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

Here we investigate the roles of human testicular orphan receptors, TR2 and TR4, on the gene regulation of the long-terminal repeat of the human immunodeficiency virus type 1 (HIV-LTR). In gel-retardation assays, a palindromic element at the 5'-end of HIV-LTR,5'-AGGGGTCAGATATCCACTGACCTTT-3',showed high affinity to TR2 and TR4 with an equilibrium dissociation constant (Kd) of 1.11 +/- 0.48 (n = 3) and 0.52 +/- 0.12 nM (n = 3), respectively. Interestingly, each half-site of the palindromic element is sufficient to compete with the binding of the labeled palindromic element to TR2 or TR4 with an equilibrium inhibition constant (ki) around 10 nM. However, the transiently expressed TR2 or TR4 in Chinese hamster ovary (CHO) cells or Japanese quail muscle myoblasts (QM7) cells showed no activity in regulating the transcriptional activity of the chloramphenicol acetyltransferase (CAT) reporter gene inserted downstream of the HIV-LTR promoter. Although both TR2 and TR4 showed no effect on CAT activity by itself, our data showed only the TR4 could crosstalk to the chicken ovalbumin upstream protein-transcription factor (COUP-TF1) and thyroid hormone receptor (TR alpha 1), and potentiated the transcriptional activity of HIV-LTR on the CAT reporter gene regulated by COUP-TF1 and TR alpha 1. These results indicate that TR4, but not TR2, may couple to other nuclear receptors in the upregulation of the HIV replication.
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PMID:TR4 orphan receptor crosstalks to chicken ovalbumin upstream protein-transcription factor and thyroid hormone receptor to induce the transcriptional activity of the human immunodeficiency virus type 1 long-terminal repeat. 970 74

The 9,000 Mr calcium-binding protein calbindin-D9k (CaBP9k) is markedly induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in mammalian intestine. However, although a vitamin D response element (VDRE) has been reported in the promoter of the rat CaBP9k gene (at -490/-472), the CaBP9k promoter is weakly transactivated by 1,25-(OH)2D3. Previous studies indicated that when MCF-7 cells are transfected with the rat CaBP9k VDRE ligated to the thymidine kinase promoter and treated with both 1,25-(OH)2D3 and T3 there is an enhancement of the response observed with 1,25-(OH)2D3 alone, suggesting direct cross-talk between thyroid hormone and the vitamin D endocrine system and activation via the formation of vitamin D receptor (VDR)-thyroid hormone receptor (TR) heterodimers. To determine whether the weak response of the rat CaBP9k natural promoter to 1,25-(OH)2D3 could be enhanced by T3, CaBP9k promoter/reporter chloramphenicol acetyltransferase constructs were transfected in MCF-7 cells, and the cells were treated with the two hormones alone or in combination. No induction with T3 alone and no enhancement of reporter activity in the presence of both hormones was observed. To determine whether a lack of effect by T3 was specific for the CaBP9k promoter and to further examine the possibility of cross-talk between the TR- and VDR-signaling pathways, the 1,25-(OH)2D3-responsive rat 24 hydroxylase [24(OH)ase] promoter and the rat osteocalcin VDRE (-457/-430), both fused to reporter genes were similarly examined in MCF-7 cells. Again, no enhancement of the response to 1,25-(OH)2D3 was observed in the presence of T3. In addition, a similar lack of response to T3 but responsiveness to 1,25-(OH)2D3 was observed when UMR106-01 osteosarcoma cells [which, like MCF-7 cells, express VDR, TR, and the retinoid X receptor (RXR) endogenously] were transfected with a 1,25-(OH)2D3 responsive mouse osteopontin promoter reporter. In vitro DNA binding assays were carried out using purified human VDR, human RXRalpha, and chick T3Ralpha and 24(OH)ase, osteocalcin, osteopontin, and CaBP9k VDRE oligonucleotide probes. No VDR-TR heterodimer binding on any of these VDREs was observed, although, as expected, there was binding by the VDR-RXR complex and strong TR-RXR binding to a consensus thyroid hormone response element. Simultaneous gel retardation assays using similar and lower concentrations of TR with RXR showed strong binding of TR-RXR on a 32P-labeled thyroid response element. Studies using the yeast two-hybrid system also did not provide evidence for the formation of a VDR-TR protein-protein interaction. In addition, in vivo data showed that transfection of TR, in fact, repressed VDR-mediated transcription and that the repression could be reversed by the addition of RXR. Thus, in vitro and in vivo experiments do not support ligand-sensitive transactivation mediated by VDR-TR heterodimer formation but rather suggest that TR expression can repress 1,25-(OH)2D3-induced transcription predominantly by sequestering RXR.
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PMID:Thyroid hormone receptor does not heterodimerize with the vitamin D receptor but represses vitamin D receptor-mediated transactivation. 973 5

The purpose of this study was to gain insights into the underlying mechanism of myocardial fibrosis during hypothyroidism. Treatment of cardiac fibroblasts with a medium lacking thyroid hormone led to a 47% increase in [3H]thymidine incorporation into the cell nuclei compared with that in untreated cells. Northern blot analysis of RNA from cardiac fibroblasts grown in a thyroid hormone depleted medium resulted in a 38% increase in the abundance of mRNA for pro-alpha1(I) collagen. At the protein level, the amount of type I collagen, as determined by immunoprecipitation, was increased either in the cell lysate (46%) of cardiac fibroblasts grown in a thyroid hormone depleted medium or in the medium (44%). The chimeric plasmid, ColCAT 3.6, contains the 5'-flanking region of the rat pro-alphal(I) collagen gene (from bases -3520 to +115) fused to the chloramphenicol acetyltransferase (CAT) gene. The plasmid was cotransfected with thyroid hormone receptor (TR) expression plasmid into rat cardiac fibroblasts and COS-l cells (monkey mesangial cells). Cells transfected with the ColCAT plasmid in the presence of thyroid hormone (100 nM T(3)) had a significant decrease (39% in fibroblasts, P<0.01; 52% in COS-1 cells, P<0.001) in CAT activity when compared to cells not exposed to thyroid hormone. Transient co-transfection of TR with various pro-alphal(I) collagen/CAT deletion constructs showed that T(3)-dependent repression was preserved with the deletion from 3520 bp of the flanking sequence to a 5' end point at position -224, indicating that a thyroid hormone-response element (TRE) was localized at the region -224 to +115. The TR-DNA binding assays demonstrated binding of the human TRbeta1 to a fragment containing a proposed TRE located between position -35 and +115 in the 5'-flanking region of the rat pro-alphal(I) collagen gene.
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PMID:Molecular characterization of myocardial fibrosis during hypothyroidism: evidence for negative regulation of the pro-alpha1(I) collagen gene expression by thyroid hormone receptor. 1085 97

The molecular mechanism involved in the liganded thyroid hormone receptor suppression of the TSHbeta (thyroid-stimulating hormone beta, or thyrotropin beta) gene transcription is undetermined. One of the main reasons is the limitation of useful cell lines for the experiments. We have developed an assay system using non-pituitary CV1 cells and studied the negative regulation of the TSHbeta gene. In CV1 cells, the TSHbeta-CAT (chloramphenicol acetyltransferase) reporter was stimulated by Pit1 and GATA2 and suppressed by T3 (3,3',5-tri-iodothyronine)-bound thyroid hormone receptor. The suppression was dependent on the amounts of T3 and the receptor. Unliganded receptor did not stimulate TSHbeta activity, suggesting that the receptor itself is not an activator. Analyses using various receptor mutants revealed that the intact DNA-binding domain is crucial to the TSHbeta gene suppression. Co-activators and co-repressors are not necessarily essential, but are required for the full suppression of the TSHbeta gene. Among the three receptor isoforms, beta2 exhibited the strongest inhibition and its protein level was the most predominant in a thyrotroph cell line, TalphaT1, in Western blotting. The dominant-negative effects of various receptor mutants measured on the TSHbeta-CAT reporter were not simple mirror images of those in the positive regulation under physiological T3 concentration.
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PMID:Thyroid-hormone-dependent negative regulation of thyrotropin beta gene by thyroid hormone receptors: study with a new experimental system using CV1 cells. 1461 44

Previously we reported that the negative regulation of the TSHbeta gene by T(3) and its receptor [thyroid hormone receptor (TR)] is observed in CV1 cells when GATA2 and Pit1 are introduced. Using this system, we further studied the mechanism of TSHbeta inhibition. The negative regulatory element (NRE), which had been reported to mediate T(3)-bound TR (T(3)-TR)-dependent inhibition, is dispensable, because deletion or mutation of NRE did not impair suppression. The reporter construct, TSHbeta-D4-chloramphenicol acetyltransferase, which possesses only the binding sites for Pit1 and GATA2, was activated by GATA2 alone, and this transactivation was specifically inhibited by T(3)-TR. The Zn finger region of GATA2 interacts with the DNA-binding domain of TR in a T(3)-independent manner. The suppression by T(3)-TR was impaired by overexpression of a dominant-negative type TR-associated protein (TRAP) 220, an N- and C-terminal deletion construct, indicating the participation of TRAP220. Chromatin immunoprecipitation assays with a thyrotroph cell line, TalphaT1, revealed that T(3) treatment recruited histone deacetylase 3, reduced the acetylation of histone H4, and caused the dissociation of TRAP220 within 15-30 min. The reduction of histone H4 acetylation was transient, whereas the dissociation of TRAP220 persisted for a longer period. In the negative regulation of the TSHbeta gene by T(3)-TR we report that 1) GATA2 is the major transcriptional activator of the TSHbeta gene, 2) the putative NRE previously reported is not required, 3) TR-DNA-binding domain directly interacts with the Zn finger region of GATA2, and 4) histone deacetylation and TRAP220 dissociation are important.
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PMID:Essential role of GATA2 in the negative regulation of thyrotropin beta gene by thyroid hormone and its receptors. 1724 62


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