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Thyroid Hormones Regulate Rat Thyrotropinβ Gene Promoter Activity Expressed in GH₃ Cells

Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Howard Hughes Medical Institute and Harvard Medical School Boston, Massachusetts 02115
Department of Clinical Investigation, Kyle Metabolic Unit, Walter Reed Army Medical Center Washington, DC 20307

Address requests for reprints to: Dr. Frances E. Carr, Department of Clinical Investigation, Kyle Metabolic Unit, Walter Reed Army Medical Center, Washington, DC 20307.

Abstract

Thyroid hormones suppress the synthesis of TSH in part by decreasing the rate of and TSHβ gene transcription. Cis-acting DNA sequences present in the rat TSHβ subunit gene that are induced in transcriptional regulation by thyroid hormone have been identified by deletion-mutation and transient expression studies. Plasmid expression vectors were constructed including 2900, 900, 204, 77, 17 base pairs (bp) of 5'-flanking sequence and exon (5'-untranslated sequence, transcriptional start sites) fused to the coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene. The transfected chimaeric plasmids demonstrated expression (with TSHβ DNA sequences in the 5'- to -3'-but not 3'- to -5'-orientation) in both a clonal pituitary cell line, GH₃, and primary pituitary cell cultures, both of which are responsive to thyroid hormones. T₃ (10^–11 M to 10^–7 M) treatment of transfected cells produced a dose-dependent decrease in CAT expression with a maximal 70% decrease at 10^–8 M. While a decrease in the basal level of expression was noted with progressive removal of both 5'-flanking and intronic sequences adjacent to exon 1, the fold-decrease in response to T₃ was equivalent even in the 57 bp construct. In contrast, T₃ had no effect on CAT expression directed by the promoter of the herpes simplex virus thymidine kinase gene. Thus, the rat TSHβ gene 5'-flanking region can direct heterologous gene expression in GH₃ cells and contains sequences which have properties of a putative cis-active T₃ responsive regulatory element(s). The cis-element(s) resides within a 57 bp DNA fragment which includes 17 bases of 5'-flanking sequence, exon 1, and 13 bases of the first intron.

FOOTNOTES

The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.

Received for publication October 31, 1988. Accepted for publication January 24, 1989.

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