Thyroid hormones regulate rat thyrotropin beta gene promoter activity expressed in GH3 cells.
Thyroid hormones suppress the synthesis of TSH in part by decreasing the rate of alpha and TSH beta gene transcription. Cis-acting DNA sequences present in the rat TSH beta 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 TSH beta 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 TSH beta 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).2+he
Carr
F E
FE
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
Burnside
J
J
Chin
W W
WW
D000818:Animals; D002478:Cells, Cultured; D002678:Chimera; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002872:Chromosome Deletion; D004720:Endonucleases; D005786:Gene Expression Regulation; D009154:Mutation; D010902:Pituitary Gland / Q000166:cytology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013972:Thyrotropin / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014284:Triiodothyronine / Q000502:physiology*
D014284:Triiodothyronine; D013972:Thyrotropin; D015500:Chloramphenicol O-Acetyltransferase; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases