Thyroglobulin Promoter Research Articles

Multiple mechanisms of interference between transformation and differentiation in thyroid cells.

Transformation of the thyroid cell line FRTL-5 results in loss or reduction of differentiation as measured by the expression of thyroglobulin and thyroperoxidase, two proteins whose genes are exclusively expressed in thyroid follicular cells. The biochemical mechanisms leading to this phenomenon were investigated in three cell lines obtained by transformation of FRTL-5 cells with Ki-ras, Ha-ras, and polyomavirus middle-T oncogenes. With the ras oncogenes, transformation leads to undetectable expression of the thyroglobulin and thyroperoxidase genes. However, the mechanisms responsible for the extinction of the differentiated phenotype seem to be different for the two ras oncogenes. In Ki-ras-transformed cells, the mRNA encoding TTF-1, a transcription factor controlling thyroglobulin and thyroperoxidase gene expression, is severely reduced. On the contrary, nearly wild-type levels of TTF-1 mRNA are detected in Ha-ras-transformed cells. Furthermore, overexpression of TTF-1 can activate transcription of the thyroglobulin promoter in Ki-ras-transformed cells, whereas it has no effect on thyroglobulin transcription in the Ha-ras-transformed line. Expression of polyoma middle-T antigen in thyroid cells leads to only a reduction of differentiation and does not severely affect either the activity or the amount of TTF-1. Another thyroid cell-specific transcription factor, TTF-2, is more sensitive to transformation, since it disappears in all three transformed lines, and probably contributes to the reduced expression of the differentiated phenotype.

Pax-8, a paired domain-containing protein, binds to a sequence overlapping the recognition site of a homeodomain and activates transcription from two thyroid-specific promoters.

The Pax-8 gene, a member of the murine family of paired box-containing genes (Pax genes), is expressed in adult thyroid and in cultured thyroid cell lines. The Pax-8 protein binds, through its paired domain, to the promoters of thyroglobulin and thyroperoxidase, genes that are exclusively expressed in the thyroid. In both promoters, the binding site of Pax-8 overlaps with that of TTF-1, a homeodomain-containing protein involved in the activation of thyroid-specific transcription. Pax-8 activates transcription from cotransfected thyroperoxidase and thyroglobulin promoters, indicating that it may be involved in the establishment, control, or maintenance of the thyroid-differentiated phenotype. Thus, the promoters of thyroglobulin and thyroperoxidase represent the first identified natural targets for transcriptional activation by a paired domain-containing protein.

Pax-8, a paired domain-containing protein, binds to a sequence overlapping the recognition site of a homeodomain and activates transcription from two thyroid-specific promoters.

The Pax-8 gene, a member of the murine family of paired box-containing genes (Pax genes), is expressed in adult thyroid and in cultured thyroid cell lines. The Pax-8 protein binds, through its paired domain, to the promoters of thyroglobulin and thyroperoxidase, genes that are exclusively expressed in the thyroid. In both promoters, the binding site of Pax-8 overlaps with that of TTF-1, a homeodomain-containing protein involved in the activation of thyroid-specific transcription. Pax-8 activates transcription from cotransfected thyroperoxidase and thyroglobulin promoters, indicating that it may be involved in the establishment, control, or maintenance of the thyroid-differentiated phenotype. Thus, the promoters of thyroglobulin and thyroperoxidase represent the first identified natural targets for transcriptional activation by a paired domain-containing protein.

Insulin and insulin-like growth factor I regulate a thyroid-specific nuclear protein that binds to the thyroglobulin promoter.

The mechanism responsible for the stimulation of thyroglobulin (Tg) gene expression by insulin and insulin-like growth factor I (IGF-I) in rat thyroid FRTL-5 cells has been investigated. Both insulin and IGF-I stimulate transcription from the Tg promoter in a transient transfection assay demonstrating that the promoter used contains the DNA signals necessary for insulin and IGF-I regulation. Promoter mutations that interfere with the binding of thyroid transcription factor 1 (TTF-1), TTF-2, and the ubiquitous transcription factor abolish the insulin/IGF-I response, indicating that the three factors may be involved in the observed transcriptional control. Protein-DNA binding studies did not reveal any effect of insulin/IGF-I on the ubiquitous transcription factor and the TTF-1 binding capacity. Instead, TTF-2 is absent in nuclear extracts from cells depleted of serum and insulin. Addition of insulin or IGF-I restores the TTF-2 concentration to normal levels and requires ongoing protein synthesis. The insulin effect was maximal at 24 h and at a concentration of 1 microgram/ml. The same effect was observed with a 10-fold lower concentration of IGF-I. These results suggest that insulin (probably through the IGF-I receptor) and IGF-I modulate the levels of TTF-2, which results in an increased expression of the Tg gene.

Insulin and insulin-like growth factor I regulate a thyroid-specific nuclear protein that binds to the thyroglobulin promoter

Insulin and insulin-like growth factor I regulate a thyroid-specific nuclear protein that binds to the thyroglobulin promoter

Functional role of TTF-1 binding sites in bovine thyroglobulin promoter

We have studied the binding of purified TTF-1 on the bovine thyroglobulin gene promoter. DNase I footprinting experiments revealed three binding sites which corresponded in location to the A, B and C sites found in the rat thyroglobulin promoter. Mutants in the A and C regions showing reduced binding of TTF-1, also exhibited largely decreased promoter activity in transient expression experiments in primary-cultured dog thyrocytes. Two mutants in the B site that exhibited a reduced capacity to bind TTF-1 also displayed a drastically affected transcriptional activity in transient assays. As in the rat, sites A and C only are critical for promoter activity, these results suggest that full occupancy of the B site is required for thyroglobulin promoter activity in the cow only.

Functional study of the human thyroid peroxidase gene promoter

Structure/function relationships in the human thyroid peroxidase gene promoter have been studied by deletion and mutation analyses and confronted with footprint patterns obtained with thyroid nuclear extracts and the purified thyroid transcription factor TTF-1. Crude nuclear extracts from dog thyroid primary cultures were shown to contain a binding activity recognizing the -119 to -105 segment of the promoter (coordinates relative to the transcriptional start site). Deletion, or site-directed mutagenesis of this segment dramatically reduced transcriptional activity in transient expression experiments on gene fusions of the thyroid peroxidase promoter and the growth hormone reporter. This binding activity was increased in nuclear extracts from thyrocytes cultured in the presence of the cAMP-agonist forskolin. A mutation that decreased the promoter function in forskolin-stimulated thyrocytes resulted in weakening of the corresponding footprint. The binding site displays no significant sequence similarities with known cAMP-responsive elements. Mutagenesis of another region of the promoter (-99 to -94) induced the binding of an additional factor, resulting in a dramatically enhanced promoter activity. We show that the thyroid-specific transcriptional factor TTF-1 is not directly involved in the above-mentioned interactions and provide evidence suggesting that, in spite of displaying a similar binding pattern to thyroperoxidase and thyroglobulin promoters in vitro, TTF-1 plays a less important role in the former. Altogether, our data delineate the minimal thyroid peroxidase gene promoter in the human and identify the binding sites of two trans-activating factors, one of them being potentially the mediator of a non-conventional cAMP control, independent of the cAMP-responsive element and factor AP-2.

Overexpression of transgenic class I MHC alloantigen (H-2Kb) in the thyroid: acquired tolerance and nonimmune thyroid atrophy

We previously demonstrated that overexpression of transgenic class I MHC proteins in the beta cells of mice leads to non-immune beta cell destruction and insulin-dependent diabetes. Overexpression of MHC, an early feature of autoimmune diabetes, may therefore directly impair endocrine cell function. To test whether this is a general phenomenon we expressed class I MHC transgenically in the thyroid. The rat thyroglobulin promoter was subcloned into a vector containing the mouse class I MHC (H-2K b ) genomic clone. This fusion construct was injected into the pronuclei of (CBA x BIO.BR) F 2 mice (H-2K k haplotype) and transferred to oviducts of (CBA x C57) pseudo-pregnant mice. Transgenic offspring were identified by probing mouse tail DNA with the 32 P-labelled thyroglobulin promoter. Despite expression of the alloantigen in the thyroid cells there was no inflammatory infiltration of the thyroid. Heterozygous mice had no histologic changes in the thyroid but developed modest hypothyroxinaemia. Homozygous mice developed neonatal growth retardation (~ 50% of littermates’ weight), biochemical hypothyroidism and death by 18-21 days of age. Histology revealed reduction in follicular size and in thyrocyte height, but again no cellular infiltrate. To further study the mechanisms of tolerance in these animals we examined the ability of spleen cells from transgenic mice and their non-transgenic littermates to destroy target cells syngeneic and allogeneic to the transgene. H-2K b spleen cells (from BIOA (5R) mice) were irradiated and transgenic and non-transgenic spleen cells were primed against them for 5 days. Transgenic spleen cells were tolerant to H-2K b bearing target cells (EL4), but not to H-2 d (P815) cells, as determined by release of 51 Cr from labelled targets. We conclude that these transgenic mice have developed tolerance to the peripheral expression of an alloantigen, whose overexpression leads to non-immune mediated thyroid destruction and hypothyroidism. MHC class I overexpression, an early feature of autoimmune thyroid disease, may thus play a direct role in thyrocyte destruction.

Isolation and characterization of the canine thyroglobulin gene promoter region

The 5' flanking sequences from the canine thyroglobulin gene were isolated by homology screening with the evolutionary conserved sequence from the bovine thyroglobulin promoter and sequenced. Transient expression in primary cultured dog thyrocytes demonstrated that the canine clone contains a functional promoter inducible by cAMP. DNAse I footprinting assays showed that the thyroid-specific transcription factor TTF-1, purified from bovine thyroid, also recognizes the canine thyroglobulin promoter. Similar footprints were obtained with crude nuclear extracts from primary cultured dog thyrocytes.

Reversible inhibition of a thyroid-specific trans-acting factor by Ras.

Exposure of rat thyroid cells for 1 week to a temperature-sensitive variant of Kirsten murine sarcoma virus (KiMSV) Ras inactivated the thyroglobulin promoter (pTg). Cellular dedifferentiation was paralleled by the loss of the thyroid-specific trans-acting factor, TgTF1, which binds to pTg. When Ras was denatured by shifting cells to 39 degrees C, TgTF1 binding and pTg function recovered rapidly without the synthesis of new protein. TgTF1 could be reactivated in vitro by treating nuclear extracts with protein kinase A. After 4 weeks of exposure to the oncogene, denaturation of Ras no longer restored TgTF1 binding or reactivated pTg. Incubation of nuclear extracts with protein kinase A likewise did not reactivate TgTF1. Cells chronically exposed to Ras did, however, yield differentiated clones after treatment with 5-azacytidine. We suggest that Ras induces dedifferentiation in two sequential steps: (1) Ras reduces PKA activity; TgTF1 (or an auxiliary protein) becomes dephosphorylated, and binding to pTg is abolished. (2) The effects of Ras become imprinted by methylation, possibly of the TgTF1 gene.

The Deoxyribonucleic Acid Regions Involved in the Hormonal Regulation of Thyroglobulin Gene Expression*

Transcription of the thyroglobulin (TG) gene is stimulated by TSH via cAMP. We have characterized the sequence elements responsible for the hormone-dependent expression of TG gene in rat thyroid FRTL-5 cells using internal deletion and linker-scanning mutants of the minimal TG promoter (-170 basepairs) fused with the bacterial chloramphenicol acetyltransferase reporter gene. The TG gene is regulated by at least two regions located between -165 and -140 bp (TG-III) and between -95 and -65 bp (TG-I) from the transcription initiation site. The intervening region can be deleted without significant effect on the promoter activity. Either of the two regions alone does not promote hormone-dependent transcription. A DNase footprinting assay showed that TG-I and TG-III are the principal protein-binding sites and that the proteins interacting with these two regions are induced by TSH or cAMP. These results suggest that the hormone-dependent expression of TG gene may be achieved by cooperative interaction of the proteins bound to TG-I and TG-III.

Extinction and activation of the thyroglobulin promoter in hybrids of differentiated and transformed thyroid cells.

Thyroglobulin gene expression was repressed in a rat thyroid cell line transformed with Kirsten murine sarcoma virus. Expression of a dominant selectable marker driven by the thyroglobulin promoter was also inhibited. Somatic cell hybridization of transformed and differentiated thyroid cells resulted in extinction of thyroglobulin gene expression. When transformed cells carrying a dominant selectable marker driven by the thyroglobulin promoter were fused to differentiated cells and expression of this marker was selected, we obtained stable hybrid cell lines expressing both the endogenous and the exogenous thyroglobulin promoters. Although the expression of v-ras remained unchanged compared with expression in the parental transformed cells, transformation was suppressed in the hybrid cell lines. The other thyroid differentiation markers, iodide uptake and thyroid-stimulating hormone-dependent growth, were inhibited in all the hybrids tested. We show that activity of the thyroglobulin promoter correlates with the presence of a thyroid nuclear factor that binds the promoter at position -60 from the transcription start site. Loss of this factor accompanies the extinction of thyroglobulin gene expression in hybrids selected for expression of a non-thyroid-specific promoter.

Extinction and Activation of the Thyroglobulin Promoter in Hybrids of Differentiated and Transformed Thyroid Cells

Thyroglobulin gene expression was repressed in a rat thyroid cell line transformed with Kirsten murine sarcoma virus. Expression of a dominant selectable marker driven by the thyroglobulin promoter was also inhibited. Somatic cell hybridization of transformed and differentiated thyroid cells resulted in extinction of thyroglobulin gene expression. When transformed cells carrying a dominant selectable marker driven by the thyroglobulin promoter were fused to differentiated cells and expression of this marker was selected, we obtained stable hybrid cell lines expressing both the endogenous and the exogenous thyroglobulin promoters. Although the expression of v-ras remained unchanged compared with expression in the parental transformed cells, transformation was suppressed in the hybrid cell lines. The other thyroid differentiation markers, iodide uptake and thyroid-stimulating hormone-dependent growth, were inhibited in all the hybrids tested. We show that activity of the thyroglobulin promoter correlates with the presence of a thyroid nuclear factor that binds the promoter at position -60 from the transcription start site. Loss of this factor accompanies the extinction of thyroglobulin gene expression in hybrids selected for expression of a non-thyroid-specific promoter.

