... more A rat thyroglobulin promoter fragment, capable of directing thyroid-specific transcription, binds at least three different factors, TTF-1, TTF-2 and UFA, which are all present in nuclear extracts of the differentiated rat thyroid cell line FRTL-5. TTF-1 and TTF-2 are FRTL-5 specific, as demonstrated by their absence in nuclear extracts prepared from cell lines that do not express any thyroid-differentiated function, while UFA is present in all cell lines tested. TTF-1 has been extensively purifled. It binds to the rat throyglobulin promoter at three diffrerent sites which share sequence homology. Mutations in two of the three sites decrease both binding of TTF-1 in vitro and promoter function in vivo. This suggests that the tissue-specific expression of the thyroglobulin genes is mediated, at least in part, by the presence of a transcription factor exclusively in thyroid cells.

Research paper thumbnail of Thyroid transcription factor 1 activates the promoter of the thyrotropin receptor gene

Molecular Endocrinology, Dec 1, 1993

The TSH receptor (TSHr) is one of the most important thyroid differentiation markers. The binding... more The TSH receptor (TSHr) is one of the most important thyroid differentiation markers. The binding of the TSH hormone to its receptor is an essential step in the modulation of thyroid function and differentiation. Here we report that the thyroid transcription factor 1 (TTF1), a transcription factor essential for thyroid-specific gene expression, binds to the TSHr minimal promoter. The promoter, when mutated at this binding site, shows a decreased activity in thyroid cells. In cotransfection experiments in nonthyroid cells, TTF1 is able to trans-activate the TSHr minimal promoter. This finding strengthens the importance of TTF1 in the maintenance of thyroid differentiation. The promoters of the main thyroid differentiation markers thyroglobulin, thyroperoxidase, and now TSHr, are regulated by TTF1.

Research paper thumbnail of PAX 8 activates the enhancer of the human thyroperoxidase gene

Biochemical Journal, Apr 1, 1998

In this study we report on a novel natural target of the paired domain transcription factor PAX 8... more In this study we report on a novel natural target of the paired domain transcription factor PAX 8 in the enhancer element of the human thyroperoxidase gene, one of the most important thyroid differentiation markers. It is the primary enzyme involved in thyroid hormone synthesis and PAX 8 has been previously identified as an activating factor of the rat thyroperoxidase gene promoter. In itro, PAX 8 binds a cis element of the human

Research paper thumbnail of Partial purification of a thyroid specific nuclear protein recognizing the thyroglobulin promoter

PubMed, 1987

We have used a gel retardation assay to follow the purification of a calf thyroid nuclear protein... more We have used a gel retardation assay to follow the purification of a calf thyroid nuclear protein that binds to the -70 region of the rat thyroglobulin promoter. The activity producing the observed band shift is thyroid specific. The same shift is in fact observed with extracts prepared from a differentiated rat thyroid cell line which synthesizes and secretes thyroglobulin, while no similar shift is detected when cell unable to express their endogenous thyroglobulin gene or tissues different from thyroid are used as a source of nuclear extract. Competition experiments suggest that the same protein may bind at two different sites within the promoter. The two sites display considerable sequence homology. Sequence comparisons between the rat, calf and human promoter suggest that more than the sequence is the geometry of the promoter which is conserved.

Research paper thumbnail of The Thyroid Hormone Inhibits the Thyrotropin Receptor Promoter Activity: Evidence for a Short Loop Regulation

Biochemical and Biophysical Research Communications, Nov 1, 1994

Research paper thumbnail of The Synergistic Activity of Thyroid Transcription Factor 1 and Pax 8 Relies on the Promoter/Enhancer Interplay

Molecular Endocrinology, Apr 1, 2002

The transcription factors, thyroid transcription factor 1 (TTF-1) and Pax 8, play a pivotal role ... more The transcription factors, thyroid transcription factor 1 (TTF-1) and Pax 8, play a pivotal role in the transcriptional regulation of the thyroid differentiation marker genes and in the differentiation of the thyroid follicular cells. They have a very restricted tissue distribution, and the thyrocyte is the only cell type with the simultaneous expression of these factors. Here we show that TTF-1 and Pax 8 cooperatively activate their target genes and that their synergistic activity requires the cross-talk between enhancer and gene promoter. We have characterized the cis and trans requirements of the TTF1/Pax 8 synergistic activity on the thyroperoxidase gene. We show that their synergy is also important for thyroglobulin gene transcription.

