Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells

Abstract

Several reports suggest that CmCWGG methylation tends not to co-exist with mCG methylation in human cells. We have asked whether or not methylation at CCWGG sites can influence CG methylation. DNA from cells expressing an M.EcoRII–GFP fusion was actively methylated at CCWGG sites. CG methylation as measured by R.HpaII/R.MspI ratios was unchanged in cells expressing the transgene. Cloned representatives of CmCWGG methylated DNA often contained, or were adjacent to an ALU repeat, suggesting that M.EcoRII-GFP actively methylated gene-rich R-band DNA. The transgenic methyltransferase applied CmCWGG methylation to a representative human promoter that was heavily methylated at CG dinucleotides (the SERPINB5 promoter) and to a representative promoter that was essentially unmethylated at CG dinucleotides (the APC promoter). In each case, the CG methylation pattern remained in its original state, unchanged by the presence of neighboring CmCWGG sites. Q-PCR measurements showed that RNA expression from the APC gene was not significantly altered by the presence of CmCWGG in its promoter. Kinetic studies suggested that an adjacent CmCWGG methylation site influences neither the maintenance nor the de novo methylation activities of purified human Dnmt1. We conclude that CmCWGG methylation does not exert a significant effect on CG methylation in human kidney cells.

Distribution of CWG and CCWGG in the Human Genome

“…1,18 Further, since its frequency is not underrepresented throughout the genome or in putative control regions adjacent to genes it would appear that the methylation of the CWG sites may not have a specific biological function in human DNA that involves high levels of 5-methylcytosine. This possibility is supported by previous evidence, 11 although further experimentation will be required to answer this question. On the other hand the data does allow us to conclude that site specific systems apply methylation to CG sites in the human genome with high frequency while site specific methylation systems apply cytosine methylation to CWG and CCWGG only at very low frequency if at all.…”

“…This stands in stark contrast to methylation at CG sites which is correlated with a variety of clastic events including viral integration, genetic recombination, gene expansion, as well as transcriptional silencing. [2][3][4][5][6][7][8][9] Consistent with this contrast are the findings 10 that the expression of the bacterial G M CGC methyltransferase M•HhaI resulted in increased numbers of soft agar foci, formed by Mouse 3T3 cells, while the expression of the bacterial C M CWGG methyltransferase M•EcoRII had no detectable biological effect 11 on human HK293 cells.…”

Global Leukocyte DNA Methylation is Similar in African American and Caucasian Women Under Conditions of Controlled Folate Intake

“…4 During the period of folate restriction (wk 0-7), the subjects consumed a low folate diet providing 135 mg DFE/d. During the period of folate treatment (wk [8][9][10][11][12][13][14], the subjects continued to consume the folate restricted diet providing 135 mg DFE/d plus 156 or 391 mg/d supplemental folic acid for total folate intakes of 400 or 800 mg DFE/d, respectively.…”

Choline status is not a reliable indicator of moderate changes in dietary choline consumption in premenopausal women

“…The cytosine extension assay [33] with minor modifications [34] was used to assess global DNA methylation. DNA was extracted from mononuclear cells, and genomic DNA (0.75 μg) was digested with an excess of methylationsensitive HpaII restriction endonuclease (New England Biolabs, Beverly, MA) according to the manufacturer's protocol.…”