Two evolutionarily conserved sequence elements for Peg3/Usp29 transcription.
ABSTRACT: Two evolutionarily Conserved Sequence Elements, CSE1 and CSE2 (YY1 binding sites), are found within the 3.8-kb CpG island surrounding the bidirectional promoter of two imprinted genes, Peg3 (Paternally expressed gene 3) and Usp29 (Ubiquitin-specific protease 29). This CpG island is a likely ICR (Imprinting Control Region) that controls transcription of the 500-kb genomic region of the Peg3 imprinted domain.The current study investigated the functional roles of CSE1 and CSE2 in the transcriptional control of the two genes, Peg3 and Usp29, using cell line-based promoter assays. The mutation of 6 YY1 binding sites (CSE2) reduced the transcriptional activity of the bidirectional promoter in the Peg3 direction in an orientation-dependent manner, suggesting an activator role for CSE2 (YY1 binding sites). However, the activity in the Usp29 direction was not detectable regardless of the presence/absence of YY1 binding sites. In contrast, mutation of CSE1 increased the transcriptional activity of the promoter in both the Peg3 and Usp29 directions, suggesting a potential repressor role for CSE1. The observed repression by CSE1 was also orientation-dependent. Serial mutational analyses further narrowed down two separate 6-bp-long regions within the 42-bp-long CSE1 which are individually responsible for the repression of Peg3 and Usp29.CSE2 (YY1 binding sites) functions as an activator for Peg3 transcription, while CSE1 acts as a repressor for the transcription of both Peg3 and Usp29.
Project description:The imprinting of the mouse Peg3 domain is controlled through a 4-kb genomic region encompassing the bidirectional promoter and 1st exons of Peg3 and Usp29. In the current study, this ICR was inverted to test its orientation dependency for the transcriptional and imprinting control of the Peg3 domain. The inversion resulted in the exchange of promoters and 1st exons between Peg3 and Usp29. Paternal transmission of this inversion caused 10-fold down-regulation of Peg3 and 2-fold up-regulation of Usp29 in neonatal heads, consistent with its original promoter strength in each direction. The paternal transmission also resulted in reduced body size among the animals, which was likely contributed by the dramatic down-regulation of Peg3. Transmission through either allele caused no changes in the DNA methylation and imprinting status of the Peg3 domain except that Zfp264 became bi-allelic through the maternal transmission. Overall, the current study suggests that the orientation of the Peg3-ICR may play no role in its allele-specific DNA methylation, but very critical for the transcriptional regulation of the entire imprinted domain.
Project description:The ICR (Imprinting Control Region) of the Peg3 (Paternally Expressed Gene 3) domain contains an unusual cluster of YY1 binding sites. In the current study, these YY1 binding sites were mutated to characterize the unknown roles in the mouse Peg3 domain. According to the results, paternal and maternal transmission of the mutant allele did not cause any major effect on the survival of the pups. In the mutants, the maternal-specific DNA methylation on the ICR was properly established and maintained, causing no major effect on the imprinting of the domain. In contrast, the paternal transmission resulted in changes in the expression levels of several genes: down-regulation of Peg3 and Usp29 and up-regulation of Zim1. These changes were more pronounced during the neonatal stage than during the adult stage. In the case of Peg3 and Zim1, the levels of the observed changes were also different between males and females, suggesting the different degrees of YY1 involvement between two sexes. Overall, the results indicated that YY1 is mainly involved in controlling the transcriptional levels, but not the DNA methylation, of the Peg3 domain.
Project description: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.
Project description:Mammalian imprinted domains are regulated through small genomic regions termed Imprinting Control Regions (ICRs). In the current study, the evolution patterns of the ICRs of Peg3 and H19-imprinted domains were analyzed using the genomic sequences derived from a large number of mammals. The results indicated that multiple YY1 and CTCF binding sites are localized within the Peg3 and H19-ICR in all the mammals tested. The numbers of YY1 and CTCF binding sites are variable among individual species, yet positively correlate with the presence of tandem repeats within the Peg3 and H19-ICRs. Thus, multiple YY1 and CTCF binding sites within the respective ICRs may have been maintained through tandem repeats/duplications. The unit lengths of tandem repeats are also non-random and locus-specific, 140 and 400?bp for the Peg3 and H19-ICRs. Overall, both Peg3 and H19-ICRs may have co-evolved with two unique features, multiple transcription factor binding sites and tandem repeats.
Project description:Imprinting control regions (ICRs) often harbor tandem arrays of transcription factor binding sites, as demonstrated by the identification of multiple YY1 binding sites within the ICRs of Peg3, Nespas, and Xist/Tsix domains. In the current study, we have sought to characterize possible roles for YY1 in transcriptional control and epigenetic modification of these imprinted domains. RNA interference-based knockdown experiments in Neuro2A cells resulted in overall transcriptional up-regulation of most of the imprinted genes within the Peg3 domain and also, concomitantly, caused significant loss in the DNA methylation of the Peg3 differentially methylated region. A similar overall and coordinated expression change was also observed for the imprinted genes of the Gnas domain: up-regulation of Nespas and down-regulation of Nesp and Gnasxl. YY1 knockdown also resulted in changes in the expression levels of Xist and Snrpn. These results support the idea that YY1 plays a major role, as a trans factor, in the control of these imprinted domains.
Project description:In this study, we performed the first systematic survey of DNA methylation status of the CpG islands of the PEG3 (Paternally expressed gene 3) imprinted domain in the mouse, cow, and human genomes. Previous studies have shown that the region surrounding the first exon of PEG3 contains a differentially methylated CpG island. In addition, we have discovered two previously unreported differentially methylated regions (DMR): one in the promoter region of mouse Zim3 and another in the promoter region of human USP29. In the cow, the Peg3-CpG island was the only area that showed DMR status. We have also examined the methylation status of several CpG islands in this region using human tumor-derived DNA. The CpG islands near PEG3 and USP29 both showed hypermethylation in DNA derived from breast and ovarian tumors. Overall, this study shows that the PEG3 imprinted domain of humans, cows, and mice contains differing numbers of DMRs, but the PEG3-CpG island is the only DMR that is conserved among these three species.
Project description: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. Gene Usp29 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 mouse Usp29 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).
Project description:The imprinting of the mouse Peg3 domain is controlled through the Peg3-DMR, which obtains its maternal-specific DNA methylation during oogenesis. In the current study, we deleted an oocyte-specific alternative promoter, termed U1, which is localized 20 kb upstream of the Peg3-DMR. Deletion of this alternative promoter resulted in complete removal of the maternal-specific DNA methylation on the Peg3-DMR. Consequently, the imprinted genes in the Peg3 domain become biallelic in the mutants with maternal transmission of the deletion. Expression levels of the imprinted genes were also affected in the mutants: 2-fold upregulation of Peg3 and Usp29 and downregulation of Zim1 to basal levels. Breeding experiments further indicated under-representation of females among the surviving mutants, a potential sex-biased outcome from the biallelic expression of the Peg3 domain. Overall, the results suggest that U1-driven transcription may be required for establishing oocyte-specific DNA methylation on the Peg3 domain.
Project description:The biological impetus for gene dosage and allele specificity of mammalian imprinted genes is not fully understood. To address this, we generated and analyzed four sets of mice from a single breeding scheme with varying allelic expression and gene dosage of the Peg3 domain. The mutants with abrogation of the two paternally expressed genes, Peg3 and Usp29, showed a significant decrease in growth rates for both males and females, while the mutants with biallelic expression of Peg3 and Usp29 resulted in an increased growth rate of female mice only. The mutant cohort with biallelic expression of Peg3 and Usp29 tended to have greater numbers of pups compared to the other genotypes. The mutants with switched active alleles displayed overall similar phenotypes to the wild type, but did show some differences in gene expression, suggesting potential non-redundant roles contributed by the maternal and paternal alleles. Overall, this study demonstrates a novel in vivo approach to investigate the allele and dosage specificity of mammalian imprinted domains.
Project description:Usp29 (Ubiquitin-specific protease 29) is a paternally expressed gene located upstream of another imprinted gene Peg3. In the current study, the transcription of this long coding gene spanning a 250-kb genomic distance was truncated using a knockin allele. According to the results, paternal transmission of the mutant allele resulted in reduced body and litter sizes whereas the maternal transmission caused no obvious effects. In the paternal mutant, the expression levels of Usp29 were reduced to 14-18% level of the wild-type littermates due to the Poly-A signal included in the knockin cassette. Expression analyses further revealed an unusual female-specific up-regulation of the adjacent imprinted gene Zfp264 in the mutant. Consistent with this, the promoter of Zfp264 was hypomethylated only in the female mutant. Interestingly, this female-specific hypomethylation by the knockin allele was not detected in the offspring of an interspecific crossing, indicating its sensitivity to genetic background. Overall, the results suggest that the transcription of Usp29 may be involved in DNA methylation setting of Zfp264 promoter in a sex-specific manner.