Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We previously reported a family with generalized resistance to thyroid hormone (GRTH) which had a point mutation with codon 448 CCT (proline) being converted to ACT (threonine) in the thyroid hormone receptor (TR) beta. To characterize functional properties of the mutant TR beta, transient expression studies were performed in COS cells. A double stranded oligonucleotide encompassing thyroid hormone response element (TRE) derived from the rat GH gene was synthesized. We constructed chloramphenicol acetyl transferase (CAT) plasmid containing the thymidine kinase promoter under the control of the rat GH TRE. T3 induction of CAT activity by the mutant TR beta was significantly reduced as compared with that of the normal TR beta. This was observed in the presence of 0.5-50 nM T3, but not at 500 nM T3. When the normal and mutant TR beta were cotransfected, the mutant TR beta inhibited gene activation regulated by the normal TR beta. However, a high molar excess was necessary to significantly inhibit the function of the normal receptor. Additionally, the binding of in vitro synthesized mutant TR beta to TRE was preserved.
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PMID:Transcriptional activity of a mutant thyroid hormone receptor beta in a family with generalized resistance to thyroid hormone. 130 92

The epidermal growth factor (EGF) receptor (EGFR) promoter is negatively regulated by thyroid hormone and retinoic acid. This regulation can be mapped to a 36-basepair GC-rich region of the promoter (EGFR P/E) that functions autonomously as a promoter and an enhancer when placed in front of the thymidine kinase gene TATA element. Direct high affinity binding of the thyroid hormone receptor (T3R) to this element requires a nuclear protein. Through ion exchange chromatography and gel filtration of HeLa nuclear extract, this activity was identified as a protein of approximately 67 kilodaltons. This protein did not bind to DNA alone, but greatly augmented T3R binding to the EGFR P/E sequence in gel mobility shift and DNA precipitation assays. When combined with the T3R auxillary protein (TRAP), the T3R migrated as a larger complex on the DNA. Chemical cross-linking identified this complex as a heterodimer between T3R and TRAP. T3R-TRAP binds to a 7-basepair site in the EGFR P/E (GGGACTC) that has weak homology to a consensus thyroid response element half-site. Thus, on this element, T3R-TRAP heterodimers contact the DNA primarily on a single site that comprises an inhibitory thyroid response element.
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PMID:A nuclear protein is required for thyroid hormone receptor binding to an inhibitory half-site in the epidermal growth factor receptor promoter. 158 25

In the presence of its ligand, thyroid hormone receptor (T3R) binds specifically to DNA sequences near a number of genes and induces their expression. We show that in the absence of the hormone, a T3R binding site acts in cis to decrease expression from such genes. The endogenous T3 receptors in rat pituitary cell lines are sufficient to mediate this effect, as shown by comparisons of basal levels of expression directed by transiently transfected plasmids containing the rat growth hormone promoter with wild-type or point-mutated T3 response elements (T3RE). The magnitude of the negative effect is increased by increasing the strength of the T3RE or by raising intracellular levels of T3R by appropriate transfections. T3REs exert a similar negative effect on the herpes virus thymidine kinase (TK) promoter; this effect is dependent on expression of functional T3 aporeceptor (apoT3R). Analysis of a set of T3REs of increasing strength inserted upstream of the TK promoter showed a strong correlation between the level of induced expression in the presence of hormone and the level of repressed expression in the absence of hormone. These results show that, unlike other members of the nuclear hormone receptor family, T3R binds to specific DNA sequences in the absence of hormone and exerts a negative effect on expression of linked genes. The apparent affinity of apoT3R and hormone-bound T3R for a T3RE was assessed by using varying amounts of T3R expression vector in a transfection dose response assay.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thyroid hormone aporeceptor represses T3-inducible promoters and blocks activity of the retinoic acid receptor. 256 25

Thyroid hormone regulation of the human thyrotropin beta-subunit gene (TSH beta) was examined in a human embryonal cell line (293). Transient expression studies were performed with chimeric plasmids containing the reporter gene, chloramphenicol acetyltransferase. Sequences in the first exon between +9 and +37 base pairs (bp) enhanced gene expression from the human TSH beta promoter in the absence of thyroid hormone as well as mediated a concentration-dependent triiodothyronine (L-T3) decrease in gene expression. Thyroid hormone inhibition of expression was also conferred to the herpes simplex virus thymidine kinase promoter by inserting +3 to +37 bp of the human TSH beta gene downstream from the start of transcription. Primer extension analysis of RNA from transfected cell cultures revealed accurate transcription initiation in only those constructs which contained sequences between +9 and +37 bp. Moreover, RNA analysis confirmed that L-T3 inhibition of chloramphenicol acetyltransferase activity from chimeric pTSH beta CAT constructs occurred at a pretranslational level. In addition, a nuclear thyroid hormone receptor, c-erbA-beta, bound to this region in an avidin-biotin DNA binding assay. These data suggest that L-T3, bound to its receptor, may inhibit human TSH beta expression by interfering with an element that functions to enhance gene expression.
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PMID:Thyroid hormone inhibition of human thyrotropin beta-subunit gene expression is mediated by a cis-acting element located in the first exon. 276 33

We have extensively characterized the sequences of the rat growth hormone (rGH) promoter required for induction by T3 (thyroid hormone, 3,5,3'-L-triiodothyronine) in a transient transfection system. Oligonucleotides containing portions of the rGH promoter sequence with various deletions and point mutations were placed upstream of the first 137 base pairs of the rGH promoter or the heterologous herpes virus thymidine kinase promoter in chloramphenicol acetyltransferase expression vectors. The rGH137 and thymidine kinase promoters show no or minimal response to T3 in the basal state. The constructs were tested in GH4C1 rat pituitary cells and COS cells (functionally deficient in thyroid hormone receptor) with and without a co-transfected plasmid expressing a beta type c-erbA gene coding for a functional T3 receptor. Oligonucleotides containing the T3 receptor binding site confer hormone-dependent induction in a manner that is independent of either orientation or variation in position on the helix relative to the promoter. Point mutations in the sequence -189 to -173 result in loss of T3 induction, and bases between -173 and -167 were also required for a full T3 response. The minimal length to confer T3 induction to the rGH promoter was 23 base pairs (-190 to -167). Point mutations creating a perfect duplication of 7 base pairs within the receptor binding site conferred 12-fold T3 response to the rGH137 promoter, 3-fold greater than the wild type rGH237 construct. T3 inductibility was also transferred to the thymidine kinase promoter by an oligonucleotide containing the sequence -200 to -157, demonstrating that cell type specific elements located 3' to 157 of the rGH promoter are not required for thyroid hormone responsiveness.
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PMID:Functional characterization of the rat growth hormone promoter elements required for induction by thyroid hormone with and without a co-transfected beta type thyroid hormone receptor. 290 14

The substance 3,5,3-triiodothyronine (T3) stimulates growth hormone gene transcription in rat pituitary tumour cells. This stimulation is thought to be mediated by the binding of nuclear T3 receptors to regulatory elements 5' to the transcriptional start site. Understanding of the mechanism by which thyroid hormone activates gene transcription has been limited by failure to purify nuclear T3 receptors because of their low abundance, and by the absence of defined T3 receptor-DNA binding sites affecting T3 regulation. Recently, human and avian c-erb-A gene products have been shown to bind thyroid hormone with high affinity and to have a molecular weight and nuclear association characteristic of the thyroid hormone receptor. In the present report, we describe the development of an avidin-biotin complex DNA-binding assay which can detect specific, high-affinity binding of rat pituitary cell T3 receptors to the sequence 5'CAGGGACGTGACCGCA3', located 164 base pairs 5' to the transcriptional start site of the rat growth hormone gene. An oligonucleotide containing this sequence transferred T3 regulation to the herpes simplex virus thymidine kinase promoter in transfected rat pituitary GC2 cells, and specifically bound an in vitro translation product of the human placental c-erb-A gene. The data provide supporting evidence that the human c-erb-A gene product mediates the transcriptional effects of T3 and also that GC2 cell nuclear extracts contain additional factors that modify the binding of pituitary T3 receptors to the rat growth hormone gene T3 response element.
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PMID:A c-erb-A binding site in rat growth hormone gene mediates trans-activation by thyroid hormone. 331 46

3,5,3,'-Triiodothyroacetic acid (Triac) has been used in therapy of resistance to thyroid hormone on an empirical basis and appears beneficial in some studies. We observed that the T3 analogs, Triac and 3,5,3'-triiodothyropropionic acid (Triprop), have a higher affinity for the thyroid hormone receptor-beta 1 (TR beta 1) than does T3 (2.7- and 1.8-fold, respectively), whereas the affinities of the three compounds for TR alpha 1 are the same. To evaluate whether T3 analogs would have a differential effect on TR beta 1 and TR beta 1 mutants and thus be a specific treatment for patients with resistance to thyroid hormone, we examined the induction of the transcriptional activation of wild-type (wt) TR alpha 1, TR beta 1, and mutant TR beta 1s by T3, Triac, and Triprop. The dose response of transcriptional activation by T3 analogs was measured by transient cotransfections with TRs and a rat malic enzyme-TRE fused to thymidine kinase (TK)-chloramphenicol acetyltransferase (CAT) in COS-1 cells. For TR alpha 1 wt, induction of CAT activity by T3 and Triac occurred at the same concentration. For TR beta 1 wt, Triac and Triprop showed a higher maximal activity than T3 (Tripro > Triac > T3) and reached 50% induction at a lower concentration than T3 (Tripro < Triac < T3). Induction of CAT activity in five mutant TR beta 1s (kindreds Mh, Mc, CL, Mf, and GH) was also analyzed. Even high levels of T3 analogs could not restore CAT activity to that of TR beta 1wt for any mutant. A dominant negative effect was produced by Mh, Mc, and Mf. Mutants CL and GH had a mild dominant negative effect depending on T3 analog concentrations and TREs. Cotransfection studies were performed using a rat malic enzyme-TK-CAT reporter plasmid to analyze the effects of hormones at near-physiological concentrations of T3 and Triac. Triac had a significantly higher transcriptional activation than T3 in Mc, CL, and GH, suggesting that Triac would have a beneficial effect to different degrees for different mutant TR beta 1s. Using mutants Mc and GH, further studies were carried out using rat GH and double palindromic and inverted palindromic TREs in COS-1 cells. On each TRE, 10 nmol/L Triac induced higher transcriptional activation in TR beta 1wt, mutant TR beta 1s, and TR beta 1wt plus mutant TR beta 1s (1:1 ratio) than the same dose of T3.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Triiodothyroacetic acid has unique potential for therapy of resistance to thyroid hormone. 760 51

Transcription of the glycoprotein hormone alpha gene is repressed by the thyroid hormone receptor (TR) in a hormone dependent manner. Previous studies identified a TR binding site immediately downstream of the TATA box. Site directed mutagenesis and transient gene expression studies were used to evaluate the role of this TR binding site as a negative thyroid response element (nTRE). Mutagenesis of the putative negative thyroid response element (nTRE) site eliminated TR binding but failed to eliminate negative regulation by T3. A mutation which converted the putative nTRE to a higher affinity palindromic element did not enhance repression, but rather eliminated thyroid hormone dependent negative regulation. Proximal alpha promoter sequences between -100 and +44 were replaced with a heterologous thymidine kinase promoter resulting in a construct that was not repressed by T3 treatment. This finding confirmed that repression required proximal alpha promoter sequences and also indicated that repression did not occur by interference with the function of upstream the alpha gene enhancers. These studies indicate that TR binding adjacent to the TATA box is not required for T3 mediated repression of the alpha promoter and suggest that negative regulation may involve protein-protein interactions with promoter-specific transcription factors.
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PMID:Negative regulation of the glycoprotein hormone alpha gene promoter by thyroid hormone: mutagenesis of a proximal receptor binding site preserves transcriptional repression. 769 Jul 22

The functionally inactive thyroid hormone receptor splicing variant-alpha 2 (TRv alpha 2) can inhibit transcriptional activation by TR alpha 1 or beta 1, demonstrating a dominant negative effect (DNE). We examine here the three commonly proposed mechanisms, namely, competition for binding to thyroid hormone response elements (TREs), formation of inactive heterodimers, and squelching. A mutation introduced into the DNA-binding domain (DBD) of the TRv alpha 2 was designed to prevent its binding to TREs. In transient cotransfection studies, the DBD mutant has nearly the same DNE as does TRv alpha 2 on three different TRE-containing reporter genes. The DNE of TRv alpha 2 is also not reversed by cotransfection with excess retinoid X receptor-alpha. Extracts of COS cells cotransfected with TR alpha 1 and either TRv alpha 2 or DBD mutant at different ratios were analyzed by gel shift assays. Neither TRv alpha 2 or the mutant altered binding of TR alpha 1 to four radiolabeled TREs. TRv alpha 2 itself can inhibit constitutive transactivation by a thymidine kinase promoter-driven reporter construct. Our results suggest that TRv alpha 2 can function in a dominant negative manner without binding to a TRE, at least for certain TREs. It is concluded that the DNE of TRv alpha 2 may occur through another unrecognized mechanism, perhaps by binding to basal transcription factors.
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PMID:The dominant negative effect of thyroid hormone receptor splicing variant alpha 2 does not require binding to a thyroid response element. 776 Aug 53

Rat Rev-erbA alpha (rRev), which is related to thyroid hormone receptor (TR), is a conserved member of the nuclear hormone receptor superfamily whose physiological roles are unknown ("orphan" receptor). We studied DNA binding of rRev in vitro by electrophoretic mobility shift assay. A fusion protein was constructed, called NGR.Rev, containing part of the N terminus of the glucocorticoid receptor fused to nearly full-length rRev. Inasmuch as rRev and TR share homology in their DNA-binding domains, we tested binding to three different thyroid hormone response elements (TREs) in which the half-sites are arranged in different orientations. NGR.Rev bound direct repeats (DR4), but not palindromic (TREpal) or inverted palindromic (F2H) repeats. Also, transfection of CV1 cells with a reporter gene containing the luciferase gene under control of the inducible thymidine kinase promoter resulted in an increase in luciferase activity when NGR.Rev was cotransfected and when the thymidine kinase promoter contained DR4. In addition, a series of deletions in the ligand-binding domain of NGR.Rev revealed regions that can modulate DNA binding. Finally, we studied DNA binding of bacterially produced fusion proteins that contain the DNA-binding domains of rRev or rTR alpha fused to glutathione S-transferase, to a panel of natural TREs. Our results indicate that Rev binds DNA with a different specificity than TR alpha-1 and might be involved in the regulation of a subset of thyroid hormone-regulated genes.
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PMID:Rat Rev-erbA alpha, an orphan receptor related to thyroid hormone receptor, binds to specific thyroid hormone response elements. 801 47


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