Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 6.3-kbp segment of DNA, upstream of the human thyroid peroxidase gene, and various deletions thereof were linked to a promoterless bacterial chloramphenicol acetyltransferase reporter gene. These constructs were analyzed by transfection and expression in rat FRTL-5 thyroid cells and in human hepatoma HepG2 cells to localize sequences that are important for thyroid cell-specific expression of the thyroid peroxidase gene. A thyroid-specific enhancer element, capable of activating enhancerless simian virus 40 promoter expression in FRTL-5 cells, was localized to a 230-bp region approximately 5.5 kbp upstream of the human thyroid peroxidase gene transcription start site. DNase I footprinting, using nuclear extracts prepared from FRTL-5 cells, revealed three regions within the 230-bp fragment; none of these regions were protected by nuclear extracts from HepG2 cells. Gel mobility shift assays, using double-stranded oligonucleotides corresponding to the three protected regions, further confirmed the existence of factors in FRTL-5 cells, but not HepG2 cells, able to specifically bind to the enhancer sequences. These results suggest the presence of three cis-acting DNA elements in the human thyroid peroxidase gene enhancer that interact with thyroid-specific trans-acting factors.
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PMID:Characterization of a thyroid-specific enhancer located 5.5 kilobase pairs upstream of the human thyroid peroxidase gene. 217 2

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)
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PMID:Characterization of an up-stream thyroid transcription factor-1-binding site in the thyrotropin receptor promoter. 782 40

The chimeric chloramphenicol acetyltransferase (CAT) construct, pTRCAT5'-199, containing the TSH receptor (TSHR) minimal promoter, -199 to -39 base pairs (bp), exhibits the thyroid specificity and TSH/cAMP autoregulation evident in TSHR gene expression. The present report shows that a cis-acting element between -189 and -175 bp, which binds thyroid transcription factor-1 (TTF-1), is involved in both activities. The 22 bp between -199 and -178 contains a positive element important for expression of the TSHR minimal promoter in rat FRTL-5 thyroid cells. DNAase I footprinting shows that extracts from functioning FRTL-5, but not non-functioning FRT thyroid or Buffalo rat liver (BRL) cells, protect a region between -189 and -175 bp. The protection is duplicated by TTF-1, and the protected element has only a two-base mismatch from the consensus TTF-1 element identified in the thyroglobulin (TG) and thyroid peroxidase minimal promoters. Gel mobility shift analyses reveal that FRTL-5 thyroid cell nuclear extracts form a specific protein/DNA complex with this region, which is prevented by the TTF-1 binding element from the TG promoter; FRT and BRL cell nuclear extracts do not have TTF-1 and do not form this complex. A role for the TSHR/TTF-1 binding element in thyroid-specific expression of the TSHR gene is evidenced as follows. Overexpression of TTF-1 in FRT or BRL cells, which have no TTF-1, increased the activity of pTRCAT5'-199, but not pTRCAT5'-177, which has no TTF-1 binding element. A nonsense mutation of the TTF-1 binding element eliminated TTF-1-induced activation of TSHR promoter activity in FRT or BRL cells and reduced TSHR promoter activity in FRTL-5 thyroid cells. In contrast, mutation of this element to the TTF-1 consensus sequence of the TG or thyroid peroxidase promoter had no significant influence on TSHR promoter activity. The activity of the TSHR/TTF-1 binding element requires a functioning cAMP response element (CRE). Thus, TTF-1 activity is lost when the CRE site is mutated to a nonfunctional, nonpalindromic sequence; it is, in contrast, maximized when CRE activity is maximized by its mutation to a consensus AP1 element. TTF-1 phosphorylation is important for binding and activity. Thus, binding of TTF-1 to the TSHR/TTF-1 element is phosphatase-sensitive and is increased by treating nuclear extracts with the catalytic subunit of protein kinase A. Overexpression of the catalytic subunit of PKA enhances TTF-1-increased activity of the TSHR minimal promoter.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Thyroid-specific expression and cyclic adenosine 3',5'-monophosphate autoregulation of the thyrotropin receptor gene involves thyroid transcription factor-1. 799 32

Interferon-gamma (IFN gamma) is known to suppress the expression of thyroid-specific genes, such as thyroglobulin, thyroid peroxidase, and the TSH receptor (TSHR). In the present study, we show that this reflects, in part, a transcriptional action mediated by thyroid transcription factor-1 (TTF-1). Thus, transfected into rat FRTL-5 cells, the activity of reporter plasmids, containing rat TSHR promoter ligated to a chloramphenicol acetyltransferase gene, was significantly suppressed in the presence of rat IFN gamma. A -199-bp promoter construct showed the greatest suppression by IFN gamma whereas a -177-bp construct, in which the TTF-1 binding site was deleted, showed less suppressibility. The suppressive effect was rat IFN gamma-specific, since human IFN alpha, -beta, and -gamma exhibited no significant effects. The effect was concentration-dependent from 3-50 U/ml. In FRT rat thyroid cells that do not express TTF-1, IFN gamma-induced suppression on the promoter activity was not observed. In addition, when the TTF-1 binding site was mutated so that TTF-1 can not bind, IFN gamma-induced suppression was significantly reduced. In gel mobility shift analyses, a protein-DNA complex formed by TTF-1 was reduced when the nuclear extract prepared from IFN gamma-treated FRTL-5 cells was used; however, expression of TTF-1 mRNA and TTF-1 protein, which were assessed by Northern blot analysis and Western blot analysis, respectively, were not affected by IFN gamma treatment of FRTL-5 cells. Instead, reduction of DNA-binding affinity of TTF-1 was evident when competition analysis was performed in gel mobility shift analysis. From these results, we conclude that IFN gamma suppresses TSHR promoter activity, in part, by reducing TTF-1 binding to its recognition site. We also raise the possibility that the suppressive effect of IFN gamma on promoter activity is mediated by additional element(s) and factor(s) downstream of the TTF-1 site.
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PMID:Interferon-gamma suppresses thyrotropin receptor promoter activity by reducing thyroid transcription factor-1 (TTF-1) binding to its recognition site. 881 23