Transcriptional control in the thyrocyte
IRIBHM within the Institute of Molecular Biology and Medicine in Charleroi (Gosselies)
IBMM, rue des Profs Jeener et Brachet 12, 6041 Gosselies (Belgium), rooms AE-3-109, AE-3-110 & AE-3-311
Web : http://www.ulb.ac.be/medecine/iribhm/ & http://www.ulb.ac.be/ibmm/
Our group investigates the mechanisms of transcriptional control in the thyrocyte, essentially that of genes involved in the cell-specific function, that is the production of the thyroid hormones T4 and T3. These genes can be classified into two classes: true thyroid-specific genes, as are the thyroglobulin and thyroperoxidase genes, and genes that are also expressed in other tissues, often at lower levels than in the thyrocyte, as are the iodide symporter, thyrotropin receptor, ThOXs and DUOXAs genes. Thyroid-specific promoter and enhancer elements have been identified, in front of the former genes, whereas in the case of the iodide symporter gene for example a more or less ubiquitous promoter and a thyroid-specific enhancer were identified. Specific transcription factors that bind these promoters in the thyrocyte are mainly TTF-1 and -2 (Thyroid Transcription Factor-1 and -2) and Pax 8. TTF-1 is a homeodomain-containing protein, TTF-2 belongs to the forkhead family of transcription factors, and Pax 8 contains a paired-box domain.
Our main current research lines are summarized below.
-1) identification of the whole sets of genes that are controlled by the transcription factors TTF-1, TTF-2 and Pax 8 within the thyrocyte
Background: From studies performed in other labs, it appeared that the roles of these transcription factors in fully differentiated thyrocytes could not be revealed in K.O. mice as none of these mice develop a normal mature thyroid. In the case of TTF-1 a conditional K.O. has also been produced but as only a partial deletion of the gene could be achieved the information gained in this approach was very limited.
Our current approach relies on our earlier observation that when produced in the cell the isolated TTF-1 homeodomain is able to compete with the endogenous protein for promoter binding (see C. CHRISTOPHE-HOBERTUS, P. VAN RENTERGHEM, B. PICHON and D. CHRISTOPHE: Expression of a transactivation-deficient form of thyroid transcription factor I decreases the activity of co-transfected thyroglobulin and thyroperoxidase promoters. FEBS Letters (1996), 399, 140-142). In order to improve the transcriptional effect on TTF-1 dependent genes, the homeodomain was fused to the strong repressive domain from the Drosophila engrailed protein (“eng-HD”). As a control, another fusion protein containing point mutations in the homeodomain that abolish DNA binding was also constructed (“eng-HDm”). In transient transfection experiments in PCCl3 cells, eng-HD expression specifically and dramatically reduced the activity of the co-transfected thyroglobulin promoter as compared to that of a control SV40 promoter, whereas eng-HDm expression did not affect the activity of these promoters. PCCl3 cell lines expressing eng-HD or eng-HDm conditionally (using the Tet-off system) were constructed. Induction of eng-HD expresion entailed a clear change in the morphology of the cells. The consequences on gene expression were investigated in microarray hybridization experiments. Genes linked to thyrocyte differentiation were down-regulated, whereas genes linked to loss of epithelial phenotype and growth arrest were up-regulated. The most significant microarray results were validated by performing Q-PCR experiments. ChIP experiments will now be performed in order to identify relevant genes that constitute direct targets for TTF-1 transcriptional activity.
Similar work will also be developed in the future using the Eng-PD and Eng-PDm fusion proteins (containing the Pax 8 paired domain), and also the Eng-FK and Eng-FKm fusion proteins (containing the Forkhead domain of TTF-2) that were constructed in the lab on the basis of the same concept as that used in the case of the eng-HD and eng-HDm fusions, in order to identify the sets of genes controlled by transcriptions factors Pax 8 and TTF-2 respectively.
-2) characterization of the promoter regions of the ThOXs and DUOXAs genes
Background: Both ThOX-1 and DUOXA-1 genes on one side, and ThOX-2 and DUOXA-2 genes on the other side are coupled in a head to head configuration and only a very limited space is left between the supposed transcription starts in each of the couples.
The precise transcription start sites of all four genes were recently identified using RLM-RACE on mRNA preparations from both rat thyroid and PCCl3 cells separately (see
C. CHRISTOPHE-HOBERTUS and D. CHRISTOPHE: Delimitation and functional characterization of the bidirectional THOX-DUOXA promoter regions in thyrocytes; Mol. Cell. Endocrinol. (2010), 317, 161-167). It revealed that only 63pb separates the transcription starts of ThOX-1 and DUOXA-1 genes, whereas in the case of the ThOX-2 and DUOXA-2 genes the distance is 170pb. Using a bidirectional reporter construct we showed that the corresponding DNA sequences act as bidirectional promoters. In vitro DNA binding experiments and functional mutagenesis assays revealed that a unique Sp1 binding site plays a significant role in the control of both ThOX-1 and DUOXA-1 transcription in PCCl3 cells. Further functional characterization of these promoters and the search for the existence of thyroid-specific enhancer sequence(s) involved in the control of their activity are currently in progress.
-3) construction of a thyroid cell line containing a reporter allele for the thyroglobulin gene
Aim: to create a cell line for the easy investigation of the transcriptional control of this gene in conditions where the normal genomic and chromatin contexts are preserved as far as possible. This experimental model should allow us to address questions relating to epigenetic modifications implicated in the control of thyroglobulin gene transcription for example.
We are presently trying to obtain a knock-in cell line in which the thyroglobulin coding sequence is replaced by that of the reporter protein SEAP on one of the two alleles of the gene in rat thyroid PCCl3 cells. In order to preserve the sequences surrounding the transcription start site, the coding sequence of SEAP devoid of the part coding the signal peptide was fused to that of the amino-terminal end of thyroglobulin in exon 2 of the gene. The resulting fusion protein is secreted in the culture medium and exhibits detectable phosphatase activity. To avoid the insertion of another promoter close to that of the thyroglobulin gene itself, the expression of resistance to neomycin was coupled to that of the thyroglobulin-SEAP fusion by using an IRES element. The construct proved to be functional in transfected PCCl3 cells. The screening for cell clones in which the expected homologous recombination event occurred is currently in progress.