An epigenetic mechanism regulates germ cell-specific expression of the porcine Deleted in Azoospermia-Like (DAZL) gene.
The Deleted in Azoospermia-Like (DAZL) gene is specifically expressed in fetal and adult gonads. While DAZL is known to play a role during gametogenesis, the mechanisms governing its germ cell-specific expression remain unclear. We identified the 5' untranslated region (UTR) of the porcine DAZL gene and cloned and characterized 2 kilobase pairs of its TATA-less 5' flanking region, identifying CpG-rich regions within the proximal promoter. Nine of 18 CpG sites in proximity to one region were largely unmethylated in germ cells but hypermethylated in somatic cells, suggesting that DNA methylation may regulate DAZL promoter activity. Furthermore, DAZL expression was induced in fibroblasts treated with a demethylating agent. Deletion analyses revealed that the minimal 149 base pair promoter region was sufficient to activate transcription. In vitro methylation of a reporter construct corresponding to these 149 base pairs resulted in complete suppression of DAZL promoter activity in primordial germ cells, further supporting a role for methylation in regulating DAZL expression. Interestingly, the differentially methylated region was shown to harbor several putative Sp1-binding sites. Mutation of only the most highly conserved site significantly reduced promoter activity in a reporter assay. Furthermore, gel shift assays revealed that Sp1 was able to specifically bind to this site, and that complex formation was inhibited when CpG dinucleotides within this region were methylated. Chromatin immunoprecipitation (ChIP) assays revealed that in vivo Sp1 binding to the core DAZL promoter region was enriched in germ cells but not in fibroblasts. Our data suggests that DNA methylation may suppress DAZL expression in somatic cells by interfering with Sp1 binding. This study provides insights into the potential mechanisms underlying the regulation of germ cell-specific gene expression.
Linher
Katja
K
Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Cheung
Queenie
Q
Baker
Perrin
P
Bedecarrats
Gregoy
G
Shiota
Kunio
K
Li
Julang
J
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D044127:Epigenesis, Genetic; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D013552:Swine / Q000235:genetics* / Q000378:metabolism*
D020413:3' Untranslated Regions; C101358:DAZL protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor