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)

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

Uncoupling protein (UCP) is essential to brown adipose tissue (BAT) thermogenesis and, hence, to cold adaptation and energy balance. The sympathetic nervous system, via norepinephrine and cAMP, and thyroid hormone seem to be the major regulators of UCP expression. T3 potentiates the effect of norepinephrine and is essential for the adaptive response of this protein to cold. The goal of the present studies was to investigate whether T3 directly stimulates the transcription of the rat UCP gene, as suggested by in vivo results, and if so, to identify and characterize the sequences involved. We examined the gene sequence between 114 and -3623 by transient transfection analysis in JEG-3 and HIB-1B cells, a BAT-derived cell line. This 3.7-kilobase UCP insert makes the reporter gene responsive to cAMP (4-fold), T3 (4-fold), or both combined (12-fold). We identified an 82-basepair (bp) restriction fragment between -2317 and -2399, which we called thyroid hormone response sequence (THRS), that conferred T3 responsiveness to the UCP minimal promoter (4- to 12-fold) as well as to the thymidine kinase promoter (3- to 6-fold). T3 receptor bound to THRS in vitro, retarding its migration in electrophoretic mobility shift assays. Footprinting of THRS revealed two potential thyroid hormone response elements (TRE) separated by 27 bp: upTRE, -2391/-2376, 5'ACCCCTACTGAGGCAA; and dnTRE, -2348/-2334, 5'AGGGCAGCAAGGTCA. The mutation of these putative TREs caused loss of both T3 receptor binding and transactivation by T3. The analysis of the mutants also demonstrated that both TREs contribute in similar proportion to the T3 responsiveness of the UCP gene and that dnTRE is necessary for the potentiation of the cAMP effect by T3. Both TREs are located within a previously identified 212-bp enhancer element, flanked by sequences considered essential for BAT expression and norepinephrine responsiveness. Although they do not mediate thyroid hormone responsiveness, the sequences flanking THRS increase basal reporter expression and enhance the responses to T3. In conclusion, our results indicate that T3 can stimulate the transcription of the UCP gene and amplify the effect of cAMP acting directly on the gene. The presence of two functional TREs in a location critical to the control of the gene supports the importance of thyroid hormone for its expression and suggests the potential for interactions at the gene level that may explain the complexity of UCP regulation in vivo.
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PMID:Delineation of thyroid hormone-responsive sequences within a critical enhancer in the rat uncoupling protein gene. 786 54

We constructed a recombinant adenovirus carrying the firefly luciferase gene driven by the thymidine kinase promoter and controlled by the palindromic thyroid hormone/retinoic acid-responsive element. The same adenovirus vector without the hormone-responsive element was used as control. Regulation of the luciferase gene expression was tested in pituitary-derived GH cells and hepatoma-derived HepG2 cells infected with the recombinant adenoviruses. Administration of triiodothyronine to GH cells and all-trans-retinoic acid to HepG2 cells resulted in 8.0 +/- 0.3-fold and 4.6 +/- 0.5-fold induction of luciferase activity, respectively. No significant increase was observed with the control adenovirus. Hormonal regulation was also examined in adult mice. Mice depleted of thyroid hormone were injected intravenously with the recombinant adenoviruses and given 4 times the replacement dose of triiodothyronine or vehicle only for 4 days. Hormone administration resulted in 4.2-fold increase of luciferase activity in liver homogenates. No significant effect was observed in animals injected with the control adenovirus. This recombinant adenovirus provides a new experimental system in the study of thyroid hormone and retinoic acid action and offers the potential to regulate by physiological means the expression of genes transferred for the purpose of therapy.
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PMID:Expression of a thyroid hormone-responsive recombinant gene introduced into adult mice livers by replication-defective adenovirus can be regulated by endogenous thyroid hormone receptor. 792 32

The herpes simplex type 1 virus thymidine kinase (HSV1-TK) reporter gene was coupled to a bovine thyroglobulin promoter (TG-tk construct). Within the thyroid glands of transgenic mice expression was confined to thyroid follicle cells. Infusion of Ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine) to 8 to 12 week transgenic females led to the complete loss of thyroid HSV1-TK activity (at 3 to 4 days) and thyroid follicles (between 7 and 14 days). During the first 5 days of treatment a single reciprocal oscillation in circulating thyroxine (T4) and TSH levels occurred. By 14 days the circulating tri-iodothyronine (T3) and T4 levels of all treated animals were below the detection limits of the assays, while TSH levels were elevated ten-fold and continued to increase thereafter. During 14 days of treatment the thyroids regressed, protein content fell by 80-90% and the C cells, normally dispersed within the central region of each gland, came together in aggregates. Pituitary GH levels in females rose and fell back to normal within 14 days and between 14 and 28 days fell to a level comparable with that of GH-deficient lit/lit mice. The levels of hepatic GH receptor mRNA and the predominant 6.6 kb T3 receptor mRNA were unaffected by thyrocyte ablation. Thyrocyte ablation had no effect on the level of prolactin (Prl) receptor mRNA in females, but increased Prl receptor mRNA levels in males and eliminated group 1 major urinary protein (MUP).mRNA in females. T4 replacement reversed the effects of thyrocyte ablation on MUP mRNA in females and on Prl receptor mRNA in males.2+ Despite the many physiological changes induced by thyrocyte ablation, ablated mice have been maintained for up to 1 year without thyroid hormone supplementation. T4-deficient females were normally fertile and carried pups to term. Although transgenic males expressed HSV1-TK ectopically in spermatids and spermatozoa at levels similar to thyrocyte levels, a rate of Ganciclovir infusion which successfully ablated the thyrocytes did not affect the testis. As an alternative to infusion by minipump, thyrocyte ablation could be achieved by 6 twice-daily injections of Ganciclovir, at a level of 112 micrograms Ganciclovir/g body weight per day, and fetuses in utero could be thyrocyte ablated by administering 50 or 15 micrograms/g body weight per day to pregnant females between days 14 and 18 of gestation. These data demonstrate the potential value of transgenic thyrocyte ablation in the study of the effects of thyroid hormone deprivation.
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PMID:Consequences of thyroid hormone deficiency induced by the specific ablation of thyroid follicle cells in adult transgenic mice. 796 9

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

To identify sequences responsible for the muscle-specific expression of the rat GLUT4/muscle-fat gene, we examined the transcriptional regulation of this gene in the differentiating murine C2C12 skeletal muscle cell line. Differentiated myofibers displayed a 4-5-fold increase in GLUT4 mRNA compared with undifferentiated myoblasts which paralleled the conversion from non-muscle beta-actin mRNA to muscle-specific alpha-actin mRNA expression. Transient transfection of progressive 5' and 3' deletions of the GLUT4 5'-flanking DNA identified a 281-base pair region located between -517 and -237 relative to the transcription start site which conferred myotube-specific expression. This region increased reporter activity in the context of the GLUT4 minimal promoter in an orientation-independent manner and, in addition, onto the heterologous thymidine kinase promoter. Myotube-specific expression of both GLUT4 reporter constructs and the endogenous mouse GLUT4 mRNA was also observed to be thyroid hormone-dependent. Further, cotransfection of reporter constructs containing the 281-base pair GLUT4 differentiation-specific enhancer with the thyroid hormone receptor specifically increased luciferase activity in myotubes approximately 12-fold. Thus, these data demonstrate the presence of a proximal skeletal muscle-specific activation domain that is necessary for both myotube-specific GLUT4 expression and thyroid hormone responsiveness.
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PMID:Identification of a skeletal muscle-specific regulatory domain in the rat GLUT4/muscle-fat gene. 840 39

