Daniel Christophe

Nuclear targeting of proteins: how many different signals?

The nuclear import of proteins into the cell nucleus involves the recognition of a nuclear localization signal sequence, borne by the protein to be transported, by complex molecules called importins, that will subsequently mediate the crossing over of the nuclear envelope. The most frequently encountered signal sequence is made up of short stretches of basic amino acid residues and is recognized by importins alpha and/or beta. Other signal sequences have been described, and some have been shown to mediate the association with importins other than importin alpha or beta. Recently, approaches have been developed that allow the cloning, on a functional basis, of sequences able to specify the nuclear localization of proteins. A variety of peptidic motifs of limited size which do not contain previously described signal sequences were isolated in such assays. It reveals that the spectrum of sequences that are able to target a protein to the cell nucleus may be wider than currently expected. It will probably also lead to the identification of novel target sequences for importins and will demonstrate the implication of additional members of this family of proteins in nuclear transport.

Structural and functional characterization of the two human ThOX/Duox genes and their 5'-flanking regions.

A crucial step in thyroid hormone synthesis is the oxidative coupling of iodide to thyroglobulin that is catalyzed by thyroperoxidase. The limiting factor of this reaction is the supply of hydrogen peroxide. The generation of hydrogen peroxide has been linked to an enzymatic system located at the apical pole of thyrocytes. This enzymatic activity is assumed to be exerted by NADPH oxidases encoded by two recently cloned genes hThOX1 and hThOX2. Both genes are expressed at high levels in thyrocytes. In this study we report the chromosomal organization of these two genes and the functional characterization of their respective promoter regions. The two human ThOX genes are arranged in a head to head configuration and are separated by a 16 kb-long region. Human ThOX1 and ThOX2 genes span 75 kb and are composed of 35 and 34 exons, respectively. The promoters of both genes do not resemble each other and differ from promoters of other known thyroid-specific genes. No TATA box is present in either ThOX gene promoter. Functional studies confirm that both promoters display significant transcriptional activities after transfection in differentiated thyroid cell lines. However, in contrast to that of thyroglobulin or Na(+)/I(-) symporter gene promoter, hThOX promoter activity is not restricted to thyroid cells. Additionally, functional studies show that both hThOX promoters are not positively controlled by cAMP.

Pax 8 expression in primary cultured dog thyrocyte is increased by cyclic AMP

Pax 8 proteins are paired domain-containing transcription factors expressed in thyroid, kidney, ovary, placenta and developing brain. Thyroglobulin (Tg) and thyroperoxidase (TPO) genes, which are specifically expressed in thyroid follicular cells, both harbor a Pax 8 binding site in their proximal promoter region. The transcription of these genes is, as is the expression of most of the other differentiated functions of the thyrocyte, positively regulated by thyrotropin (TSH) via a cyclic-AMP (cAMP)-dependent mechanism. However, no typical cAMP-responsive element has been detected in the promoter region of Tg and TPO genes. We therefore investigated whether Pax 8 activity itself could be regulated by cAMP, which would support a role for these factors in the cAMP-dependent expression of differentiation in thyroid cells. In this paper we show that the expression of Pax 8 mRNA and proteins are increased by treatment of the thyrocyte with forskolin. This suggests that Pax 8 could indeed participate in the mediation of the transcriptional activation of thyroid specific genes by cAMP. We also show that Pax 8 are nuclear phosphoproteins, although neither their phosphorylation, nor their nuclear translocation seem to be highly regulated by cAMP. During the course of this study, a new splicing variant of dog Pax 8, termed Pax 8g, has been isolated.

Transcriptional regulation of the thyroperoxydase gene by thyrotropin and forskolin.

The expression of the gene coding for thyroperoxydase, the main enzyme involved in the synthesis of the thyroid hormones, is controlled by thyroid stimulating hormone. In vitro transcription assays performed on nuclei isolated from dog thyroid cells in primary culture showed that this control is at the transcription level, takes place rapidly (1 h) and is cyclic AMP-dependent as it is mimicked by forskolin. Insulin does not seem to be an important modulator of the thyroperoxydase gene expression.

Study of TTF-1 gene expression in dog thyrocytes in primary culture

TTF-1 is a homeodomain-containing transcription factor mainly expressed in the thyroid where it controls the tissue-specific expression of the thyroglobulin, thyroperoxidase and TSH receptor genes. It is therefore potentially implicated in the hormonal control exerted by thyrotropin via the second messenger cyclic AMP on the transcription of these genes in thyrocytes. In order to investigate whether there exists a relationship between the stimulation of the cAMP pathway and TTF-1 gene expression in these cells, we have compared the amounts of TTF-1 protein, its state of phosphorylation and its subcellular distribution in control and cAMP-stimulated dog thyrocytes in primary culture. Dog TTF-1 was expressed in bacteria as a fusion protein and antibodies were raised against the dog TTF-1 moiety. Stimulation of the thyrocytes by cyclic AMP agonist only marginally increased TTF-1 gene expression as shown for the mRNA by RNase protection assay and for the protein by immunoblotting and immunoprecipitation of extracts from 35S-methionine labelled cells. The phosphorylation state of TTF-1 was investigated by immunoprecipitation of extracts from 32P-labelled thyrocytes. Phosphorylation level appeared to be essentially unaffected by forskolin treatment of the cells. We also looked for differences in the use of phosphorylation sites by partial proteolytic digestion of immunoprecipitated 32P-labelled TTF-1 with Glu-C and Asp-N endoproteases. Comparison of radioactivity distribution amongst the generated fragments did not reveal any difference in the pattern of TTF-1 phosphorylation in control and forskolin conditions. Lastly, in situ detection of TTF-1 by immunofluorescence demonstrated that the protein was localized in the nucleus of the cells, irrespective of the culture conditions. No major change in TTF-1 gene expression upon stimulation of the thyrocyte with a cAMP agonist could thus be detected in this study. The absence of an obvious modification of the TTF-1 protein itself in response to cAMP stimulation may indicate that other transcription factor(s) or co-factor(s) are involved in the control exerted by cAMP on the expression of thyroid-specific genes.

Structure, expression and regulation of the thyroglobulin gene

de Recherche Interdisciplinaire and Service Chimie, FactP transcription control; cyclic AMP; congenital goitre; chromosome 8.

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.

Improved synthesis of DBM paper.

Abstract A modified synthesis of DBM paper is described wich is simple and easily reproducible. The method previously published has been improved in order to optimize the reaction conditions of each step. The paper obtained in this way shows a very high binding capacity for denatured DNA.

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.

Identification of a transcriptional enhancer upstream from the bovine thyroglobulin gene

The DNA sequences corresponding to a DNaseI-hypersensitive region identified previously in bovine thyroglobulin gene chromatin (Hansen et al. (1988) Eur. J. Biochem. 178, 387-393) exhibited the properties of a transcriptional enhancer in a transient assay in primary cultured dog thyrocytes, but did not so in transfected HeLa cells. By contrast to the thyroglobulin proximal promoter, the enhancer element did not require cyclic AMP stimulation of the thyrocytes to be active. Using a bi-directional deletion approach, the minimal region displaying enhancer activity has been localized between positions -1906 and -1744 relative to the thyroglobulin gene transcription start. DNA-footprinting experiments revealed the presence of several binding sites for the thyroid-specific transcription factor TTF-1 within the enhancer sequence.