The 5'-ends of two paternally expressed mouse genes, Peg3 and Usp29, are jointly associated with a CpG island that exhibits allele-specific methylation. Sequence comparison of the regions derived from human, mouse and cow revealed the presence of two evolutionarily conserved sequence motifs including one that is repeated multiple times within the first intron of Peg3 in all three mammals. DNA mobility shift and chromatin immunoprecipitation (ChIP) assays clearly demonstrated that this motif is an in vivo binding site for the Gli-type transcription factor YY1. The YY1-binding site contains one CpG dinucleotide, and methylation of this CpG site abolishes the binding activity of YY1 in vitro. The Peg3 YY1-binding sites are methylated only on the maternal chromosome in vivo, and ChIP assays confirmed that YY1 binds specifically to the paternal allele of the gene. Promoter, enhancer and insulator assays with deletion constructs of sequence surrounding the YY1-binding sites indicate that the region functions as a methylation-sensitive insulator that may influence the imprinted expression of Peg3 and neighboring genes. The current study is the first report demonstrating the involvement of YY1 in methylation-sensitive insulator activity and suggests a potential role of this highly conserved protein in mammalian genomic imprinting.
Using mouse BAC clones spanning an imprinted interval of proximal mouse chromosome 7 and the genomic sequence of the related interval of human chromosome 19q13.4, we have identified a novel mouse gene,Usp29 (ubiquitin-specific processing protease 29), near two known imprinted genes, Peg3 and Zim1. GeneUsp29 is located directly adjacent to Peg3 in a “head-to-head” orientation, and comprises exons distributed over a genomic distance of at least 400 kb. A similar human gene is also found in the homologous location in human chromosome 19q13.4. The mouseUsp29 gene is also imprinted and is transcribed mainly from the paternal allele with highest expression levels in adult brain, especially in the cerebral cortex and hippocampus, and in the forebrain, face, and limb buds of midgestation mouse embryos. Analysis of a full-length 7.6-kb cDNA clone revealed that Usp29 encodes an 869-amino-acid protein that displays significant homology with yeast and nematode ubiquitin carboxyl-terminal hydrolases. These data suggest that, like the candidate Angelman syndrome gene Ube3a(ubiquitin ligase), Usp29 may represent another imprinted gene involved in the ubiquitination pathway. This identification of a third imprinted gene, Usp29, from the Peg3/Zim1-region confirms the presence of a conserved imprinted domain spanning at least 500 kb in the proximal portion of mouse chromosome 7 (Mmu7).[The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF229257 andAF229438.]
Mammalian genomic imprinting is regulated by imprinting control regions (ICRs) that are usually associated with tandem arrays of transcription factor binding sites. In this study, the sequence features derived from a tandem array of YY1 binding sites of Peg3-DMR (differentially methylated region) led us to identify three additional clustered YY1 binding sites, which are also localized within the DMRs of Xist, Tsix, and Nespas. These regions have been shown to play a critical role as ICRs for the regulation of surrounding genes. These ICRs have maintained a tandem array of YY1 binding sites during mammalian evolution. The in vivo binding of YY1 to these regions is allele specific and only to the unmethylated active alleles. Promoter/enhancer assays suggest that a tandem array of YY1 binding sites function as a potential orientation-dependent enhancer. Insulator assays revealed that the enhancer-blocking activity is detected only in the YY1 binding sites of Peg3-DMR but not in the YY1 binding sites of other DMRs. Overall, our identification of three additional clustered YY1 binding sites in imprinted domains suggests a significant role for YY1 in mammalian genomic imprinting.
Using multiple mammalian genomic sequences, we have analyzed the evolution and imprinting of several genes located in the Peg3 domain, including Mim1 (approved name, Mimt1), Usp29, Zim3, and Zfp264. A series of comparative analyses shows that the overall genomic structure of this 500-kb imprinted domain has been well maintained throughout mammalian evolution but that several lineage-specific changes have also occurred in each species. In the bovine domain, Usp29 has lost its protein-coding capability, Zim3 has been duplicated, and the expression of Zfp264 has become biallelic in brain and testis, which differs from paternal expression of mouse Zfp264 in brain. In contrast, the two transcript genes of cow, Mim1 and Usp29, both lacking protein-coding capability, are still expressed mainly from the paternal allele, indicating the imprinting of these two genes in cow. The imprinting of Mim1 and Usp29 along with Peg3 is the most evolutionarily selected feature in this imprinted domain, suggesting significant function of these three genes, either as protein-coding or as untranslated transcript genes.
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