GATA-6 activates transcription of thyroid transcription factor-1.

Thyroid transcription factor-1 (TTF-1) is expressed in respiratory epithelial cells, where it regulates the transcription of target genes expressed in a cell-selective manner. GATA-5 and -6, members of the zinc finger family of transcription factors, are also expressed in various cell types within in the developing lung. In the present work, GATA-6 mRNA was detected in adult mouse lung, purified mouse type II epithelial cells, and differentiated mouse pulmonary adenocarcinoma cells (MLE-15 cells), being co-expressed with TTF-1 mRNA. In order to test whether GATA factors regulated TTF-1 gene transcription, GATA-5 and -6 expression vectors were co-transfected with TTF-1 luciferase expression vector. GATA-6, but not GATA-5, markedly activated TTF-1 gene transcription in HeLa cells. EMSA and supershift analysis with GATA-6 antiserum demonstrated that GATA-6 in MLE-15 cell nuclear extracts bound to an element located 96-101 base pairs from major start of TTF-1 gene transcription. Site directed mutagenesis of the GATA element in the TTF-1 promoter region inhibited transactivation by GATA-6 in HeLa cells. GATA-6 is co-expressed with TTF-1 in the respiratory epithelium in vivo and respiratory epithelial cells in vitro. GATA-6 strongly enhanced activity of the human TTF-1 gene promoter in vitro. These findings support the concept that GATA-6 may play an important role in lung cell differentiation and gene expression, at least in part by altering the expression of TTF-1 and its potential targets.

Hepatocyte nuclear factor 3 activates transcription of thyroid transcription factor 1 in respiratory epithelial cells.

Thyroid transcription factor 1 (TTF-1), hepatocyte nuclear factor 3alpha (HNF-3alpha), and HNF-3beta regulate the transcription of genes expressed in the respiratory epithelium. To test whether members of the HNF-3/forkhead family influence TTF-1 gene expression, deletion constructs containing the 5' region of the human TTF-1 gene were transfected into immortalized mouse lung epithelial (MLE) cells. DNase I protection and electrophoretic mobility shift assays identified elements in the 5' region of the TTF-1 gene that bound MLE cell nuclear proteins consistent with the binding of HNF-3 to sites at positions -135 to -124 and -14 to -3. In MLE cells, TTF-1-luciferase reporter constructs were activated by cotransfection with HNF-3beta, activated to a lesser extent by HNF-3alpha, but not activated by HFH-8. HNF-3alpha. and HFH-8 inhibited the activation of TTF-1-luciferase by HNF-3beta. Site-specific mutagenesis of each of the HNF-3 binding sites in the human TTF-1 gene inhibited the binding of MLE cell nuclear proteins and inhibited transactivation of the TTF-1-luciferase constructs after cotransfection with HNF-3beta. Immunohistochemical staining demonstrated that both HNF-3beta and TTF-1 were detected in bronchiolar and alveolar type II cells in the human lung. Modulation of TTF-1 gene expression by members of the HNF-3/forkhead family members may provide a mechanism by which distinct HNF-3/forkhead family members influence respiratory epithelial cell gene expression and cell differentiation.

Gene structure and expression of human thyroid transcription factor-1 in respiratory epithelial cells.

The human gene encoding thyroid transcription factor-1 (TTF-1), a homeodomain-containing nuclear transcription protein of the Nkx2 gene family, was isolated and characterized. Human TTF-1 was encoded by a single gene locus spanning approximately 3.3 kilobases and consisted of two exons and a single intron. The TTF-1 cDNA and polypeptide of 371 amino acids have been highly conserved, sharing 98% identity with the rat TTF-1 polypeptide. Human TTF-1 mRNA and polypeptide were selectively expressed in human and mouse pulmonary adenocarcinoma cell lines. In addition to its presence in thyroid gland epithelium, the human TTF-1 protein was detected by immunohistochemistry in human fetal lung as early as 11 weeks of gestation, being localized in the nuclei of epithelial cells of the developing airways. After birth, TTF-1 was selectively expressed in Type II epithelial cells in the alveoli and in subsets of bronchiolar epithelial cells in the conducting regions of the lung. The 5'-flanking region of the human TTF-1 gene directed transcription of luciferase cDNA in a lung epithelial cell-selective manner. The conservation and distribution of TTF-1 in the human respiratory tract support its role in the regulation of lung development and surfactant homeostasis.

Expression of the lacZ gene targeted to the HPRT locus in embryonic stem cells and their derivatives.

Transgenes in mice often exhibit different expression patterns in different transgenic lines. While the basis for this phenomenon is not understood, it is widely believed that the site at which the transgene becomes integrated into the mouse genome is a major factor in determining the pattern of expression. Most transgenic mice have been produced by microinjection of DNA into the male pronucleus, which results in integration of tandem arrays of the transgene at random chromosomal sites. In the experiments described in this report, electroporation of embryonic stem (ES) cells was used to place single copies of a lacZ transgene into either random sites or into the HPRT (hypoxanthine phosphoribosyl transferase) locus of the mouse genome. Expression of lacZ was assayed by histochemical staining for Escherichia coli beta-galactosidase activity in ES cells and in differentiated derivatives obtained by teratocarcinoma formation. Several of the randomly integrated cell lines expressed lacZ at high levels in a variety of cell types present in the tumours, but most notably in epithelial cells. Targeted cell lines with lacZ in opposite orientation to the direction of HPRT gene transcription also expressed well in epithelial cells, but the targeted cell lines did not express in a wider variety of cell types than some of the nontargeted cell lines. Targeted cell lines transcribing lacZ in the same orientation as HPRT transcription did not express high levels of lacZ in any differentiated cell type. Analysis of transcripts suggested that this orientation effect may have been the result of transcriptional interference perpetrated by the HPRT gene promoter.