Identification of a novel insulin-responsive element in the rat thyrotropin receptor promoter.
By transfecting TSH receptor (TSHR)-chloramphenicol acetyltransferase (CAT) chimeras into FRTL-5 thyroid cells in the presence or absence of insulin, we identify an insulin-responsive element (IRE) between -220 and -190 bp of the TSHR 5'-flanking region. The region between -220 and -192 bp is footprinted by nuclear extracts from FRTL-5 cells and, coupled to a heterologous SV40-CAT chimera, an oligonucleotide containing the protected region induces insulin responsiveness in FRTL-5 cells. FRTL-5 cell nuclear extracts form two groups of protein-DNA complexes, A and B, in gel shift assays using an oligonucleotide having the protected sequence; mutation data indicate only the A complexes are increased by exposure of FRTL-5 cells to insulin; TSH can also increase A complex formation, but the TSH action is insulin-dependent. The nuclear factor(s) in FRTL-5 cells that interact with the TSHR IRE are distinct from thyroid transcription factor-2 (TTF-2), the insulin regulatory factor of the thyroglobulin promoter, as evidenced by the absence of competition in gel shift assays; there is no apparent sequence similarity of this region with other known IREs. The IRE is immediately upstream of a thyroid transcription factor-1 (TTF-1) binding site, -189 to -175 bp; mutation of the TTF-1 site causing a loss of TTF-1 activity also causes a loss of insulin responsiveness when the TSHR-CAT chimera at -220 bp is transfected into FRTL-5 cells and an altered IRE footprint by nuclear extracts. The TSHR appears, therefore, to contain a novel IRE whose activity depends at least in part on TTF-1, a thyroid-specific, homeodomain-containing transcription factor important both for thyroid-specific TSHR gene expression and TSH/cAMP autoregulation of the TSHR.
Shimura
Y
Y
Section on Cell Regulation, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
Shimura
H
H
Ohmori
M
M
Ikuyama
S
S
Kohn
L D
LD
D000818:Animals; D001483:Base Sequence; D000242:Cyclic AMP / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D007328:Insulin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011989:Receptors, Thyrotropin / Q000235:genetics*; D016415:Sequence Alignment; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship; D013954:Thyroglobulin / Q000235:genetics; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D004268:DNA-Binding Proteins; D007328:Insulin; C000614416:Nkx2-1 protein, rat; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011989:Receptors, Thyrotropin; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; D013954:Thyroglobulin; D000242:Cyclic AMP