Research paper thumbnail of Absence of Mutations in the Gene EncodingThyroid Transcription Factor-1 (TTF-1) in Patients with Thyroid Dysgenesis

Thyroid, Jun 1, 1997

Thyroid transription factor-1 (TTF-1) is a homeodomain-containing nuclear transcription factor, i... more Thyroid transription factor-1 (TTF-1) is a homeodomain-containing nuclear transcription factor, important in reg¬ ulation of the thyroid-specific genes thyroglobulin (Tg), thyroperoxidase (TPO), and thyrotropin receptor (TSHR). TTF-1 is an early biochemical marker of thyroid differentiation, essential for thyroid development and mainte¬ nance of the thyroid differentiated state. It is possible that mutations in titfl gene encoding TTF-1 could result in failure of the thyroid gland to develop. Single strand conformation polymorphism (SSCP) was used to detect the presence of titfl gene mutation in a group of 15 patients with congenital hypothyroidism. The etiology of the con¬ genital hypothyroidism included thyroid agenesis (9), sublingual ectopie thyroid (4), and severe hypoplasia (2). The analysis did not identify any titfl gene mutation, among these patients. These results rule out the presence of titfl mutations, at least in the coding region, in our thyroid dysgenesis patients. Mutations in titfl coding region may be an extremely rare event, and was not detected in our small sample size or, alternatively, such a mutant might even be viable since TTF-1 plays an important role in lung, brain, and pituitary development.

Research paper thumbnail of A thyroid-specific nuclear protein essential for tissue-specific expression of the thyroglobulin promoter

The EMBO Journal, Sep 1, 1989

A rat thyroglobulin promoter fragment, capable of directing thyroid-specific transcription, binds... more A rat thyroglobulin promoter fragment, capable of directing thyroid-specific transcription, binds at least three different factors, TTF-1, TTF-2 and UFA, which are all present in nuclear extracts of the differentiated rat thyroid cell line FRTL-5. TTF-1 and TTF-2 are FRTL-5 specific, as demonstrated by their absence in nuclear extracts prepared from cell lines that do not express any thyroid-differentiated function, while UFA is present in all cell lines tested. TTF-1 has been extensively purifled. It binds to the rat throyglobulin promoter at three diffrerent sites which share sequence homology. Mutations in two of the three sites decrease both binding of TTF-1 in vitro and promoter function in vivo. This suggests that the tissue-specific expression of the thyroglobulin genes is mediated, at least in part, by the presence of a transcription factor exclusively in thyroid cells.

Research paper thumbnail of Role for p300 in Pax 8 Induction of Thyroperoxidase Gene Expression

Journal of Biological Chemistry, Nov 1, 2000

Research paper thumbnail of Large scale analysis of transcription factor TTF-1/NKX2.1 target genes in GnRH secreting cell line GT1-7

Molecular and Cellular Endocrinology, Jul 1, 2010

TTF-1/Nkx2.1 is a homeodomain-containing transcription factor required for the proper development... more TTF-1/Nkx2.1 is a homeodomain-containing transcription factor required for the proper development of ventral forebrain, including some structures of the hypothalamus. TTF-1/Nkx2.1 remains expressed in the hypothalamus after birth and it plays a crucial role during sexual development. To identify putative TTF-1/Nkx2.1 target genes in GnRH neurons, we have studied the gene expression profile of the GT1-7 cells exogenously expressing TTF-1/Nkx2.1 coding gene. Our transcriptome analysis confirms that TTF-1/Nkx2.1 is involved in neuron morphogenesis and differentiation. Many of the newly identified TTF-1/Nkx2.1 target genes have a direct involvement with the central regulation of sexual maturity. In particular, we have identified Sparc as a gene directly regulated by TTF-1/Nkx2.1 at the promoter level. To further support the role of TTF-1 in GnRH neurons, we show that Sparc is involved in the regulation of the GnRH secretion in GT1-7 cells.

