title: Expression patterns and miRNA regulation of DNA methyltransferases in chicken primordial germ cells.
abstract: DNA methylation is widespread in most species, from bacteria to mammals, and is crucial for genomic imprinting, gene expression, and embryogenesis. DNA methylation occurs via two major classes of enzymatic reactions: maintenance-type methylation catalyzed by DNA (cytosine-5-)-methyltransferase (DNMT) 1, and de novo methylation catalyzed by DNMT 3 alpha (DNMT3A) and -beta (DNMT3B). The expression pattern and regulation of DNMT genes in primordial germ cells (PGCs) and germ line cells has not been sufficiently established in birds. Therefore, we employed bioinformatics, RT-PCR, real-time PCR, and in situ hybridization analyses to examine the structural conservation and conserved expression patterns of chicken DNMT family genes. We further examined the regulation of a candidate de novo DNA methyltransferase gene, cDNMT3B by cotransfection of cDNMT3B 3'UTR- and cDNMT3B 3'UTR-specific miRNAs through a dual fluorescence reporter assay. All cDNMT family members were differentially detected during early embryonic development. Of interest, cDNMT3B expression was highly detected in early embryos and in PGCs. During germ line development and sexual maturation, cDNMT3B expression was reestablished in a female germ cell-specific manner. In the dual fluorescence reporter assay, cDNMT3B expression was significantly downregulated by four miRNAs: gga-miR-15c (25.82%), gga-miR-29b (30.01%), gga-miR-383 (30.0%), and gga-miR-222 (31.28%). Our data highlight the structural conservation and conserved expression patterns of chicken DNMTs. The miRNAs investigated in this study may induce downregulation of gene expression in chicken PGCs and germ cells.
authors.affiliation: WCU Biomodulation Major, Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Korea.
authors.forename: Deivendran
authors.initials: D
authors.lastname: Rengaraj
authors.forename: Bo Ram
authors.initials: BR
authors.lastname: Lee
authors.forename: Sang In
authors.initials: SI
authors.lastname: Lee
authors.forename: Hee Won
authors.initials: HW
authors.lastname: Seo
authors.forename: Jae Yong
authors.initials: JY
authors.lastname: Han
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D002460:Cell Line; D002645:Chickens; D019295:Computational Biology; D018899:CpG Islands; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D005260:Female; D005456:Fluorescent Dyes / Q000494:pharmacology; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D005854:Germ Cells / Q000378:metabolism*; D017403:In Situ Hybridization; D035683:MicroRNAs / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000097584:DNA Methyltransferase 3B
chemical_list: D020413:3' Untranslated Regions; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D005456:Fluorescent Dyes; D035683:MicroRNAs; D000097584:DNA Methyltransferase 3B