Nucleotide sequence of a putative transcription factor recognizing the thyroglobulin promoter

Nucleotide sequence of a putative transcription factor recognizing the thyroglobulin promoter

Reactivation of thyroglobulin gene expression in transformed thyroid cells by 5-azacytidine

Transformed rat thyroid cells fail to express thyroglobulin. Cells transformed with a Kirsten murine sarcoma virus carrying a temperature-sensitive ras allele lose their transformation phenotype when shifted to the nonpermissive (39°C) temperature. The thyroglobulin promoter, however, remains inactive. Similarly, transfection of these cells with a thyroglobulin promoter fused to a neomycin resistance reporter gene does not produce clones resistant to G418. Treatment of the transfected cells with the DNA demethylating agent 5-azacytidine reactivates the thyroglobulin promoter and yields stable G418-resistant clones. We show that thyroglobulin promoter activity is correlated with the presence of a thyroid-specific nuclear factor, TgTF1. TgTF1 cannot be detected in transformed cells but reappears after treatment with 5-azacytidine at 39°C. Restoration of Ras activity at 33°C leads to the rapid loss of TgTF1 and G418 resistance.

A thyroid-specific nuclear protein essential for tissue-specific expression of the thyroglobulin promoter.

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 purified. It binds to the rat thyroglobulin promoter at three different 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.

Protein binding domains of the rat thyroglobulin promoter

We have previously shown that DNA elements controlling tissue specific expression of the rat thyroglobulin gene extend 170 bp upstream of the cap site and have identified a thyroid specific nuclear factor which binds the promoter in the −60 region (site C). Here we report that the distal portion of the promoter, extending from −160 to −120, contains two contiguous DNA elements (sites A and B) which interact with the same thyroid-specific factor binding to proximal site C. A second nuclear factor, ubiquitously distributed, binds to the distal site A. Transient cotransfection-competition studies show that all the three binding sites A, B and C titrate a trans-acting factor(s) which is necessary for the transcription of the thyroglobulin gene.

CAMP-dependent binding of a trans-acting factor to the thyroglobulin promoter

We have investigated the interaction of a nuclear factor(s) with the promoter region of the thyroglobulin (Tg) gene, which is only expressed in differentiated thyroid cells under the positive control of the pituitary hormone thyrotropin (TSH) via a cAMP-dependent pathway. Using the mobility shift assay, we first demonstrated that a thyroid nuclear factor interacts with a short segment of 60 bp (−136 – −77) which is conserved among species in the regulatory region of the Tg gene. A specific binding site was then localized in a subfragment of 20 bp located between −126 bp and −107 bp relative to the transcription initiation site. The corresponding nuclear factor is absent in a tissue which does not express the Tg gene. This factor differs from previously identified factors shown to mediate a direct cAMP response since the observed binding is neither competed out by the cAMP responsive element (CRE) nor by the activator protein 2 (AP2) binding site. This trans-acting factor represents a new candidate intermediate in the regulation of transcription by a cAMP dependent mechanism.

Neoplastic transformation inactivates specific trans-acting factor(s) required for the expression of the thyroglobulin gene.

The expression of rat thyroglobulin gene is repressed following the transformation of rat thyroid cells with Kirsten murine sarcoma virus. The expression of a reporter gene fused to the thyroglobulin promoter is down-regulated in transformed thyroid cells in transient or in stable transfection assays. DNase and exonuclease III cleavage-protection analysis reveals that a promoter binding activity located at -60 base pairs from the transcription start site is substantially reduced in transformed thyroid cells. The repression in the transformed cells of the reporter gene joined to the thyroglobulin promoter can be reversed by fusion with normal differentiated thyroid cells. Fusion of transformed thyroid cells to liver cells does not reactivate the reporter under control of the thyroglobulin promoter.

A cell type specific factor recognizes the rat thyroglobulin promoter.

We have fused a 900 base pair long DNA segment containing the transcriptional start site of the rat thyroglobulin (Tg) gene to the bacterial gene for chloramphenicol acetyltransferase (cat). The fusion gene has been introduced into three different cell lines derived from the rat thyroid gland and into a rat liver cell line. Expression of the fusion gene was detected only in the one thyroid cell line that is able to express the endogenous Tg gene. The minimum DNA sequence required for the cell type specific expression was determined by deletion analysis; it extends 170 nucleotides upstream of the transcription initiation site. The Tg promoter contains a readily detectable binding sites for a factor present in salt extracts of thyroid cell nuclei. This binding site is not recognized by the nuclear extracts of any other cell type that we have tested, suggesting that it may help mediate the cell type specific expression of the Tg gene.