Tissue-specific influences affect glucocorticoid regulatory element actions on the human growth hormone gene promotor.
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Animals↗
Biomedical subjects
M Karin
Publications and source records attributed to M Karin.
Mechanisms of glucocorticoid hormone action.
This work summarizes some of our studies of the mechanisms of glucocorticoid action, including aspects of steroid binding to receptors, the activation of glucocorticoid-receptor complexes and the regulation of expression of endogenous and transferred glucocorticoid-responsive genes. Studies of the receptor-steroid interaction support the notion that steroid entry is passive. A comparative analysis of binding in isolated cytosol and intact cells suggests that the initial receptor-steroid binding reaction and not subsequent steps such as activation and nuclear binding, is predominantly responsible for the high-affinity state that is generated. The binding is driven by entropy and enthalpy changes at low temperature; at higher temperatures it is driven by entropy changes, with enthalpy working against it. Studies of the activation of the receptor-glucocorticoid complex with the use of highly purified receptors suggest that this step is associated with a change in charge of the receptor-glucocorticoid complex (such as would occur with a dephosphorylation reaction), whereas the data do not support the notion that dissociation of a bound RNA or of receptor oligomers is responsible for generating the nuclear- and DNA-binding activity of the complex. Studies of the regulation by glucocorticoids of expression of the endogenous rat growth hormone (rGH) gene in cultured rat pituitary tumor (GC, GH3D6) cells suggest that glucocorticoids increase the expression of this gene by multiple mechanisms. First, there is a modest direct stimulation of transcription by a mechanism(s) that does not depend on protein synthesis; however, if the cells have been exposed to thyroid hormone for several hours, the steroid exerts a much greater increase in rGH pre-mRNA levels. Secondly, the steroid appears to stimulate some relatively stable function or functions that increase the ability of thyroid hormone to increase rGH levels. Thirdly, the steroid probably increases rGH mRNA stability, since the fold-increases in rGH mRNA exceed those of transcription. Finally, the steroid may, by unknown mechanisms, affect rGH mRNA polyadenylation. The gene transfer experiments utilized the rat and human (h) GH genes and hybrid genes containing either rGH and Herpes Simplex virus thymidine kinase (TK) gene sequences or the human metallothionein-IIA (hMT-IIA) and TK gene sequences. The steroid was found to regulate hMT-IIA gene expression in all glucocorticoid-responsive cell types tested by actions on its 5'-flanking DNA. By contrast, the glucocorticoid regulated GH gene expression in some but not all glucocorticoid-responsive cell types.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Activation of a heterologous promoter in response to dexamethasone and cadmium by metallothionein gene 5'-flanking DNA.
Human metallothionein-IIA (hMT-IIA) gene expression is regulated by heavy metals and glucocorticoids. When the cloned hMT-IIA gene or its 5'-flanking DNA structure fused to herpes simplex virus thymidine kinase (HSV-TK) structural gene sequences were transferred into TK- Rat 2 fibroblasts, both genes were inducible by Cd++ and/or dexamethasone. Placement of the hMT-IIA gene 5'-flanking region, either intact of deleted in its TATA box and cap site, upstream of the HSV-TK gene promoter rendered the latter both glucocorticoid- and heavy metal-inducible. Thus the structure that mediates both Cd++ and glucocorticoid responsiveness is present in the hMT-IIA gene 5'-flanking DNA, does not require its TATA box or cap site, and can activate a heterologous promoter.
Animals↗
Structural and functional analysis of the human metallothionein-IA gene: differential induction by metal ions and glucocorticoids.
We describe a region of human DNA containing four metallothionein (hMT) genes. One of these genes, hMT-IA, was found to encode a functional protein that confers heavy metal resistance to NIH 3T3 cells after transfer on a bovine papilloma virus-derived vector. This gene is expressed in cultured human cell lines, but at a lower basal level than the hMT-IIA gene; it shows a different induction response to heavy metals and glucocorticoids than the hMT-IIA gene. Induction of the human MT family therefore does not represent an equivalent elevation in the level of expression of individual genes, but is the sum of the differential responses of active members. The differential response is due to functional differences of the respective promoter/regulatory regions of the genes as shown by gene-fusion experiments. While the hMT-IIA promoter is responsive to Cd++, Zn++, and glucocorticoids, the hMT-IA promoter mediates response only to Cd++.
Animals↗
Human metallothionein genes are clustered on chromosome 16.
The metallothioneins are a family of heavy-metal binding proteins of low molecular weight. They function in the regulation of trace metal metabolism and in the protection against toxic heavy metal ions. In man, the metallothioneins are encoded by at least 10-12 genes separated into two groups, MT-I and MT-II. To understand the genomic organization of these genes and their involvement in hereditary disorders of trace metal metabolism, we have determined their chromosomal location. Using human-mouse cell hybrids and hybridization probes derived from cloned and functional human MT1 and MT2 genes, we show that the functional human genes are clustered on human chromosome 16. Analysis of RNA from somatic cell hybrids indicated that hybrids that contained human chromosome 16 expressed both human MT1 and MT2 mRNA, and this expression is regulated by both heavy metal ions and glucocorticoid hormones.
Animals↗
Primary structure and transcription of an amplified genetic locus: the CUP1 locus of yeast.
Copper resistance in yeast is controlled by the CUP1 locus. The level of resistance is proportional to the copy number of this locus, which can be found in up to 15 tandemly iterated copies. To elucidate the molecular mechanisms controlling the amplification and expression of the CUP1, locus, we determined its full nucleotide sequence. We have also identified and mapped two transcription units within the basic amplification unit of CUP1 in laboratory yeast strains. One of those transcription units is inducible by copper and encodes a low molecular weight copper binding protein--copper chelatin. The increased production of chelatin, due to both gene amplification and induction of transcription, leads to increased resistance of yeast cells to copper ions.
Amino Acid Sequence↗
Nucleotide sequence requirements for transient expression of human metallothionein-IIA-thymidine kinase fusion genes.
The transient expression of the human metallothionein-IIA (hMT-IIA) gene was examined after introduction into NIH-3T3 cells. A series of deletion mutants within the 5' flanking region of the hMT-IIA gene was constructed and fused to the structural sequences of the Herpes simplex thymidine kinase (TK) gene to generate hMTK chimeric genes. During transient expression, the hMTK gene is responsive to both ligands. In addition we find at least 92 bp of 5' flanking DNA are required for both transient expression and induction by Cd2+, in contrast to only 50 bp required for induction by the heavy metal ions of the same gene in stable transformants. This difference is due to a deletion of a regulatory element, located between nucleotides -70 to -90 of the hMT-IIA gene, which functions to maintain basal expression in the absence of inducers. This element is absolutely required for transient expression of the hMT-IIA gene.
Base Sequence↗
Expression and hormonal regulation of the rat growth hormone gene in transfected mouse L cells.
Expression of the rat growth hormone (rGH) gene in the pituitary and in cultured pituitary tumor cells is regulated by glucocorticoid hormones. After co-transfer of cloned DNA containing the rGH gene with the herpes simplex virus (HSV) thymidine kinase (tk) gene into mouse Ltk- cells, rGH gene transcripts were detected in eight of fifteen tk+ cell lines. However, in all eight clones, the predominant rGH gene transcript was only about 0.75 kb, 0.3 kb shorter than pituitary rGH mRNA. The 0.75-kb transcripts, examined from one clone, L-rGH-4, lacked sequences derived from exons 1 and 2 of the rGH gene. Although transcripts larger than 0.75 kb were detected, the normal 2.2-kb rGH gene primary transcript was present only at very low levels. Nuclease mapping studies also failed to reveal transcripts initiated at the normal rGH gene promoter, but instead revealed transcripts with 5' termini arising within intron B of the gene. These data suggest either that transcripts arise from internal promoters within the rGH gene or that a transcript initiated upstream from the normal promoter was processed abnormally. Dexamethasone increased the levels of the 0.75-kb rGH gene transcripts about fourfold in all eight clones expressing rGH mRNA. These data suggest that structural elements important for glucocorticoid-mediated influences on regulation of GH gene expression are contained within the transferred rGH gene fragment and can function even when the normal rGH gene promoter is not used and the pattern of expression is grossly abnormal.
Animals↗
The human metallothionein gene family: structure and expression.
Metallothioneins (MTs) are encoded by a multigene family in man. We have isolated those genes and analyzed the structure of some of them. The MT-II variant is encoded by a single functional gene: MT-IIA. The MT-IIB gene is a processed pseudogene derived from a reverse transcript of MT-II mRNA. On the other hand, the MT-I class of variants are encoded by a large number of genes, arranged in tandem. The MT-IIA and the MT-IA genes show a differential response to glucocorticoid hormones and heavy metals, yet they are both expressed in primary human fibroblasts and in HeLa cells. Expression of both of those genes, in high level after transfer on bovine papilloma virus vectors, leads to increased resistance of the host cells to cadmium-induced toxicity.
Cadmium↗
Expression and regulation of a human metallothionein gene carried on an autonomously replicating shuttle vector.
A human metallothionein (MT) gene was inserted into a bovine papillomavirus (BPV) vector. The chimeric vector (pMTII-BPV) transforms rodent fibroblasts to a cadmium-resistant phenotype. The resistance is due to the high level of expression of human MT-II in those cells. The vector is maintained in the cells as a free replicating plasmid, present at about 10--15 copies per cell. Transcription of the episomal human MT-IIA gene is initiated from its authentic start sites and is regulated by the level of cadmium in the growth medium. The presence of the human MT-IIA gene allows the BPV replicon to function even though it is ligated to an intact copy of pBR322. Due to the presence of plasmid origins of replication and dominantly acting selective markers functional in both Escherichia coli and mammalian cells, pMTII-BPV can be used as a shuttle vector.
Animals↗
A method for isolation of intact, translationally active ribonucleic acid.
A method for isolation of large, translationally active RNA species is presented. The procedure involves homogenization of cells or tissues in 5 M guanidine monothiocyanate followed by direct precipitation of RNA from the guanidinium by 4 M LiCl. Modifications are described for use with tissue culture cells, yeast, tissues, or isolated nuclei. The advantages of the procedure include speed, simplicity, avoidance of an ultracentrifugation, and its applicability to large numbers of small samples. The procedure yields large mRNA precursors up to 10 kb and mRNA species which translate very well. However, small (less than 300 nucleotides) RNA species are recovered with a poor yield.
Chemical Precipitation↗
Industrial yeasts display tandem gene iteration at the CUP1 region.
The gene copy number at the CUP1 locus and the resistance level to external copper was directly correlated in five wild-type commercial Saccharomyces strains. An increased copy number of the CUP1 gene leads to increased accumulation of chelatin mRNA, which codes for a low-molecular-weight, copper-binding protein. The enhanced production of this rapidly inducible protein mediates resistance of the cell to copper. Industrial yeasts exhibit homologies to the amplified copper resistance repeat unit found in laboratory strains. However, the extent of tandem iteration is strain dependent, and the repetitious unit is either 1.7 or 1.5 kilobases in length compared with the 2.0-kilobase unit in laboratory strains. Strain 522 (Montrachet) contains two chromosome VIII segments distinguishable by their numbers of repeat units (2 and 11) and the size of the units (1.5 and 1.7 kilobases). Distillers yeast 513 carries a 1.5-kilobase repeat unit on each homologous chromosome, although they contain nine and five iterations, respectively.
Copper↗
Human metallothionein genes--primary structure of the metallothionein-II gene and a related processed gene.
The complete nucleotide sequence of two of the human metallothionein gene family has been compared. One is a functional metallothionein-II gene, the other a pseudogene, lacking introns, terminating in a poly(A) tail and flanked by two direct repeats. In addition, we have detected a size polymorphism associated with the processed gene in the population, examined, and we have observed a region of apparent secondary structure homology between of 5' flanking region of the functional metallothionein-II gene and that of a mouse metallothionein-I gene.
Base Sequence↗
Human metallothionein genes: molecular cloning and sequence analysis of the mRNA.
