title: Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells.
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.
authors.affiliation: City of Hope National Medical Center and Beckman Research Institute, 1500 E. Duarte Road, Duarte, CA 91010, USA.
authors.forename: Taras
authors.initials: T
authors.lastname: Shevchuk
authors.forename: Leo
authors.initials: L
authors.lastname: Kretzner
authors.forename: Kristofer
authors.initials: K
authors.lastname: Munson
authors.forename: John
authors.initials: J
authors.lastname: Axume
authors.forename: Jarrod
authors.initials: J
authors.lastname: Clark
authors.forename: Olga V
authors.initials: OV
authors.lastname: Dyachenko
authors.forename: Marie
authors.initials: M
authors.lastname: Caudill
authors.forename: Yaroslav
authors.initials: Y
authors.lastname: Buryanov
authors.forename: Steven S
authors.initials: SS
authors.lastname: Smith
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D003596:Cytosine / Q000378:metabolism; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006147:Guanine / Q000032:analysis; D006801:Humans; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D011401:Promoter Regions, Genetic; D012313:RNA / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000032:analysis; D019076:Transgenes
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006147:Guanine; D012313:RNA; D003596:Cytosine; C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human