Regulation of cytosolic aspartate aminotransferase mRNAs in the Fao rat hepatoma cell line by dexamethasone, insulin and cyclic AMP

Glucocorticoid hormones increase the activity of cytosolic aspartate aminotransferase (cAspAT) in the Fao rat hepatoma cell line. Maximal increase (6-10-fold) was observed 48 h following the addition of the glucocorticoid agonist dexamethasone at a concentration of 0.1 microM. The effect of dexamethasone was specific since it was not mimicked by sex steroids and was inhibited by the glucocorticoid antagonist RU 486. Insulin (0.1 microM) inhibited by more than 50% the induction of cAspAT by glucocorticoids. The cAMP analog, 8-bromoadenosine 3',5'-monophosphate (Br8cAMP, 0.5 mM), potentiated the effect of dexamethasone (2-3-fold) and partially relieved the inhibitory effect of insulin on the induction by dexamethasone. Both insulin and Br8-cAMP had no significant effect on basal activity. The mitochondrial isoenzyme was insensitive to the various hormonal treatments. Northern blot analysis revealed the presence of two major (2.1-kb and 1.8-kb) and one minor (4-kb) mRNA species hybridizing with a rat cAspAT probe. The regulation of these mRNAs by glucocorticoids, insulin and cAMP correlated with the variation of the cAspAT activity, suggesting that these hormones act at the pretranslational level. We compared the regulation of cAspAT mRNAs with those of tyrosine aminotransferase mRNA. Both were similarly increased by dexamethasone but the latter was also increased by cAMP even in the absence of the glucocorticoid agonist. In addition, the increase in tyrosine aminotransferase mRNA was inhibited by cycloheximide whereas the increase in cAspAT mRNAs was not. These results show that there are significant differences in the regulation of cAspAT and tyrosine aminotransferase by glucocorticoids and other hormones, although both enzymes probably contribute to the same metabolic pathway.     

The mouse L-histidine decarboxylase gene: structure and transcriptional regulation by CpG methylation in the promoter region

To investigate the regulation of mouse L-histidine decarboxylase (HDC) gene expression, we isolated genomic DNA clones encoding HDC. Structural analysis revealed that the mouse HDC gene was composed of 12 exons, spanning approximately 24 kb. Northern blotting analysis indicated that, among the cell lines examined, a high level of HDC gene expression was restricted to mature mast cell lines and an erythroblastic cell line. The gene was induced strongly in the mouse immature mast cell line P815 after incubation in the peritoneal cavity of BDF1 mice. We observed that the promoter region was demethylated in the HDC-expressing cell lines and in induced P815 cells. Interestingly, forced demethylation by 5-azacytidine (5-azaC) treatment induced high expression of HDC mRNA in P815 cells. The activity of a mouse HDC promoter-reporter construct stably transfected in P815 cells was repressed by in vitro patch-methylation. This low promoter activity of the patch-methylated reporter construct was restored after 5-azaC treatment, which demethylated the patch-methylated promoter. These results indicate that DNA methylation state of the promoter region controls HDC gene expression.     

Effect of 5-azacytidine on expression of DNA sequences homologous to ENV gene of murine mammary tumor virus in normal human lymphocytes

Expression of DNA sequences, related to MMTV env gene, in peripheral blood lymphocytes, which was strictly specific for human mammary carcinoma, has been previously reported. These sequences (homologous to env gene site coding for MMTV gp52 envelope antigen) expressed in T cells can play the key role in virus infection transmission and propagation. In order to elucidate the possible routes of env MMTV-homologous sequences expression, we tried to induced it in donot T lymphocytes by various methods: hormone and virus treatment (related genome "saving" at the expense of the added virus envelope), T cell culturing with conA, interferon-2, and 5-azacytidine. RT-PCR with primers specific for the gp52-coding area of MMTV env gene showed expression of env-homologous sequences in donor T cells cultured in medium with 5-azacytidine. Indirect immunofluorescence with monospecific serum to MMTV gp52 detected gp52 analogous genes only in cultures with 5-azacytidine but not other agents. We therefore suggested that MMTV env-homologous sequences in donors are situated in the methylated promoter zone. Expression of these sequences in T cells, specific for human mammary carcinoma, can be due to demethylation of the promoter and induction of env-homologous sequences to the level of translation of gp52 analogous antigens or by initial location of some of the expressed sequences in the demethylated zone of the genome.     

Inhibition of herpes simplex thymidine kinase gene expression by DNA methylation is an indirect effect.

The biological activity of in vitro methylated HSV-TK DNA was analysed after microinjection into thymidine kinase negative rat 2 cells. It was found that the fully methylated DNA (Hpa II methylase) was as active as the non methylated control DNA for about 48 hours after injection. DNA reextraction experiments and blot analysis showed that DNA demethylation was not the reason for the observed TK activity. With prolonged cultivation time the methylated DNA becomes rapidly inactive and 100 hrs after injection thymidine incorporation was no longer detectable in the recipient cells. In transformed cells, obtained after coinjection with SV40 DNA, the HSV-DNA was partially demethylated and inactive. Addition of 5-azacytidine to the culture medium induced further demethylation and reactivation of the thymidine kinase gene.     

