DNA methylation controls the inducibility of the mouse metallothionein-I gene in lymphoid cells

The W7 mouse thymoma cell line does not express the metallothionein-I (MT-I) gene in the presence of either cadmium or glucocorticoids, unlike most other cell lines. This cell line was therefore used as a model system for studying the role of DNA methylation on MT-I gene expression. The extent of DNA methylation within the MT-I gene and its flanking regions was determined by comparing the cleavage patterns generated by the isoschizomeric restriction enzymes Hpa II and Msp I. In W7 cells, all of the Hpa II sites in the vicinity of the MT-I gene are methylated, whereas in cells that have an expressible MT-I gene (for example, Friend erythroleukemia cells) all of these Hpa II sites are unmethylated. When W7 cells are treated for a few hours with 5-azacytidine, the MT-I gene becomes inducible by both cadmium and glucocorticoids. Addition of hydroxyurea along with 5-azacytidine prevents MT-I gene induction, suggesting that incorporation of 5-azacytidine into DNA is required before this gene can be activated. To determine whether 5-azacytidine treatment changes the methylation pattern near the MT-I gene, we treated W7 cells with 5-azacytidine and selected inducible cells in 10 μM cadmium. All of the Hpa II sites within the MT-I gene are unmethylated in these cadmium-resistant W7 cells. In addition, flanking DNA sequences are also undermethylated in a pattern similar to that seen in Friend erythroleukemia cells that express the MT-I gene. The possible significance of methylation as a mechanism of gene commitment during cell differentiation is discussed.     

Extensive methylation of a part of the CpG island located 3.0-4.5 kbp upstream to the chicken alpha-globin gene cluster may contribute to silencing the globin genes in non-erythroid cells

Here, we show that in the chicken genome, the domain of alpha-globin genes is preceded by a CpG island of which the downstream part ( approximately 0.65 kbp) is heavily methylated in lymphoid cells; it is either non-methylated or undermethylated in erythroid cells. Recombinant plasmids were constructed with the corresponding DNA fragment (called "uCpG") placed upstream to a reporter CAT gene expressed from the promoter of the alpha(D) chicken globin gene. Selective methylation of CpG dinucleotides within the uCpG fragment suppressed fivefold the expression of the CAT gene, when neither this gene itself nor the alpha(D) promoter were methylated. Methylation of CpG dinucleotides within the alpha(D) gene promoter did not modify the suppression effect exerted by methylated uCpG. We interpret these results within the frame of the hypothesis postulating, that methylation of the upstream CpG island of the chicken alpha-globin gene domain may play an essential role in silencing the alpha-globin genes in non-erythroid cells.     

Site-specific DNA methylation contributes to neurotensin/neuromedin N expression in colon cancers

The neurotensin/neuromedin N (NT/N) gene is expressed in fetal colon, repressed in newborn and adult colon, and reexpressed in approximately 25% of colon cancers. Our purpose was to determine the effect of gene methylation on NT/N silencing in colon cancers. We found that the NT/N gene was expressed in human colon cancer cell line KM12C but not in KM20 colon cancer cells. Bisulfite genomic sequencing demonstrated that all CpG dinucleotides in the region from -373 to +100 of the NT/N promoter, including a CpG site in a distal consensus AP-1 site, were methylated in KM20 but unmethylated in KM12C cells. Treatment of KM20 cells with demethylating agent 5-azacytidine induced NT/N expression, suggesting a role for DNA methylation in silencing of NT/N in colon cancers. To better elucidate the mechanisms responsible for NT/N repression by DNA methylation, we performed gel shift assays using an oligonucleotide probe corresponding to the distal AP-1 consensus sequence of the NT/N promoter. Methylation of the oligonucleotide probe inhibited protein binding to the distal AP-1 site of the NT/N promoter, suggesting a potential mechanism of NT/N gene repression in colon cancers. We show that DNA methylation plays a role in NT/N gene silencing in the human colon cancer KM20 and that NT/N expression in KM12C cells is associated with demethylation of the CpG sites. DNA methylation likely contributes to NT/N gene expression noted in human colon cancers.     

Promoter methylation and transgene copy numbers predict unstable protein production in recombinant Chinese hamster ovary cell lines

Mammalian cell lines for recombinant protein production need to maintain productivity over extended cultivation times. Long-term stability studies are time and resource intensive, but are widely performed to identify and eliminate unstable candidates during cell line development. Production instability of manufacturing cell lines can be associated with methylation and silencing of the heterologous promoter. We have identified CpG dinucleotides within the human cytomegalovirus major immediate early promoter/enhancer (hCMV-MIE) that are frequently methylated in unstable antibody-producing Chinese hamster ovary (CHO) cell lines. We have established methylation-specific real-time qPCR for the rapid and sensitive measurement of hCMV-MIE methylation in multiple cell lines and provide evidence that hCMV-MIE methylation and transgene copy numbers can be used as early markers to predict production instability of recombinant CHO cell lines. These markers should provide the opportunity to enrich stable producers early in cell line development and allow developers to put more emphasis on other criteria, such as product quality and bioprocess robustness.     

Array-based analysis of genomic DNA methylation patterns of the tumour suppressor gene p16INK4A promoter in colon carcinoma cell lines

Aberrant DNA methylation at CpG dinucleotides can result in epigenetic silencing of tumour suppressor genes and represents one of the earliest events in tumourigenesis. To date, however, high-throughput tools that are capable of surveying the methylation status of multiple gene promoters have been restricted to a limited number of cytosines. Here, we present an oligonucleotide microarray that permits the parallel analysis of the methylation status of individual cytosines, thus combining high throughput and high resolution. The approach was used to study the CpG island in the promoter region of the tumour suppressor gene p16^INK4A. In total, 876 oligonucleotide probes of 21 nt in length were used to inspect the methylation status of 53 CpG dinucleotides, producing correct signals in colorectal cancer cell lines as well as control samples with a defined methylation status. The information was validated by established alternative methods. The overall methylation pattern was consistent for each cell line, while different between them. At the level of individual cytosines, however, significant variations between individual cells of the same type were found, but also consistencies across the panel of cancer cell lines were observed.