Research paper thumbnail of Cloning and sequence analysis of human thyroid transcription factor 1

Biochimica et biophysica acta (N), Apr 1, 1995

The thyroid transcription factor 1 (Iq'F-1) is a homeodomain-containing transcription factor that... more The thyroid transcription factor 1 (Iq'F-1) is a homeodomain-containing transcription factor that activates the transcriptional activity of thyroid-specific gene promoters by binding to them. Hence, qTF-1 is crucial in the maintenance of the thyroid differentiation phenotype. The authors isolated and analysed the human TIT-1 gene, which shows a striking homology with the rat T177-1 gene.

Research paper thumbnail of Synergistic transcriptional activation of the thyrotropin receptor promoter by cyclic AMP-responsive-element-binding protein and thyroid transcription factor 1

Biochemical Journal, Sep 1, 1995

In this study we have investigated the molecular mechanisms involved in hormonal induction of thy... more In this study we have investigated the molecular mechanisms involved in hormonal induction of thyroid-specific transcription of the thyrotropin receptor (TSHr). A cyclic AMP-responsive element (CRE) has been characterized in the minimal TSHr promoter, and promoter activity shown to be also induced by thyroid transcription factor 1 (TTF-1). We here describe a cooperative effect between TTF-l and CRE-binding protein on the

Research paper thumbnail of Retinoblastoma protein acts as Pax 8 transcriptional coactivator

Oncogene, Jun 27, 2005

Control of cell proliferation and differentiation by the retinoblastoma protein (pRb) depends on ... more Control of cell proliferation and differentiation by the retinoblastoma protein (pRb) depends on its interactions with key cellular substrates. Available data indicate that pRb and the transcription factor Pax 8 play a crucial role in the differentiation of thyroid follicular cells. In this study, we show that pRb takes part in the complex assembled on the thyroperoxidase gene promoter acting as a transcriptional coactivator of Pax 8. Accordingly, pRb interacts with and potentiates Pax 8 transcriptional activity. In addition, we show that the downregulation of pRb gene expression, in thyrocytes, through RNA interference results in a reduction of the thyroperoxidase gene promoter activity mediated by the Pax 8-binding site. In agreement with these results and with the ability of the adenoviral protein E1A to bind pRb, we show that E1A downregulates Pax 8 activity and that such inhibition requires the E1A-Rb interaction. Furthermore, we show that the Pax 8/pRb synergy plays a role on the sodium/ iodide symporter gene expression as well.

Research paper thumbnail of The tissue-specific expression of the thyroglobulin gene requires interaction between thyroid-specific and ubiquitous factors

European journal of biochemistry, Oct 1, 1990

Thyroid-specific expression of the rat thyroglobulin gene is mediated by transcriptional control.... more Thyroid-specific expression of the rat thyroglobulin gene is mediated by transcriptional control. Sufficient DNA sequence information to confer thyroid-specific expression to a heterologous gene is contained between positions-168 and + 39. DNA-binding studies have demonstrated that this region interacts with two thyroidspecific factors (TTF-1 and TTF-2), and a ubiquitous factor (UFA). Here we have characterized three elements within the promoter, A, K, and C, which are important for promoter activity in thyroid cells. We have shown by mutational analysis that the interaction of TTF-1 with the A and C regions, UFA with the A region, and TTF-2 with the K region are required for full promoter activity. The complex interactions in the A region can be replaced by the substitution of the UFA/TTF-1-binding site with a high-affinity TTF-1 binding site. There is a correlation between the presence of TTF-1 and TTF-2 DNA-binding activities and the expression of thyroglobulin, which implies that the mechanism restricting thyroglobulin expression to thyroid cells is mediated through the control of the expression, or the activity, of TTF-1 and TTF-2.

Research paper thumbnail of Sequence-specific DNA recognition by the thyroid transcription factor-1 homeodomain

Nucleic Acids Research, 1994

The molecular basis for the DNA binding specificity of the thyroid transcription factor 1 homeodo... more The molecular basis for the DNA binding specificity of the thyroid transcription factor 1 homeodomain (TTF-1 HD) has been investigated. Methylation and ethylation interference experiments show that the TTF-1 HD alone recapitulates the DNA binding properties of the entire protein. Studies carried out with mutant derivatives of TTF-1 HD indicate a precise correspondence of some of its amino acid residues with specific bases in its binding site, allowing a crude orientation of the TTF-1 HD within the protein-DNA complex. TTF-1 HD shows an overall geometry of interaction with DNA similar to that previously observed for Antennapedia class HDs, even though the binding specificities of these two types of HDs are distinct. We demonstrate that the crucial difference between the binding sites of Antennapedia class and TTF-1 HDs is in the motifs 5'-TAAT-3', recognized by Antennapedia, and 5'-CAAG-3', preferentially bound by TTF-1. Furthermore, the binding of wild type and mutants TTF-1 HD to oligonucleotides containing either 5'-TAAT-3' or 5'-CAAG-3' indicate that only in the presence of the latter motif the GIn50 in TTF-1 HD is utilized for DNA recognition. Since the Gin at position 50 is an essential determinant for DNA binding specificity for several other HDs that bind to 5'-TAAT-3' containing sequences, we suggest that utilization by different HDs of key residues may depend on the sequence context and probably follows a precise hierarchy of contacts.

Research paper thumbnail of TTF-1/Nkx2.1 functional connection with mutated EGFR relies on LRIG1 and β-catenin pathways in lung cancer cells

Biochemical and Biophysical Research Communications, Nov 1, 2018

In non-small lung cancer, the expression of the transcription factor TTF-1/Nkx2.1 correlates with... more In non-small lung cancer, the expression of the transcription factor TTF-1/Nkx2.1 correlates with the presence of EGFR mutations, therefore TTF-1/Nkx2.1 expression is used to optimize an EGFR testing strategy and to guide clinical treatment. We investigate the molecular mechanisms underlying the functional connection between EGFR and TTF-1/Nkx2.1 gene expression in lung adenocarcinoma. Using the H1975 cell line as a non-small cell lung cancer model system and short hairpin RNA, we have selected clones with TTF-1/Nkx2.1 silenced expression. We have found that Leucine-rich immunoglobulin repeats-1 (LRIG1) gene is a direct target of TTF-1/Nkx2.1 and the transcription factor binding to the LRIG1 genomic sequence inhibits its gene expression. In TTF-1/Nkx2.1 depleted clones, we have found high levels of LRIG1 and decreased presence of EGFR protein. Furthermore, in TTF-1/Nkx2.1 depleted clones we detected a reduced β-catenin level and we provide experimental evidence indicating that TTF-1/Nkx2.1 gene expression is regulated by β-catenin. Published studies indicate that LRIG1 triggers EGFR degradation and that mutated EGFR induces β-catenin activity. Hence, with the present study we show that mutated EGFR, enhancing β-catenin, stimulates TTF-1/Nkx2.1 gene expression and, at the same time, TTF-1/Nkx2.1, down-regulating LRIG1, sustains EGFR pathway. Therefore, LRIG1 and β-catenin mediate the functional connection between TTF-1/Nkx2.1 and mutated EGFR.

Research paper thumbnail of Functional characterization of two novel mutations in TTF-1/NKX2.1 homeodomain in patients with benign hereditary chorea

Journal of the Neurological Sciences, 2016

The thyroid transcription factor 1 (TTF-1) is encoded, on chromosome 14q13, by the gene termed TI... more The thyroid transcription factor 1 (TTF-1) is encoded, on chromosome 14q13, by the gene termed TITF-1/NKX2.1. Mutations in this gene have been associated with chorea, hypothyroidism, and lung disease, all included in the "brain-thyroid-lung syndrome." We here describe two cases of novel missense mutations [NM_003317.3:c.516GNT and c.623GNC resulting in p.(Gln172His) and p.(Trp208Ser), respectively] in TITF-1/ NKX2-1 in non-consanguineous patients. We provide a functional study of the role of the two mutations on the TTF-1 ability to bind DNA and to trans-activate both thyroid and lung specific gene promoters. Our results confirm the difficulty to correlate the TTF-1 activity with the clinical phenotype of affected patients and highlight the need to increase the limited knowledge we have on the activity of TTF-1 in neuronal cells.

Research paper thumbnail of Functional characterization of a novel mutation in TITF-1 in a patient with benign hereditary chorea

Journal of the Neurological Sciences, 2008

Benign hereditary chorea (BHC) is an autosomal dominant disorder of early onset characterised by ... more Benign hereditary chorea (BHC) is an autosomal dominant disorder of early onset characterised by non progressive choreic movements with normal cognitive function occasionally associated with hypothyroidism and respiratory problems. Numerous pieces of evidence link BHC with TITF-1/NKX2.1 gene mutations. We studied a patient with a familial benign hereditary chorea and normal thyroid and respiratory function. Sequence analysis of TITF-1 revealed the presence of a heterozygous C>T substitution at nucleotide 532, predicted to change an arginine (CGA) with a stop codon (TGA) at position 178 (R178X). A functional analysis shows that the mutated TTF-1 is not binding DNA, nor activating the canonical thyroid target gene promoter or interfering with the ability of wild type TTF-1 to activate transcription. In addition, the mutated protein is predominantly cytoplasmic, rather than nuclear as in the case of the wild type TTF-1. Thus, we have identified a new mutation in the TTF-1 coding gene in a patient with benign hereditary chorea. The results show that the mutation leads to a haploinsufficiency of TITF-1 and opens the question of genotype/phenotype correlation.

Research paper thumbnail of Adipocyte signaling affects thyroid-specific gene expression via down-regulation of TTF-2/FOXE1

Journal of Molecular Endocrinology, 2023

Obesity affects thyroid gland function. Hypothyroidism, thyroid nodules, goiter, and thyroid canc... more Obesity affects thyroid gland function. Hypothyroidism, thyroid nodules, goiter, and thyroid cancer are more frequent in patients with higher body mass index values. Although these data are supported by many clinical and epidemiological studies, very scarce is our knowledge at the molecular level. In this study, we present the first experimental evidence that adipocyte signaling down-regulates the expression of thyroid-specific transcription factor 2 (TTF-2/FOXE1). It plays a crucial role in thyroid development and thyroid homeostasis and it is strictly connected to thyroid cancer as well. We provide in vivo and in vitro evidence that inhibition of TTF-2/FOXE1 gene expression is mediated by adipocyte signaling.

Research paper thumbnail of Thyroid transcription factor 1 activates the promoter of the thyrotropin receptor gene

Molecular Endocrinology, Dec 1, 1993

The TSH receptor (TSHr) is one of the most important thyroid differentiation markers. The binding... more The TSH receptor (TSHr) is one of the most important thyroid differentiation markers. The binding of the TSH hormone to its receptor is an essential step in the modulation of thyroid function and differentiation. Here we report that the thyroid transcription factor 1 (TTF1), a transcription factor essential for thyroid-specific gene expression, binds to the TSHr minimal promoter. The promoter, when mutated at this binding site, shows a decreased activity in thyroid cells. In cotransfection experiments in nonthyroid cells, TTF1 is able to trans-activate the TSHr minimal promoter. This finding strengthens the importance of TTF1 in the maintenance of thyroid differentiation. The promoters of the main thyroid differentiation markers thyroglobulin, thyroperoxidase, and now TSHr, are regulated by TTF1.

Research paper thumbnail of A thyroid-specific nuclear protein essential for tissue-specific expression of the thyroglobulin promoter

The EMBO Journal, 1989

A rat thyroglobulin promoter fragment, capable of directing thyroid-specific transcription, binds... more A rat thyroglobulin promoter fragment, capable of directing thyroid-specific transcription, binds at least three different factors, TTF-1, TTF-2 and UFA, which are all present in nuclear extracts of the differentiated rat thyroid cell line FRTL-5. TTF-1 and TTF-2 are FRTL-5 specific, as demonstrated by their absence in nuclear extracts prepared from cell lines that do not express any thyroid-differentiated function, while UFA is present in all cell lines tested. TTF-1 has been extensively purifled. It binds to the rat throyglobulin promoter at three diffrerent sites which share sequence homology. Mutations in two of the three sites decrease both binding of TTF-1 in vitro and promoter function in vivo. This suggests that the tissue-specific expression of the thyroglobulin genes is mediated, at least in part, by the presence of a transcription factor exclusively in thyroid cells.