Reactivation of a methylation-silenced gene in adenovirus-transformed cells by 5-azacytidine or by E1A trans activation.

In the adenovirus type 2 (Ad2)-transformed hamster cell line HE3, the integrated late E2A promoter of Ad2 DNA is inactive, is methylated at all three 5'-CCGG-3' sequences, and can be reactivated by growing the cells in the presence of 50 microM 5-azacytidine (5-azaC). The three 5'-CCGG-3' sequences then become demethylated. Demethylation and reactivation are stable over 30 passages even after the removal of 5-azaC. The dormant late E2A promoter in cell line HE3 can also be reactivated by transfecting the cells with recombinant plasmids that carry the left terminal E1A and part of the E1B region of Ad2 DNA or the E1A 13S cDNA, but not with plasmids containing the E1A 12S cDNA. The E1A 13S cDNA encodes the 289-amino-acid trans-activating protein of Ad2. The E1A-mediated reactivation of the late E2A promoter is not accompanied by its demethylation in both DNA complements. Cell line HE3 produces constitutively E1A-encoded mRNAs and reactivates the methylated late E2A promoter-chloramphenicol acetyltransferase gene construct after transfection into HE3 cells. Constitutive levels of the endogenous E1A gene products in HE3 cells are detectable but, paradoxically, appear insufficient to reactivate the endogenous, chromosomally integrated E2A gene.     

DNA methylation is involved in maintenance of an unusual expression pattern of an introduced gene

In one of 30 transgenic tobacco (Nicotiana tabacum) plants, the expression of an introduced β-glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S promoter, was found to be repressed as the plant matured, whereas the endogenous GUS activity was unaffected. Plants grown from seeds or regenerated from leaf discs derived from this plant showed a similar temporal pattern of expression. Suspension-cultured cells established from nonexpressing leaves did not express the introduced gene. In these cells, the silent gene could be reactivated by treatment for 5 or 10 days with 5-azacytidine. Overall, demethylation of the genome preceded recovery of the enzyme activity. The increase in the fraction of reactivated cells progressed in two phases. Up to 8 weeks after starting the 5-azacytidine treatment, approximately 2 to 4% of the cells were expressing GUS, followed by a dramatic increase of GUS-expressing cells. Thirteen weeks after starting the 5-azacytidine treatment, the fraction of GUS-expressing cells amounted to 80%. At this time, the original overall level of DNA methylation was reestablished. The degree of DNA demethylation, as well as the magnitude of reactivation, was dependent on the duration of the 5-azacytidine treatment. These results demonstrate that DNA methylation appears to be involved in the regulation of the introduced GUS gene and that this development-dependent pattern of expression can be inherited.     

DNA methylation alters chromatin structure and regulates Thy-1 expression in EL-4 T cells

Thy-1 exhibits marked differences in expression in various tissues in many species; therefore, it is of interest to define possible mechanisms that may regulate Thy-1 expression. We produced Thy-1 negative variants of the murine T cell lymphoma EL-4 by mutagenesis with ethylmethanesulfonate (EMS), negative selection with anti-Thy-1 monoclonal antibodies (mAb) plus complement, and fluorescence-activated cell sorting (FACS). Thy-1 surface negative (Thy-1-) mutants produced in this manner were shown to produce no detectable Thy-1 mRNA, but contained an intact Thy-1 gene as determined by Southern blotting. 5' CG sequences, which had been demethylated in the parent EL-4 clone, were completely methylated in the EMS-induced Thy-1-variant. In addition, a DNase I hypersensitive site that mapped to the 5' end of the Thy-1 gene in EL-4 was absent in the Thy-1- variant. Treatment of this Thy-1- clone with 5-azadeoxycytidine (5-dAZA) resulted in re-expression of surface Thy-1, demethylation of the 5' CG sequences, and regeneration of the DNase I hypersensitive site. These studies indicate that methylation of certain critical DNA sequences in the 5' region of the Thy-1 gene can alter local chromatin structure and regulate expression of this gene.     

Genomic hypomethylation and far-5'' sequence alterations are associated with carcinogen-induced activation of the hamster thymidine kinase gene.

We have investigated the mechanism of activation of an inactive but functionally intact hamster thymidine kinase (TK) gene by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Following carcinogen treatment of TK- RJK92 Chinese hamster cells, aminopterin-resistant (HATr) colonies appeared at a frequency 50-fold higher than in untreated controls. More than 80% of these HATr variants expressed TK enzymatic activity and were divided into high- and low-activity classes. In all TK+ variants, TK expression was correlated with demethylation in the 5' region of the TK gene and the appearance a 1,400-nucleotide TK mRNA. Using high-performance liquid chromatography to measure the level of genomic methylation, we found that four of five high-activity lines demonstrated extensive genomic hypomethylation (approximately 25% of normal level) that was associated with demethylation of all TK gene copies. Restriction endonuclease analysis of 15 low-activity lines revealed four instances of sequence alterations in the far-5' region of the TK gene and one instance of a tandem low-copy amplification. In these lines, the structurally altered gene copy was demethylated. Thus, we propose that a chemical carcinogen can activate TK expression by several different mechanisms. Focal demethylation with or without gene rearrangement was associated with low TK activity, whereas demethylation throughout the genome was associated with high TK activity.     

DNA demethylation induced by 5-azacytidine does not affect fragile X expression.

Experiments were performed to determine the role of DNA demethylation in fragile X expression. Fragile X positive lymphoblastoid cells were treated with 5-azacytidine and harvested for analysis of fragile X expression both directly following treatment and after a recovery period in the absence of the drug. The effectiveness of 5-azacytidine treatment in inducing DNA demethylation was concurrently monitored by analysis of methylation changes at random autosomal loci in isolated DNA from treated cells. Under conditions where 5-azacytidine was found to inhibit fragile X expression, no DNA demethylation was observed. At the time when demethylation did occur, fragile X expression was not affected. These results strongly indicate that DNA demethylation is not involved in fragile X expression.     

Cloning and characterization of a cDNA encoding aspartate aminotransferase-P1 from Lupinus angustifolius root tips.

A root tip cDNA library, constructed in the lambda Zap II expression vector, was immunoscreened with a monoclonal antibody raised against aspartate aminotransferase-P1 from Lupinus angustifolius L. var Uniharvest. One 1452-base pair clone was isolated. The encoded protein sequence had high homology to both plant and animal aspartate aminotransferase sequences. The clone was converted to the phagemid form and expressed in Escherichia coli. The expressed protein was enzymically active and could be immunocomplexed with aspartate aminotransferase-P1-specific antibodies. The complete aspartate aminotransferase-P1 cDNA was cloned into the yeast expression vector pEMBL-yex4 and transformed into Saccharomyces cerevisiae strain BRSCS6, which possesses a mutated aspartate aminotransferase gene (the asp5 mutation). Complementation of the mutation was achieved using this construct.     

DNA demethylation in PD-1 gene promoter induced by 5-azacytidine activates PD-1 expression on Molt-4 cells

The expression of the programmed death 1 (PD-1) gene is an indicator of exhausted T-cells with decreased activation and function. It remains unknown, however, whether the methylation status of the PD-1 gene promoter is associated with PD-1 expression level. This study shows the changes of PD-1 expression levels and the demethylation status of the PD-1 promoter region in Molt-4 cells under different concentrations of 5-azacytidine (5-Zac). The result demonstrated that DNA demethylation at PD-1 promoter may contribute to PD-1 overexpression.     

Regulation of mouse mammary-gland gamma-glutamyltranspeptidase mRNA during pregnancy, lactation and weaning.

The level of gamma-glutamyltranspeptidase (GGT) activity and of its mRNA were determined in the mouse mammary gland during pregnancy, lactation and weaning. The GGT activity, which is very low in the virgin-mouse mammary gland (5 munits/mg of protein), increases progressively during pregnancy (3-fold), reaches its maximum at the onset of lactation (8-fold) and returns rapidly to basal level at weaning. Although no GGT-specific mRNA is detected in the virgin-mouse mammary gland, a single faint band of 2.2 kb in size is found during pregnancy. During lactation, an additional mRNA of 2.4 kb in size appears, and the level of both mRNAs is higher. This high level of mRNA persists during weaning as well. Southern-blot analysis of mouse mammary-gland DNA provides convincing evidence that there is only one gene which codes for the two mRNAs. The present study provides the first evidence for a physiological regulation of the two GGT mRNAs in the same tissue.     

Macronuclear DNA demethylation is involved in the encystment process of the ciliate Colpoda inflata.

Ciliate encystment is an eukaryotic cell differentiation process which involves a specific gene expression, to form the resting stage. In this study, we investigate, for first time, the DNA methylation pattern changes during encystment in the ciliate Colpoda inflata, and the 5-azacytidine effect on growing cells and encystment. Results indicate that 5-methylcytosine is present in macronuclear DNA of this ciliate and the 5-azacytidine treatment induces encystment in growth conditions. From restriction enzyme digestion and 5-azacytidine experiments, we conclude that a specific DNA demethylation is probably involved in the encystment gene expression of this ciliate.     

Macronuclear DNA demethylation is involved in the encystment process of the ciliate Colpoda inflata.

Ciliate encystment is an eukaryotic cell differentiation process which involves a specific gene expression, to form the resting stage. In this study, we investigate, for first time, the DNA methylation pattern changes during encystment in the ciliate Colpoda inflata, and the 5-azacytidine effect on growing cells and encystment. Results indicate that 5-methylcytosine is present in macronuclear DNA of this ciliate and the 5-azacytidine treatment induces encystment in growth conditions. From restriction enzyme digestion and 5-azacytidine experiments, we conclude that a specific DNA demethylation is probably involved in the encystment gene expression of this ciliate.