Characterization of an up-stream thyroid transcription factor-1-binding site in the thyrotropin receptor promoter.
A thyroid transcription factor-1 (TTF-1)-binding element in the rat TSH receptor (TSHR) promoter, between -189 and -175 basepairs (bp), is important for both thyroid-specific expression and thyroid-specific TSH/cAMP autoregulation of the TSHR. The identification of an up-stream TTF-1-binding site and its relationship to the function of the down-stream TTF-1 element are the subjects of this report. Sequence analysis identifies a potential TTF-1 site at -878 bp; deoxyribonuclease-I footprinting shows that the -881 to -866 bp region is protected by recombinant TTF-1 protein and by nuclear extracts from FRTL-5 thyroid cells that contain TTF-1, but not by extracts from nonfunctioning FRT thyroid or Buffalo rat liver (BRL) cells, which have no TTF-1, or by Pax-8. FRTL-5, but not FRT or BRL cell nuclear extracts, form a specific protein-DNA complex with this region in gel mobility shift analyses; its formation is prevented by TTF-1-binding elements from the thyroglobulin promoter. The upstream TTF-1 site acts as an enhancer when coupled to a heterologous simian virus-40 promoter-chloramphenicol acetyltransferase (CAT) chimera and transfected into FRTL-5 thyroid cells. There is a greater increase, 3-vs. 2-fold (P < 0.05), when TSHR promoter-CAT chimeras, which contain the up-stream TTF-1 element, pTRCAT5'-907 or pTRCAT5'-886, as opposed to those in which it is deleted, pTRCAT5'-907 delta USTTF-1, are transfected into FRTL-5 cells or cotransfected with a TTF-1 expression vector into BRL cells, which have no endogenous TTF-1. The TTF-1-dependent activity of pTRCAT5'-907 delta USTTF-1 is the same as that of the minimal promoter, -220 to -39 bp, containing only the down-stream TTF-1 site in both cells. Transfection of chimeric TSHR-CAT plasmids with the down- and/or up-stream TTF-1 site deleted reveals that the down-stream TTF-1 element functions in the absence of the up-stream element, but function of the up-stream site requires the down-stream TTF-1 element. Like the down-stream TSHR TTF-1-binding site, the up-stream TTF-1 site is different from TTF-1 sites in the thyroglobulin and thyroid peroxidase promoter, in that it does not interact with Pax-8.(ABSTRACT TRUNCATED AT 400 WORDS)
Ohmori
M
M
Section on Cell Regulation, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Shimura
H
H
Shimura
Y
Y
Ikuyama
S
S
Kohn
L D
LD
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000187:drug effects; D004742:Enhancer Elements, Genetic; D015870:Gene Expression / Q000187:drug effects; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism* / Q000494:pharmacology; D015345:Oligonucleotide Probes / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D011915:Rats, Inbred BUF; D011989:Receptors, Thyrotropin / Q000235:genetics*; D000074482:Thyroid Nuclear Factor 1; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism* / Q000494:pharmacology
C000614416:Nkx2-1 protein, rat; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D011989:Receptors, Thyrotropin; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors