Infection with human immunodeficiency virus type 1 upregulates DNA methyltransferase, resulting in de novo methylation of the gamma interferon (IFN-gamma) promoter and subsequent downregulation of IFN-gamma production

The immune response to pathogens is regulated by a delicate balance of cytokines. The dysregulation of cytokine gene expression, including interleukin-12, tumor necrosis factor alpha, and gamma interferon (IFN-gamma), following human retrovirus infection is well documented. One process by which such gene expression may be modulated is altered DNA methylation. In subsets of T-helper cells, the expression of IFN-gamma, a cytokine important to the immune response to viral infection, is regulated in part by DNA methylation such that mRNA expression inversely correlates with the methylation status of the promoter. Of the many possible genes whose methylation status could be affected by viral infection, we examined the IFN-gamma gene as a candidate. We show here that acute infection of cells with human immunodeficiency virus type 1 (HIV-1) results in (i) increased DNA methyltransferase expression and activity, (ii) an overall increase in methylation of DNA in infected cells, and (iii) the de novo methylation of a CpG dinucleotide in the IFN-gamma gene promoter, resulting in the subsequent downregulation of expression of this cytokine. The introduction of an antisense methyltransferase construct into lymphoid cells resulted in markedly decreased methyltransferase expression, hypomethylation throughout the IFN-gamma gene, and increased IFN-gamma production, demonstrating a direct link between methyltransferase and IFN-gamma gene expression. The ability of increased DNA methyltransferase activity to downregulate the expression of genes like the IFN-gamma gene may be one of the mechanisms for dysfunction of T cells in HIV-1-infected individuals.     

Mast cell-/basophil-specific transcriptional regulation of human L-histidine decarboxylase gene by CpG methylation in the promoter region

L-Histidine decarboxylase (HDC) catalyzes the formation of histamine from L-histidine, and in hematopoietic cell lineages the gene is expressed only in mast cells and basophils. We attempted here to discover how HDC gene expression is restricted in these cells. In the cultured cell lines tested, only the mast cells and basophils strongly transcribed the HDC gene. However, in transient transfection analysis, the reporter constructs with the HDC promoter were active not only in expressing cells but also in nonexpressing cells. Detailed analyses of the HDC promoter region revealed that the GC box is essential for transactivation. Also, the promoter region of the HDC gene proved to be sensitive to DNase I and restriction endonucleases exclusively in HDC-expressing cells, suggesting that the promoter region is readily accessible to trans-acting factor(s). Furthermore, the promoter region in HDC-expressing cell lines was found to be selectively unmethylated. The correlation between HDC expression and hypomethylation was also found in primary human mast cells. Methylation of the HDC promoter in vitro reduced the luciferase reporter activity in transient expression analysis, suggesting that methylation of the promoter region is functionally important for HDC gene expression. These results imply that alteration of DNA methylation is one of the mechanisms regulating cell-specific expression of the HDC gene.     

DNA methylation in the promoter region of the p16 (CDKN2/MTS-1/INK4A) gene in human breast tumours

The p16 (CDKN2/MTS-1/INK4A) gene is one of several tumour-suppressor genes that have been shown to be inactivated by DNA methylation in various human cancers including breast tumours. We have used bisulphite genomic sequencing to examine the detailed sequence specificity of DNA methylation in the CpG island promoter/exon 1 region in the p16 gene in DNA from a series of human breast cancer specimens and normal human breast tissue (from reductive mammaplasty). The p16 region examined was unmethylated in the four normal human breast specimens and in four out of nine breast tumours. In the other five independent breast tumour specimens, a uniform pattern of DNA methylation was observed. Of the nine major sites of DNA methylation in the amplified region from these tumour DNAs, four were in non-CG sequences. This unusual concentration of non-CG methylation sites was not a general phenomenon present throughout the genome of these tumour cells because the methylated CpG island regions of interspersed L1 repeats had a pattern of (almost exclusively) CG methylation similar to that found in normal breast tissue DNA and in DNA from tumours with unmethylated p16 genes. These data suggest that DNA methylation of the p16 gene in some breast tumours could be the result of an active process that generates a discrete methylation pattern and, hence, could ultimately be amenable to therapeutic manipulation.     

Genetic analysis of the Caenorhabditis elegans MAP kinase gene mpk-1

Differential genomic DNA methylation has the potential to influence the development of T cell cytokine production profiles. Therefore, we have conducted a clonal analysis of interferon (IFN)-gamma and interleukin (IL)-3 gene methylation and messenger (m)RNA expression in primary CD8+ T cells during the early stages of activation, growth, and cytokine expression. Despite similar distributions and densities of CpG methylation sites, the IFN-gamma and IL-3 promoters exhibited differential demethylation in the same T cell clone, and heterogeneity between clones. Methylation patterns and mRNA levels were correlated for both genes, but demethylation of the IFN-gamma promoter was widespread across >300 basepairs in clones expressing high levels of IFN-gamma mRNA, whereas demethylation of the IL-3 promoter was confined to specific CpG sites in the same clones. Conversely, the majority of clones expressing low or undetectable levels of IFN-gamma mRNA exhibited symmetrical methylation of four to six of the IFN-gamma promoter CpG sites. Genomic DNA methylation also has the potential to influence the maintenance or stability of T cell cytokine production profiles. Therefore, we also tested the heritability of IFN-gamma gene methylation and mRNA expression in families of clones derived from resting CD44(low)CD8+ T cells or from previously activated CD44(high)CD8+ T cells. The patterns of IFN-gamma gene demethylation and mRNA expression were faithfully inherited in all clones derived from CD44(high) cells, but variable in clones derived from CD44(low) cells. Overall, these findings suggest that differential genomic DNA methylation, including differences among cytokine genes, among individual T cells, and among T cells with different activation histories, is an important feature of cytokine gene expression in primary T cells.     

MUC2 gene suppression in human colorectal carcinomas and their metastases: in vitro evidence of the modulatory role of DNA methylation

The development of the majority of colorectal carcinomas is associated with a diminished expression of the intestinal mucin MUC2 in the tumor cells. The significance and the mechanism of this alteration are not yet known. We sought to determine the molecular basis of this tumor-associated change and to investigate the extent to which it might also relate to metastases. MUC2 gene expression was compared in normal (N), carcinomatous (T), and metastatic tissues (M) from nine patients by immunohistochemistry, in situ hybridization, and Northern blotting. Immunohistochemistry and in situ hybridization showed consistently lower amounts of the expressed protein and mRNA in T and in M than in N; quantitative analysis by Northern blotting confirmed that the differences between MUC2 mRNA expression between N, T, and M were significant, the expression in metastases being less than 5% of that in the normal colonic tissue. The influence of DNA methylation as a possible regulatory mechanism of MUC2 gene expression was tested after the 5' and 3'-regions flanking the first exon of MUC2 were recovered from a genomic DNA library and used as probes in Southern blot. The DNA was isolated from colon carcinoma cell lines expressing MUC2 strongly (LS174T) or moderately (T84) and from that which was nonexpressing (Colo 205), and it was digested with the methylation-sensitive enzyme HpaII. The Southern blot patterns indicated that the increased methylation in the promoter region was concomitant with the decrease of MUC2 mRNA expression. Methylation of the promoter region ligated into a reporter vector suppressed the expression of the luciferase reporter gene in the three investigated cell lines. Furthermore, the expression of MUC2 gene was enhanced by treating the MUC2-expressing colon carcinoma cells with 5-aza-2'-deoxycytidine, a methylation-inhibiting agent. To our knowledge this is the first report to show that: (a) MUC2 gene is strongly suppressed in liver and lymph node metastases of colorectal carcinomas, and (b) suppression of MUC2 gene in colon carcinoma cells in vitro is associated with methylation of the promoter region.     

Altered methylation of the human MDR1 promoter is associated with acquired multidrug resistance

One of the most important forms of drug resistance in acute myeloid leukemia is the multidrug resistance (MDR) phenotype, which is characterized by the expression of the MDR1 gene product, P-glycoprotein. Although a number of factors affect MDR1 gene expression, the genetic events that "switch on" the human MDR1 gene in tumor cells that were previously P-glycoprotein negative have remained elusive. Here, we report evidence that the methylation status of the human MDR1 promoter may serve as a basis for this "switch." Based on Southern analysis using methylation-sensitive and methylation-insensitive restriction enzymes, a tight correlation was found between MDR phenotype and demethylation of the 5' region of the MDR1 gene in a human T cell leukemia cell line. Similar results were obtained from the analysis of P-glycoprotein-positive and P-glycoprotein-negative samples of chronic lymphocytic leukemia. Treatment of the cell lines with the demethylating agent 5'-azadeoxycytidine altered the methylation pattern of the MDR1 promoter in P-glycoprotein-negative cells to resemble that of P-glycoprotein-positive cells and activated the promoter such that MDR1 mRNA was now detectable. Treatment also resulted in an increased resistance to epirubicin and decreased daunomycin accumulation, both of which were reversible by verapamil, a characteristic of the classical MDR phenotype in cells expressing P-glycoprotein. These results suggest that the MDR phenotype may be acquired as a result of changes in methylation of the MDR1 promoter.     

Regulation of tissue factor gene expression in epithelial cells. Induction by serum and phorbol 12-myristate 13-acetate

Cell-specific expression of tissue factor (TF) in vivo is consistent with its primary role in hemostasis. In addition, TF expression is induced in cultured cells by a variety of agents, including serum and growth factors, which define the TF gene as a "primary response" gene. In this study we examined the signaling pathways and cis-acting regulatory elements required for induction of TF gene expression in HeLa cells in response to serum and the tumor promoter, phorbol 12-myristate 13-acetate (PMA). TF activity and mRNA were induced greater than sixfold in quiescent HeLa cells by serum and PMA. TF mRNA induction by both agonists required intracellular Ca2+ mobilization, whereas inhibition of protein kinase C abolished induction of the TF gene by PMA but had no effect on induction by serum. Functional studies demonstrated that a region of the human TF promoter between -96 and +121 bp contained regulatory elements required for serum and PMA induction. These data indicate that different signaling pathways regulate TF gene expression in response to serum and PMA, although the same cis-acting DNA elements may mediate induction.     

Specific expression in mouse mesoderm- and neural crest-derived tissues of a human PDGFRA promoter/lacZ transgene

The platelet-derived growth factor alpha-receptor (PDGFR-alpha) displays a lineage-specific expression pattern in the mouse embryo and is required for normal development of mesoderm and cephalic neural crest derivatives. The purpose of the present study was to demonstrate the in vivo promoter function of genomic DNA fragments representing the 5'-flanking part of the human PDGFRA gene. 2.2, 0.9 and 0.4 kb PDGFRA promoter fragments, ligated to a lacZ reporter gene, were microinjected into fertilized mouse eggs and transgenic mouse lines were established. The expression patterns were basically similar in the 2.2 and 0.9 kb lines and overlapped grossly the endogenous Pdgfra gene expression pattern. The transgenic line with the highest expression level was chosen for detailed analysis. Expression was, as expected, mainly confined to tissues of mesodermal and neural crest origin. No expression was found in epithelial tissues of endo- or ectodermal origin. The promoter fragments were also active in neuroepithelium and in certain neuronal cell types that did not faithfully express PDGFR-alpha mRNA, while they failed to specify reporter expression in PDGFR-alpha expressing O-2A progenitor cells and other glial elements of the central nervous system. Thus, the isolated human PDGFRA promoter contains most but not all of the regulatory elements that are necessary to establish tissue specific gene expression during development.