It has been shown that the mouse histone H10 promoter contains a DNA element, composed of a direct repeat of the sequence GGTGACC separated by 7 nt, which is able to bind retinoic acid receptors and to modulate transcription of reporter genes following treatment with retinoic acid. We have now investigated whether this DNA motif is also responsive to thyroid hormone. We co-transfected CV-1 monkey kidney cells with chloramphenicol acetyltransferase (CAT) expression plasmids containing either 740 bp of the H10 wild-type promoter or five copies of the repeat element cloned in front of the thymidine kinase promoter and expression vectors for human thyroid hormone receptors (TRs) alpha or beta and retinoid X receptor alpha (RXR alpha). Treatment of transfected cells with triiodothyronine led to a dose-dependent increase in CAT activity. Transfection experiments with increasing amounts of expression vectors for either TR alpha or RXR alpha resulted in up to 6-fold enhancement of CAT transcription. Furthermore, point mutations within the half-sites of the response element of the H10 promoter, as well as deletions within the interspace region, lowered CAT activity to 60-80% of that of the wild-type control. Electrophoretic mobility shift assays showed that the repeat element was able to form retarded complexes with TR alpha homodimers, as well as with TR alpha-RXR alpha heterodimers. Our results suggest that thyroid hormone receptors are involved in the regulation of mouse histone H10 expression.
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PMID:Thyroid hormone receptors bind to the promoter of the mouse histone H10 gene and modulate its transcription. 855 62

We characterized the cross-talk between activators of protein kinase A (PKA) and thyroid hormone (T3) in T3 receptor (TR)-mediated transcription. U937 cells were cotransfected with a plasmid expressing the TR and a reporter plasmid containing a T3 response element (TRE) oriented either as a direct repeat or as a palindrome upstream of the thymidine kinase promoter linked to the chloramphenicol acetyltransferase gene. T3 activated transcription by 10-fold. T3 response was potentiated 2.5-3-fold by activators of PKA, but an activator of protein kinase C or of guanylate kinase was ineffective. In the absence of T3, activators of PKA had no effect on transcription. TR heterodimerization with the retinoid X receptor may facilitate T3/PKA cross-talk because coexpression of the retinoid X receptor potentiated cross-talk. Synergy was not observed in JEG-3, F9, CV-1, HeLa, L929, and HTC cells, indicating that it may require cell-specific factors. Synergy required the DNA- and ligand-binding domains, but not the amino-terminal domain, indicating that T3- and TRE-induced conformational changes on the TR are essential for cross-talk. PKA phosphorylated the TR in vitro, suggesting that, like other nuclear receptors, the TR is a target for PKA. These results imply that PKA cross-talks with T3 at the level of the TRE-bound TR, enhancing its transcriptional activity in a cell-specific manner.
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PMID:Thyroid hormone activation of transcription is potentiated by activators of cAMP-dependent protein kinase. 870

Thyroid hormone action is mediated through its nuclear receptors (TRs), which bind to target DNA sequences [thyroid hormone response element (TRE)] as a homodimer or a heterodimer with 9-cis-retinoic acid receptors. Mutations of TR beta identified in patients with resistance to thyroid hormone (RTH) cluster primarily at two areas separated by the putative dimerization region. Two TR beta mutations were newly found in patients with RTH at codon 435 histidine (H435L and H435Q) close to the dimerization region. Recent crystallographic study suggested that H435 is critical for direct contact with T3. To study how the side-chain charge of amino acids at this position affects receptor characteristics, T3-binding activity, receptor dimerization, transcriptional activity, and dominant negative action were analyzed in two RTH mutants and two additional artificial mutants (H435R and H435E). The T3 binding affinities of all four mutants were below detection. In electrophoretic mobility shift assay using TRE-DR4 or the inverted palindrome (Lap), heterodimer formation of mutant receptors with 9-cis-retinoic acid receptor was similar to that of wild type receptors. However, homodimer formation varied among mutant receptors, especially using TRE-DR4, with a rank order of wild type = H435R > H435Q > H435L > > H435E. In the presence of a basic amino acid at codon 435, homodimer formation was preserved, whereas substitution to neutral or acidic amino acids resulted in decreased homodimer formation. In transient transfection assays using reporter genes under the control of 2xPal-thymidine kinase (TK), DR4-TK, Lap-TK, or TSH alpha promoter, these four mutants were inactive in T3-dependent transcriptional activation. Dominant negative inhibition was similar for all four mutants. These results indicate that 1) newly found TR beta mutations at codon 435 are responsible for RTH; and 2) codon 435 in TR beta is located at a position that can predominantly alter homodimer formation on certain TREs, such as DR4.
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PMID:Amino acid substitutions of thyroid hormone receptor-beta at codon 435 with resistance to thyroid hormone selectively alter homodimer formation. 882 60


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