In vitro DNA cytosine methylation of cis-regulatory elements modulates c-Ha-ras promoter activity in vivo.
The effect of DNA cytosine methylation on promoter activity was assessed using a transient expression system employing pHrasCAT. This 551 bp Ha-ras-1 gene promoter region is enriched with 84 CpG dinucleotides, six functional GC boxes, and is prototypic of many genes possessing CpG islands in their promoter regions. Bacterial modification enzymes HhaI methyl transferase (MTase) and HpaII MTase, alone or in combination with a human placental DNA methyltransferase (HP MTase) that methylates CpG sites in a generalized manner, including asymmetric elements such as GC box CpG's, were used to methylate at different types of sites in the promoter. Methylation of HhaI and HpaII sites reduced CAT expression by approximately 70%-80%, whereas methylation at generalized CpG sites with HP MTase inactivated the promoter by greater than 95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in non-promoter regions.
Rachal
M J
MJ
Department of Molecular Pathology, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
Yoo
H
H
Becker
F F
FF
Lapeyre
J N
JN
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003596:Cytosine; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005809:Genes, Regulator; D011905:Genes, ras; D007700:Kinetics; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014162:Transfection
D003596:Cytosine; D015257:DNA-Cytosine Methylases; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases