Epigenetic modifications affect Dnmt3L expression.
Imprinted genes are expressed from a single allele due to differential methylation of maternal or paternal alleles during gametogenesis. Dnmt3L (DNA cytosine-5-methyltransferase 3 like), a member of de novo methyltransferase Dnmt3 protein family, is a regulator of maternal imprinting. In the present study, we have characterized the promoter region of the mouse Dnmt3L gene. Transient transfection assays performed with 5'-deletion promoter constructs indicated a minimal promoter area within 440 bp upstream from the translational start site. Longer promoter constructs showed decreased activity, suggesting the presence of repressor elements within the upstream sequences. According to electrophoretic mobility-shift assays and mutation analysis, the minimal promoter region contained four functional binding sites for the Sp1 (specificity protein 1) family of transcription factors, Sp1 and Sp3. In vitro methylation of Dnmt3L promoter constructs decreased the transcriptional activity significantly, demonstrating down-regulation by cytosine methylation. This was supported by the results from bisulphite sequencing and real-time quantitative reverse transcriptase-PCR analysis of different mouse cell lines and tissues. In testis and embryonic stem cells showing strong Dnmt3L expression, all CpG sites studied were fully unmethylated, whereas non-expressive cell lines and tissues with lesser Dnmt3L expression showed complete or diverse CpG methylation levels. Treatment of Dnmt3L non-expressive cell lines with deacetylase inhibitor trichostatin A and methyltransferase inhibitor 5-aza-2'-deoxycytidine induced the expression of Dnmt3L mRNA. Furthermore, we show that the repressional effect of longer promoter fragments was also relieved by these inhibitors, altogether indicating an epigenetic control for Dnmt3L gene regulation.
Aapola
Ulla
U
Department of Pathology, Institute of Medical Technology, Tampere University Hospital, University of Tampere, 33014 Tampere, Finland. ulla.aapola@uta.fi
Mäenpää
Katja
K
Kaipia
Antti
A
Peterson
Pärt
P
D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001483:Base Sequence / Q000235:genetics; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D000077209:Decitabine; D004622:Embryo, Mammalian / Q000166:cytology; D044127:Epigenesis, Genetic / Q000235:genetics*; D004847:Epithelial Cells / Q000737:chemistry / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics*; D006877:Hydroxamic Acids / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000235:genetics; D013234:Stem Cells / Q000737:chemistry / Q000378:metabolism; D013950:Thymus Gland / Q000166:cytology
D006877:Hydroxamic Acids; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; C012589:trichostatin A; D000077209:Decitabine; D008156:Luciferases; C498796:Dnmt3l protein, mouse; D004248:DNA (Cytosine-5-)-Methyltransferases; D001374:Azacitidine