From a cDNA clone bank prepared from cadmium-treated HeLa cells, we isolated clones representing mRNAs whose concentration is increased after cadmium induction. Several metallothionein cDNA clones were isolated by cross-hybridization to mouse metallothionein-I cDNA. The nucleotide sequence of one of these clones, containing a nearly full-length cDNA copy of human metallothionein-II mRNA, was determined. The homology between the human and mouse metallothionein sequences is strictly limited to the coding region of the mRNA. Codon usage in metallothionein mRNA is not random. Seventy-nine percent of the codons have G or C residues at the third position, resulting in a GC-rich sequence.
Amino Acid Sequence↗
Induction of metallothionein mRNA in HeLa cells by dexamethasone and by heavy metals.
Synthesis of metallothionein, a cysteine-rich heavy metal binding protein, is induced in cultured HeLa cells both by the heavy metals Cd2+, Zn2+ and Cu2+, and by the glucocorticoid hormone dexamethasone. The accumulation of [35S]cysteine-labeled metallothionein and the amount of translatable metallothionein mRNA show identical concentration dependences in response to dexamethasone treatment and in response to zinc exposure. Induction of translatable metallothionein mRNA is rapid in response to both the metal and glucocorticoid inducers. Increased synthesis and accumulation of metallothionein in response to either metal or glucocorticoid exposure is regulated by the level of translatable metallothionein mRNA in HeLa cells.
Cadmium↗
Receptor-mediated endocytosis of transferrin in developmentally totipotent mouse teratocarcinoma stem cells.
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Animals↗
Regulation of metallothionein synthesis in HeLa cells by heavy metals and glucocorticoids.
Metallothioneins (MTs) are low molecular weight, cysteine-rich proteins that bind heavy metals. MT induction occurs in liver in response to either heavy metal (Zn++ or Cd++) administration or stress. The synthesis of MT can also be induced by either heavy metals or glucocorticoid hormones in HeLa cells cultured in serum-free medium. Induction of MT by zinc is subject to "desensitization." In contrast, dexamethasone (dex) induction results in a continued elevation in the rate of MT synthesis. The stability of MT is dependent on the availability of metal; consequently, MT induced by dex is degraded much more rapidly (half-life of 11 to 12 hours) than MT induced by elevated zinc levels (half-life of 36 to 38 hours). Removal of either inducer results in biphasic degradation curves, as apothionein and zinc come into balance. In contrast, deinduction kinetics for MT synthesis following removal of the two inducers (zinc and dex) are the same, with a half-life of two and one-half hours. Inhibition of RNA synthesis blocks deinduction after removal of inducer. Induction of MT occurs in a wide variety of species, from blue-green algae to man. This system should provide an excellent model for the comparative biochemistry of regulation of gene expression.
Dexamethasone↗
Induction of metallothionein in HeLa cells by dexamethasone and zinc.
Metallothioneins are induced by both Zn2+ and dexamethasone in HeLa cells grown in serum-free medium. Dexamethasone is able to induce metallothionein synthesis in HeLa cells in virtually zinc-free medium ([Zn2+] = .01 microM). The presence of dexamethasone does not shift the dose/response curve for metallothionein induction by Zn2+, further indicating that the two inducers work through independent mechanisms. Dexamethasone stimulates Zn2+ uptake 1.7-fold over 24 h. However, there is no increase in Zn2+ uptake during the first 4 h. In contrast, metallothionein synthesis in response to either Zn2+ or dexamethasone is clearly observable within 4 h of exposure to either inducer. The increased intracellular 65Zn2+ content observed at 24 h is completely accounted for by the increased level of metal bound to metallothionein. In a continuous labeling experiment the rate of synthesis of metallothionein reached a steady state after about 4 h, in response to either inducer. The lag period was identical for both dexamethasone and Zn2+, with similarly shaped induction curves. Induction by dexamethasone, but not by Zn2+, was inhibited by progesterone. Zn2+ and dexamethasone appear to induce metallothionein synthesis in HeLa cells by mechanisms independent of one another.
Dexamethasone↗