Methylation of CpG dinucleotides in the open reading frame of a testicular germ cell-specific intronless gene, Tact1/Actl7b, represses its expression in somatic cells.
Methylation of CpG islands spanning promoter regions is associated with control of gene expression. However, it is considered that methylation of exonic CpG islands without promoter is not related to gene expression, because such exonic CpG islands are usually distant from the promoter. Whether methylation of exonic CpG islands near the promoter, as in the case of a CpG-rich intronless gene, causes repression of the promoter remains unknown. To gain insight into this issue, we investigated the distribution and methylation status of CpG dinucleotides in the mouse Tact1/Actl7b gene, which is intronless and expressed exclusively in testicular germ cells. The region upstream to the gene was poor in CpG, with CpG dinucleotides absent from the core promoter. However, a CpG island was found inside the open reading frame (ORF). Analysis of the methylation status of the Tact1/Actl7b gene including the 5'-flanking area demonstrated that all CpG sites were methylated in somatic cells, whereas these sites were unmethylated in the Tact1/Actl7b-positive testis. Trans fection experiments with in vitro-methylated constructs indicated that methylation of the ORF but not 5' upstream repressed Tact1/Actl7b promoter activity in somatic cells. Similar effects of ORF methylation on the promoter activity were observed in testicular germ cells. These are the first results indicating that methylation of the CpG island in the ORF represses its promoter in somatic cells and demethylation is necessary for gene expression in spermatogenic cells.
Hisano
Mizue
M
Department of Laboratory for Science Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamadaoka, Suita, Osaka 565-0871, Japan.
Ohta
Hiroshi
H
Nishimune
Yoshitake
Y
Nozaki
Masami
M
D000199:Actins; D000818:Animals; D018899:CpG Islands / Q000235:genetics*; D003598:Cytoskeletal Proteins; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins; D007438:Introns / Q000235:genetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D041681:NIH 3T3 Cells; D016366:Open Reading Frames / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011506:Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013087:Spermatids / Q000166:cytology / Q000378:metabolism; D013737:Testis / Q000166:cytology / Q000378:metabolism
D000199:Actins; C480961:Actl7b protein, mouse; D003598:Cytoskeletal Proteins; D008164:Luminescent Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D008156:Luciferases