PEG3 controls lipogenesis through ACLY.

Peg3 (Paternally expressed gene 3) is an imprinted gene encoding a DNA-binding protein that is a well-known transcriptional repressor. Previous studies have shown that the mutant phenotypes of Peg3 are associated with the over-expression of genes involved in lipid metabolism. In the current study, we investigated four potential downstream genes of Peg3, which were identified through ChIP-seq data: Acly, Fasn, Idh1, and Hmgcr. In vivo binding of PEG3 to the promoter region of these key genes involved in lipogenesis was subsequently confirmed through individual ChIP experiments. We observed the opposite response of Acly expression levels against the variable gene dosages of Peg3, involving 0x, 1x, and 2x Peg3. This suggests the transcriptional repressor role of Peg3 in the expression levels of Acly. Another set of analyses showed a sex-biased response in the expression levels of Acly, Fasn, and Idh1 against 0x Peg3 with higher levels in female and lower levels in male mammary glands. These results overall highlight that Peg3 may be involved in regulating the expression levels of several key genes in adipogenesis.

Allele-specific enhancer interaction at the Peg3 imprinted domain.

The parental allele specificity of mammalian imprinted genes has been evolutionarily well conserved, although its functional constraints and associated mechanisms are not fully understood. In the current study, we generated a mouse mutant with switched active alleles driving the switch from paternal-to-maternal expression for Peg3 and the maternal-to-paternal expression for Zim1. The expression levels of Peg3 and Zim1, but not the spatial expression patterns, within the brain showed clear differences between wild type and mutant animals. We identified putative enhancers localized upstream of Peg3 that displayed allele-biased DNA methylation, and that also participate in allele-biased chromosomal conformations with regional promoters. Most importantly, these data suggest for the first time that long-distance enhancers may contribute to allelic expression within imprinted domains through allele-biased interactions with regional promoters.

High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS).

High-throughput targeted repeat element bisulfite sequencing (HT-TREBS) is designed to assay the methylation level of individual retrotransposon loci of a targeted family, in a locus-specific manner, and on a genome-wide scale. Briefly, genomic DNA is sheared and ligated to Ion Torrent A adaptors (with methylated cytosines), followed by bisulfite-conversion, and amplification with primers designed to bind the targeted retrotransposon. Since the primers carry the Ion Torrent P1 adaptor as a 5'-extension, the amplified library is ready to be size-selected and sequenced on a next-generation sequencing platform. Once sequenced, each retrotransposon is mapped to a particular genomic locus, which is achieved through ensuring at least a 10-bp overlap with flanking unique sequence, followed by the calculation of methylation levels of the mapped retrotransposon using a BiQ Analyzer HT. A complete protocol for library construction as well as the bioinformatics for HT-TREBS is described in this chapter.

Evolution patterns of Peg3 and H19-ICR.

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.

Enhancer-driven alternative promoters of imprinted genes.

In the current study, we characterized the expression and histone modification profiles of the alternative promoters found within imprinted Igf2r, Mest, Zac1, Peg3, Snrpn and non-imprinted Myc loci. In terms of expression pattern, the alternative promoters are highly tissue-specific, which is in a stark contrast to the ubiquitous expression of the corresponding main promoters. The alternative promoters are associated with the histone modification mark H3K4me1, but not with H3K4me3, which is frequently associated with the main promoters. Phylogenetic analyses also indicated that the majority of the alternative promoters are unique to the mammalian lineage, further suggesting the recent formation of these promoters during mammalian evolution. Overall, this study suggests that the alternative promoters of imprinted loci may have been derived from enhancers in recent evolutionary times and co-evolved with the genomic imprinting mechanism.

Trans-allelic mutational effects at the Peg3 imprinted locus.

How one allele interacts with the other for the function of a gene is not well understood. In this study, we tested potential allelic interaction at the Peg3 imprinted locus with several mutant alleles targeting an Imprinting Control Region, the Peg3-DMR. According to the results, maternal deletion of the Peg3-DMR resulted in 2-fold up-regulation of two paternally expressed genes, Peg3 and Usp29. These trans-allelic mutational effects were observed consistently throughout various tissues with different developmental stages. These effects were also associated mainly with the genetic manipulation of the Peg3-DMR, but not with the other genomic changes within the Peg3 locus. The observed trans-allelic effects were unidirectional with the maternal influencing the paternal allele, but not with the opposite direction. Overall, the observed mutational effects suggest the presence of previously unrecognized trans-allelic regulation associated with the Peg3-DMR.

Circular RNA identified from Peg3 and Igf2r.

Circular RNA is a newly discovered class of non-coding RNA generated through the back-splicing of linear pre-mRNA. In the current study, we characterized two circular RNAs that had been identified through NGS-based 5'RACE experiments. According to the results, the Peg3 locus contains a 214-nucleotide-long circular RNA, circPeg3, that is detected in low abundance from the neonatal brain, lung and ovary. In contrast, the Igf2r locus contains a group of highly abundant circular RNAs, circIgf2r, showing multiple forms with various exon combinations. In both cases, the expression patterns of circPeg3 and circIgf2r among individual tissues are quite different from their linear mRNA counterparts. This suggests potential unique roles played by the identified circular RNAs. Overall, this study reports the identification of novel circular RNAs specific to mammalian imprinted loci, suggesting that circular RNAs are likely involved in the function and regulation of imprinted genes.

Ablation of Ezh2 in neural crest cells leads to aberrant enteric nervous system development in mice.

In the current study, we examined the role of Ezh2 as an epigenetic modifier for the enteric neural crest cell development through H3K27me3. Ezh2 conditional null mice were viable up to birth, but died within the first hour of life. In addition to craniofacial defects, Ezh2 conditional null mice displayed reduced number of ganglion cells in the enteric nervous system. RT-PCR and ChIP assays indicated aberrant up-regulation of Zic1, Pax3, and Sox10 and loss of H3K27me3 marks in the promoter regions of these genes in the myenteric plexus. Overall, these results suggest that Ezh2 is an important epigenetic modifier for the enteric neural crest cell development through repression of Zic1, Pax3, and Sox10.

Oxytocin receptor is regulated by Peg3.

Mouse Peg3 encodes a DNA-binding protein involved in the milk letdown process. In the current study, we tested whether PEG3 controls the expression of the oxytocin receptor gene. According to the results, PEG3 directly binds to a genomic region within the 3rd exon of Oxtr, which contains a DNA-binding motif for PEG3. In nursing female mice, removal of PEG3 resulted in the increased expression of Oxtr in mammary epithelial cells and also in the hypothalamus. This suggests a repressor role of PEG3 in the expression of Oxtr in these tissues. Overall, this study suggests that Peg3 may function as a direct transcriptional regulator for Oxtr expression that acts to moderate the milk letdown process.

Allele and dosage specificity of the Peg3 imprinted domain.

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.

YinYang1 deficiency ameliorates joint inflammation in a murine model of rheumatoid arthritis by modulating Th17 cell activation.

Yin Yang 1 (YY1) is a ubiquitously expressed transcription factor that functions in cooperation with various cofactors to regulate gene expression. In the immune system, YY1 enhances cytokine production and T helper (Th) 2 effector cell differentiation, resulting in the activation of inflammation. However, no studies have reported the role of YY1 in Th17 cell regulation, which is implicated in rheumatoid arthritis (RA). We investigated the expression of YY1 in Th17 cells in vitro and revealed increased levels of YY1 mRNA and protein. To elucidate the function of YY1 pathogenesis in RA, we used a collagen-induced arthritis (CIA) mouse model with YY1 deficiency. Deficiency of YY1 reduced the severity of arthritis and joint destruction. Moreover, Th17 cells were dramatically reduced in YY1-deficient mice. The cytokine interleukin (IL)-17 was decreased in YY1-deficient CD4+ T cells ex vivo and in vivo. Interestingly, the level of signal transducer and activator of transcription 3 (STAT3), tumor necrosis factor-α, IL-17, IL-6, and IL-1ß were markedly decreased in YY1-deficient mice with CIA. The cytokine-inducing function of YY1 was more specific to IL-17 than to interferon-γ. YY1 plays a role in Th17 cell differentiation and RA pathogenesis. Our findings suggest that future RA therapies should target the regulatory mechanism involved in Th17 cell differentiation, in which YY1 may cooperate with the STAT3 signaling pathway.

Intergenic and intronic DNA hypomethylated regions as putative regulators of imprinted domains.

AIM: To investigate the regulatory potential of intergenic/intronic hypomethylated regions (iHMRs) within imprinted domains. MATERIALS & METHODS: Based on the preliminary results of the histone modification and conservation profiles, we conducted reporter assays on the Peg3 and H19 domain iHMRs. The in vitro results were confirmed by the in vivo deletion of Peg3-iHMR designed to test its function in the Peg3 imprinted domain. RESULTS & CONCLUSION: Initial bioinformatic analyses suggested that some iHMRs may be noncanonical enhancers for imprinted genes. Consistent with this, Peg3- and H19-iHMRs showed context-dependent promoter and enhancer activity. Further, deletion of Peg3-iHMR resulted in allele- and sex-specific misregulation of several imprinted genes within the domain. Taken together, these results suggest that some iHMRs may function as domain-wide regulators for the associated imprinted domains.

Transcription-driven DNA methylation setting on the mouse Peg3 locus.

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.

Epigenetic response of imprinted domains during carcinogenesis.

BACKGROUND: Imprinted domains have been identified as targets for aberrant DNA methylation during carcinogenesis, but it remains unclear when these epigenetic alterations occur and how they contribute to tumor progression. Epigenetic instability at key cis-regulatory elements within imprinted domains can concomitantly activate proto-oncogenes and turn off tumor suppressor genes. Thus, to further characterize the epigenetic response of imprinted domains during carcinogenesis, we compared the stability of DNA methylation at a variety of cis-regulatory elements within imprinted domains in two fundamentally different mouse tumors, benign and malignant, induced by the KrasG12D mutation. RESULTS: We report that imprinted domains remain stable in benign processes but are highly susceptible to epigenetic alterations in infiltrative lesions. The preservation of DNA methylation within imprinted domains in benign tumors throughout their duration suggests that imprinted genes are not involved with the initiation of carcinogenesis or the growth of tumors. However, the frequent detection of DNA methylation changes at imprinting control regions in infiltrative lesions suggest that imprinted genes are associated with tumor cells gaining the ability to defy tissue boundaries. CONCLUSION: Overall, this study demonstrates that imprinted domains are targeted for DNA hypermethylation when benign tumor cells transition to malignant. Thus, monitoring DNA methylation within imprinted domains may be useful in evaluating the progression of neoplasms.

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.

Inversion of the imprinting control region of the Peg3 domain.

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.

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.

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 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 binds to H19-ICR as a transcriptional repressor.

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.