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. 2005 Oct 24;33(19):6124-36.
doi: 10.1093/nar/gki920. Print 2005.

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

Taras Shevchuk  1 , Leo Kretzner, Kristofer Munson, John Axume, Jarrod Clark, Olga V Dyachenko, Marie Caudill, Yaroslav Buryanov, Steven S Smith
Affiliations

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

Taras Shevchuk et al. Nucleic Acids Res. .

Abstract

Several reports suggest that C(m)CWGG methylation tends not to co-exist with (m)CG 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 C(m)CWGG 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 C(m)CWGG 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 C(m)CWGG sites. Q-PCR measurements showed that RNA expression from the APC gene was not significantly altered by the presence of C(m)CWGG in its promoter. Kinetic studies suggested that an adjacent C(m)CWGG methylation site influences neither the maintenance nor the de novo methylation activities of purified human Dnmt1. We conclude that C(m)CWGG methylation does not exert a significant effect on CG methylation in human kidney cells.

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Figures

Figure 1
Figure 1
Schematic of a restriction digest scan. The scan illustrates the method used in obtaining the number average molecular weight from an ethidium bromide stained gel displaying a weight average distribution of fragment sizes.
Figure 2
Figure 2
Fluorescence detection of GFP and the M.EcoRII–GFP fusion in HK293 cells. Each of the two constructs was transfected into HK293 and subjected to long-term growth under puromycin selection. GFP fluorescence was easily detected with fluorescence microscopy when either construct was used. A set of micrographs is depicted for expression of the M.EcoRII–GFP fusion: (A) light microscopy and (B) fluorescence microscopy.
Figure 3
Figure 3
Florescence cell sorting quantification of cells expressing GFP and the M.EcoRII–GFP fusion in human HK293 cells. Each of the two constructs was transfected into HK293 and subjected to long-term growth under puromycin selection. Fluorescence cell sorting at the emission maximum for GFP quantifies the number of cells expressing each protein. (A) Quantification of cells expressing the GFP construct. (B) Quantification of cells expressing the M.EcoRII–GFP fusion. In each panel background fluorescence is given in black and cellular GFP fluorescence above background is given in grey. Total counts given by the stippled line indicate that not all positive events were collected in the sort.
Figure 4
Figure 4
GFP expression during selection with puromycin. Cells plated on puromycin containing medium were sorted for GFP expression at the indicated times. The percentage of cells positive for GFP expression in pI-GFP transfected cells (GFP expressors) is given in black. The percentage of GFP expression in pI-EcoGFP transfected cells (M.EcoRII–GFP fusion expressors) is given in grey. After each round of sorting, equal numbers of GFP positive cells were re-plated for continued growth in the presence of puromycin.
Figure 5
Figure 5
Detection of M.EcoRII activity from the M.EcoRII–GFP fusion expression vector. After 21 days of selection with 0.5 µM puromycin, fluorescent cells were harvested and genomic DNA was prepared. The DNA was digested with the isoschizomers R.EcoRII and R.BstNI. In this pair, R.EcoRII is unable to cleave the CmCWGG site when the internal cytosine is methylated. While R.BstNI is unaffected by cytosine-5 methylation at this site.
Figure 6
Figure 6
Effect of CmCWGG methylation at a promoter that is heavily methylated at CG sites. Bisulfite treated DNA from the SERPINB5 promoter was amplified, cloned and sequenced as described in Materials and Methods. The 372 bp amplicon contains 19 CG dinucleotide pairs. It also contains two CCWGG sites, one in the reverse primer and one downstream from the reverse primer. Ten clones were sequenced in order to determine the methylation state at each of the 19 CG sites and the two CCWGG sites in each clone. (A) Bar graph depicting the observed fraction of methylated sequences at each site in the two types of transformed cells. (B) Those expressing GFP alone are shown in grey (left), and those expressing the M.EcoRII-GFP fusion are shown in white (right). Map of the amplicon depicting the relative position of each of the sites in the bar graph.
Figure 7
Figure 7
Effect of CmCWGG methylation at a promoter that is unmethylated at CG sites. Bisulfite treated DNA from the APC promoter was amplified cloned and sequenced as described in Materials and Methods. The 279 bp amplicon contains 19 CG dinucleotide pairs. It contains three CCWGG sites, one in the forward primer, and two downstream from the forward primer. Ten clones were sequenced in order to determine the methylation state at each of the 19 CG sites and the three CCWGG sites in each clone. (A) Bar graph depicting the observed fraction of methylated sequences at each site in the two types of transformed cells. (B) Those expressing GFP alone are shown in grey (left), and those expressing the M.EcoRII-GFP fusion are shown in white (right). Map of the amplicon depicting the relative position of each of the sites in the bar graph.
Figure 8
Figure 8
Comparison of RNA expression levels from the APC gene and the hTERT gene in cells expressing GFP alone, or the M.EcoRII–GFP fusion. Ct values obtained at each fold dilution of input RNA are plotted. Two independent RNA preparations were used, one preparation was tested in duplicate and one preparation was tested in triplicate. P-values from χ2 analyses are listed below each set of data points. Each value is >>0.05, indicating that there is no significant difference in the level of RNA expression when comparing the two cell lines, for either of the two genes tested. (A) Data for the hTERT gene. closed squares, cells expressing the M.EcoRII–GFP fusion. Closed diamonds, cells expressing the GFP alone. (B) Data for the APC gene. Closed squares, cells expressing the M.EcoRII–GFP fusion. Closed diamonds, cells expressing the GFP alone.
Figure 9
Figure 9
Kinetics of methylation by human DNMT1 at CmCWGG methylated oligodeoxynucleotide substrates from the SERPINB5 promoter. Saturation curves for SERPINB5 oligodeoxynucleotide substrates with varying states of methylation at CCWGG and adjacent CG site are depicted. Duplex 1: saturation curve for the unmethylated duplex. Duplex 2: saturation curve for the duplex carrying a hemimethylated CCWGG site. Duplex 3: saturation curve for the duplex carrying a hemimethylated CG site. Duplex 4: saturation curve for the duplex with both the CCWGG and the CG sites in the hemimethylated state.
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