Thyrotropin and adenosine 3',5'-monophosphate stimulate the activity of the ferritin-H promoter.
Fragments of the rat ferritin-H 5'-flanking region up to 1 kilobase in length were generated by the polymerase chain reaction using FRTL5 rat thyroid cell genomic DNA as template. Ferritin-H 5'-flanking region fragments of 219, 351, 666, and 1046 basepairs (bp), ligated up-stream to the reporter gene luciferase, were transiently transfected into FRTL5 thyroid cells and NIH-3T3 mouse fibroblasts. In both cell types, constitutive (nonstimulated) ferritin-H promoter activity increased progressively with constructs containing increasing lengths of 5'-flanking region. TSH or (Bu)2cAMP (dBcAMP) stimulation of FRTL5 cells transfected with the shorter (219 and 351 bp) ferritin-H 5'-flanking region fragments increased promoter activity 2- to 3-fold. However, with the longer DNA segments (666 and 1046 bp), the extent of TSH stimulation was less. Exposure of transfected NIH-3T3 cells to dBcAMP mimicked in all respects the effects of TSH and dBcAMP on ferritin-H promoter activity in FRTL5 cells. Transcription initiation sites in the luciferase reporter gene were unaffected by the length of the ferritin-H 5'-flanking region included in the construct or by dBcAMP stimulation. Plasmid constructs with 45 bp of the ferritin-H 5'-flanking region containing a potential cAMP response element did not reveal any promoter activity or dBcAMP responsiveness in this region. Gel shift mobility assays with the -219 bp ferritin-H 5'-flanking region fragment and NIH-3T3 nuclear proteins revealed specific protein-DNA interaction. Reduced DNA mobility was inhibited by excess unlabeled probe DNA, but not by DNA fragments corresponding to the recognition sites for a variety of known trans-activating factors.(ABSTRACT TRUNCATED AT 250 WORDS)
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Thyroid Molecular Biology Laboratory, Veterans Administration Medical Center, San Francisco, California 94121.
Wadsworth
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Foti
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Rapoport
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D000818:Animals; D001483:Base Sequence; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology*; D005293:Ferritins / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D016385:TATA Box; D013972:Thyrotropin / Q000494:pharmacology*; D014158:Transcription, Genetic; D014162:Transfection
D003994:Bucladesine; D013972:Thyrotropin; D005293:Ferritins; D000242:Cyclic AMP; D008156:Luciferases