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Peg3 Mutational Effects on Reproduction and Placenta-Specific Gene Families

Peg3 (paternally expressed gene 3) is an imprinted gene encoding a DNA-binding protein. This gene plays important roles in controlling fetal growth rates and nurturing behaviors. In the current study, a new mutant mouse model has been generated to further characterize the functions of this DNA-binding protein. Besides known phenotypes, this new mutant model also revealed potential roles of Peg3 in mammalian reproduction. Female heterozygotes produce a much smaller number of mature oocytes than the wild-type littermates, resulting in reduced litter sizes. According to genome-wide expression analyses, several placenta-specific gene families are de-repressed in the brain of Peg3 heterozygous embryos, including prolactin, cathepsin and carcinoembryonic antigen cell adhesion molecule (Ceacam) families. The observed de-repression is more pronounced in females than in males. The de-repression of several members of these gene families is observed even in the adult brain, suggesting potential defects in epigenetic setting of the placenta-specific gene families in the Peg3 mutants. Overall, these results indicate that Peg3 likely controls the transcription of several placenta-specific gene families, and further suggest that this predicted transcriptional control by Peg3 might be mediated through unknown epigenetic mechanisms.

DNA-Binding Motif of the Imprinted Transcription Factor PEG3.

Peg3 is an imprinted gene that is predicted to encode a DNA-binding zinc finger protein. This was previously demonstrated through Chromatin ImmunoPrecipitation-based Sequencing experiments. In the current study, we reanalyzed the previous ChIP-Seq results and further characterized the DNA-binding motif of PEG3. According to the results, PEG3 binds to the promoters and enhancers of a subset of genes that are closely associated with the known functions of Peg3. Some of these identified targets include Tufm, Mrpl45, Cry2, Per1, Slc25a29 and Slc38a2. With this set of targets, we derived a DNA-binding motif of PEG3, 5’-GTGGCAGT-3’, which also provides a tabulated matrix that can be used for predicting other unknown genomic targets. Among the newly identified targets, we analyzed in detail the two loci, Slc38a2 and Slc38a4, which are known to be involved in neutral amino acid transport. The results indicated that PEG3 likely functions as a transcriptional repressor for these two loci. Overall, the current study provides a set of genomic targets and also redefines the DNA-binding motif for the imprinted transcription factor PEG3.

Paternally expressed Peg3 controls maternally expressed Zim1 as a trans factor.

The expression of two adjacent imprinted genes, Peg3 and Zim1, is inversely correlated: down-regulation of Peg3 coinciding with up-regulation of Zim1. The current study characterized this inverse correlation using a mutant allele targeting Peg3. According to the results, the mutation on the paternal allele of Peg3 resulted in a dramatic increase in the transcription levels of the maternal allele of Zim1, suggesting the involvement of unknown trans factors in this trans-allelic event. Subsequent ChIP experiments revealed that the protein encoded by Peg3 itself binds to the zinc finger exon of Zim1, which is modified with the repression mark H3K9me3. Interestingly, the levels of H3K9me3 on Zim1 are also reduced in the mutant cells lacking the protein PEG3, suggesting potential roles for PEG3 in establishing H3K9me3 on Zim1. Reintroducing PEG3 into the mutant cell restored down-regulation of Zim1, confirming the predicted repressor role for Peg3 on Zim1. Overall, these results demonstrated that paternally expressed Peg3 controls maternally expressed Zim1 as a trans factor. The current study also provides the first case for the trans-allelic interaction of two oppositely imprinted genes through their gene products.

Parental and sexual conflicts over the Peg3 imprinted domain

In the current study, the imprinting control region of the mouse Peg3 domain was deleted to test its functional impact on animal growth and survival. The paternal transmission of the deletion resulted in complete abolition of the transcription of two paternally expressed genes, Peg3 and Usp29, causing the reduced body weight of the pups. In contrast, the maternal transmission resulted in the unexpected transcriptional up-regulation of the remaining paternal allele of both Peg3 and Usp29, causing the increased body weight and survival rates. Thus, the imprinted maternal allele of the ICR may be a suppressor antagonistic to the active paternal allele of the ICR, suggesting a potential intralocus allelic conflict. The opposite outcomes between the two transmissions also justify the functional compromise that the maternal allele has become epigenetically repressed rather than genetically deleted during mammalian evolution. The mice homozygous for the deletion develop normally but with a skewed sex ratio, one male per litter, revealing its sex-biased effect. Overall, the Peg3 locus may have evolved to an imprinted domain to cope with both parental and sexual conflicts driven by its growth-stimulating paternal versus growth-suppressing maternal alleles.

PEG3 binds to H19-ICR as a transcriptional repressor

ABSTRACT Paternally expressed gene 3 (Peg3) encodes a DNA-binding protein with 12 C2H2 zinc finger motifs. In the current study, we performed ChIP-seq using mouse embryonic fibroblast (MEF) cells. This experiment identified a set of 16 PEG3 genomic targets, the majority of which overlapped with the promoter regions of genes with oocyte expression. These potential downstream genes were upregulated in MEF cells lacking PEG3 protein, suggesting a potential repressor role for PEG3. Our study also identified the imprinting control region (ICR) of H19 as a genomic target. According to the results, PEG3 binds to a specific sequence motif located between the 3rd and 4th CTCF binding sites of the H19-ICR. PEG3 also binds to the active maternal allele of the H19-ICR. The expression levels of H19 were upregulated in MEF cells lacking PEG3, and this upregulation was mainly derived from the maternal allele. This suggests that PEG3 may function as a transcriptional repressor for the maternal allele of H19. Overall, the current study uncovers a potential functional relationship between Peg3 and H19, and also confirms PEG3 as a transcriptional repressor for the identified downstream genes.

Phylogenetic and Epigenetic Footprinting of the Putative Enhancers of the Peg3 Domain

The Peg3 (Paternally Expressed Gene 3) imprinted domain is predicted to be regulated through a large number of evolutionarily conserved regions (ECRs) that are localized within its middle 200-kb region. In the current study, we characterized these potential cis-regulatory regions using phylogenetic and epigenetic approaches. According to the results, the majority of these ECRs are potential enhancers for the transcription of the Peg3 domain. Also, these potential enhancers can be divided into two groups based on their histone modification and DNA methylation patterns: ubiquitous and tissue-specific enhancers. Phylogenetic and bioinformatic analyses further revealed that several cis-regulatory motifs are frequently associated with the ECRs, such as the E box, PITX2, NF-κB and RFX1 motifs. A series of subsequent ChIP experiments demonstrated that the trans factor MYOD indeed binds to the E box of several ECRs, further suggesting that MYOD may play significant roles in the transcriptional control of the Peg3 domain. Overall, the current study identifies, for the first time, a set of cis-regulatory motifs and corresponding trans factors that may be critical for the transcriptional regulation of the Peg3 domain.

YY1’s role in the Peg3 imprinted domain

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.

Transcriptional Truncation of the Long Coding Imprinted Gene Usp29

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.

PEG3 Interacts with KAP1 through KRAB-A.

Peg3 (Paternally Expressed Gene 3) is an imprinted gene that encodes a zinc finger DNA-binding protein. Peg3 itself is localized in the middle of a KRAB-A (Kruppel-Associated Box) zinc finger gene cluster. The amino acid sequence encoded by its exon 7 also shows sequence similarity to that of KRAB-A, suggesting Peg3 as a KRAB-containing zinc finger gene. As predicted, the PEG3 protein was co-immunoprecipitated with KAP1, a co-repressor that interacts with KRAB-A. A series of follow-up experiments further demonstrated that the exon 7 of PEG3 is indeed responsible for its physical interaction with KAP1. ChIP and promoter assays also indicated that PEG3 likely controls its downstream genes through the KAP1-mediated repression mechanism. Overall, the current study identifies PEG3 as a KRAB-containing zinc finger protein that interacts with the co-repressor protein KAP1.

PEG3 control on the mammalian MSL complex

Peg3 (paternally expressed gene 3) encodes a DNA-binding protein that functions as a transcriptional repressor. Recent studies revealed that PEG3 binds to Msl1 (male-specific lethal 1) and Msl3, the two main components of the MSL complex. In the current study, we investigated potential roles of Peg3 in controlling its downstream genes through H4K16ac, the histone modification by the MSL complex. According to the results, complete removal of PEG3 resulted in up-regulation of Msl1 and Msl3, and subsequently an increase in the global levels of H4K16ac, confirming PEG3 as a transcriptional repressor for MSL during mammalian development. Genome-wide analyses further revealed that about 10% of the entire gene catalogue was affected in the MEF cells lacking PEG3, displaying the increased levels of H4K16ac in their promoter regions. The expression levels of a small subset of the affected genes were up-regulated in the MEF cells lacking PEG3. Interestingly, three Hox clusters also exhibited changes in the levels of H4K16ac, suggesting potential roles of PEG3 and MSL in the regulation of Hox clusters. Overall, the current study reports that Peg3 may control its downstream genes through mammalian MSL.