Differential differences in methylation status of putative imprinted genes among cloned swine genomes

DNA methylation is a major epigenetic modification in the mammalian genome that regulates crucial aspects of gene function. Mammalian cloning by somatic cell nuclear transfer (SCNT) often results in gestational or neonatal failure with only a small proportion of manipulated embryos producing live births. Many of the embryos that survive to term later succumb to a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Aberrant methylation patterns of imprinted genes in cloned cattle and mice have been elucidated, but few reports have analyzed the cloned pig genome. Four surviving cloned sows that were created by ear fibroblast nuclear transfer, each with a different life span and multiple organ defects, such as heart defects and bone growth delay, were used as epigenetic study materials. First, we identified four putative differential methylation regions (DMR) of imprinted genes in the wild-type pig genome, including two maternally imprinted loci (INS and IGF2) and two paternally imprinted loci (H19 and IGF2R). Aberrant DNA methylation, either hypermethylation or hypomethylation, commonly appeared in H19 (45% of imprinted loci hypermethylated vs. 30% hypomethylated), IGF2 (40% vs. 0%), INS (50% vs. 5%), and IGF2R (15% vs. 45%) in multiple tissues from these four cloned sows compared with wild-type pigs. Our data suggest that aberrant epigenetic modifications occur frequently in the genome of cloned swine. Even with successful production of cloned swine that avoid prenatal or postnatal death, the perturbation of methylation in imprinted genes still exists, which may be one of reason for their adult pathologies and short life. Understanding the aberrant pattern of gene imprinting would permit improvements in future cloning techniques.     

Characterization of the methylation status of five imprinted genes in sheep gametes

Genomic imprinting is a mammalian developmental process that uses epigenetic mechanisms to induce monoallelic and parental-specific expression of particular autosomal genes. A crucial epigenetic event consists of DNA methylation of CpG-islands, which become differentially methylated regions (DMRs) on the maternal and paternal alleles during oogenesis or spermatogenesis (germline DMRs). By contrast, somatic DMRs are acquired after fertilization. While there are several studies referring to methylation acquisition within germline DMRs in the mouse and human, a comparable methylation analysis of orthologous sequences is still lacking in sheep. To identify germline DMRs, this study analysed the methylation status of the available CpG-islands of five ovine imprinted genes ( H19, IGF2R, DLK1, DIO3 and BEGAIN ) in mature spermatozoa and in female gametes at different stages of their follicle growth, including in vitro matured oocytes. The 5'-end CpG-island of H19 showed a full methylation in spermatozoa and an absent methylation in growing and fully grown oocytes. The intron 2 CpG-island of IGF2R was unmethylated in male gametes, while it showed a high level of methylation in early stages of oogenesis. The promoter CpG-islands of DLK1 and DIO3 were found to be unmethylated both in spermatozoa and oocytes. Finally, the exon 9 CpG-island of BEGAIN was hypermethylated in mature male gametes, while it showed an almost complete methylation only in late stages of oocyte development. Our findings suggest that DNA methylation establishment during early stages of sheep oogenesis and subsequent in vitro maturation is gene-specific and that, of the five genes investigated, only the CpG-islands of H19 and IGF2R might represent ovine germline DMRs.     

Oppositely imprinted genes H19 and insulin-like growth factor 2 are coexpressed in human androgenetic trophoblast.

Human uniparental gestations such as gynogenetic ovarian teratomas and androgenetic complete hydatidiform moles provide a model to evaluate the integrity of parent-specific gene expression--i.e., imprinting--in the absence of a complementary parental genetic contribution. We studied expression, in these tissues, of the oppositely imprinted genes H19, which is an embryonic nontranslated RNA, and insulin-like growth factor type 2 (IGF2). Normal gestations only express H19 from the maternal allele and express IGF2 from the paternal allele, whereas neither is expressed from the maternal genome of gynogenetic gestations, and both are expressed from the paternal genome of androgenetic gestations. Coexpression of H19 and IGF2 in the androgenetic tissues was in a single population of cells, mononuclear trophoblast--the same cell type expressing these genes in biparental placentas. These results demonstrate that a biparental genome may be required for expression of the reciprocal IGF2/H19 imprint. Alternatively, biparental expression may be a normal feature of some imprinted genes in specific cell types. Additional experiments with other imprinted genes will clarify whether this reflects global failure of the imprinting process or a change specific to the IGF2/H19 locus.     

Epigenetic states and expression of imprinted genes in human embryonic stem cells

AIM: To investigate the epigenetic states and expression of imprinted genes in five human embryonic stem cell (hESC) lines derived in Taiwan.METHODS: The heterozygous alleles of single nucleotide polymorphisms (SNPs) at imprinted genes were analyzed by sequencing genomic DNAs of hESC lines and the monoallelic expression of the imprinted genes were confirmed by sequencing the cDNAs. The expression profiles of 32 known imprinted genes of five hESC lines were determined using Affymetrix human genome U133 plus 2.0 DNA microarray.RESULTS: The heterozygous alleles of SNPs at seven imprinted genes, IPW, PEG10, NESP55, KCNQ1, ATP10A, TCEB3C and IGF2, were identified and the monoallelic expression of these imprinted genes except IGF2 were confirmed. The IGF2 gene was found to be imprinted in hESC line T2 but partially imprinted in line T3 and not imprinted in line T4 embryoid bodies. Ten imprinted genes, namely GRB10, PEG10, SGCE, MEST, SDHD, SNRPN, SNURF, NDN, IPW and NESP55, were found to be highly expressed in the undifferentiated hESC lines and down-regulated in differentiated derivatives. The UBE3A gene abundantly expressed in undifferentiated hESC lines and further up-regulated in differentiated tissues. The expression levels of other 21 imprinted genes were relatively low in undifferentiated hESC lines and five of these genes (TP73, COPG2, OSBPL5, IGF2 and ATP10A) were found to be up-regulated in differentiated tissues.CONCLUSION: The epigenetic states and expression of imprinted genes in hESC lines should be thoroughly studied after extended culture and upon differentiation in order to understand epigenetic stability in hESC lines before their clinical applications.     

Epigenetic asymmetry in the mammalian zygote and early embryo: relationship to lineage commitment?

Epigenetic asymmetry between parental genomes and embryonic lineages exists at the earliest stages of mammalian development. The maternal genome in the zygote is highly methylated in both its DNA and its histones and most imprinted genes have maternal germline methylation imprints. The paternal genome is rapidly remodelled with protamine removal, addition of acetylated histones, and rapid demethylation of DNA before replication. A minority of imprinted genes have paternal germline methylation imprints. Methylation and chromatin reprogramming continues during cleavage divisions, but at the blastocyst stage lineage commitment to inner cell mass (ICM) or trophectoderm (TE) fate is accompanied by a dramatic increase in DNA and histone methylation, predominantly in the ICM. This may set up major epigenetic differences between embryonic and extraembryonic tissues, including in X-chromosome inactivation and perhaps imprinting. Maintaining epigenetic asymmetry appears important for development as asymmetry is lost in cloned embryos, most of which have developmental defects, and in particular an imbalance between extraembryonic and embryonic tissue development.     

Non-random, individual-specific methylation profiles are present at the sixth CTCF binding site in the human H19/IGF2 imprinting control region

Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR--target of the CTCF insulator--is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.     

Influence of in vitro manipulation on the stability of methylation patterns in the Snurf/Snrpn-imprinting region in mouse embryonic stem cells

Recent work on embryonic stem (ES) cells showed that stem cell-derived tissues and embryos, cloned from ES cell nuclei, often fail to maintain epigenetic states of imprinted genes. This deregulation is frequently associated with in vitro manipulations and culture conditions which might affect the cells potential to develop into normal fetuses. Usually, epigenetic instability is reported in differentially methylated regions of mostly growth-related imprinted genes. However, little is known about the epigenetic stability of genes that function late in organogenesis. Hence, we set out to investigate the epigenetic stability of neuronal genes and analyzed DNA methylation patterns in the Snurf/Snrpn imprinted cluster in several cultured mouse ES cell lines. We also determined the effects of in vitro stress factors such as consecutive passaging, trypsination, mechanical handling, single cell cloning, centrifugation, staurosporine-induced neurogenesis and the insertion of viral (foreign) DNA into the host genome. Intriguingly, none of these in vitro manipulations interfered with the stability of the methylation patterns in the analyzed neuronal genes. These data imply that, in contrast to growth-related genes like Igf2, H19, Igf2r or Grb10, the methylation imprints of the analyzed neuronal genes in the Snurf/Snrpn cluster may be particularly stable in manipulated ES cells.     

The Testis-Specific Factor CTCFL Cooperates with the Protein Methyltransferase PRMT7 in H19 Imprinting Control Region Methylation

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation—DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation.     

The testis-specific factor CTCFL cooperates with the protein methyltransferase PRMT7 in H19 imprinting control region methylation

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation—DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation.     

Active and repressive chromatin are interspersed without spreading in an imprinted gene cluster in the mammalian genome

The Igf2r imprinted cluster is an epigenetic silencing model in which expression of a ncRNA silences multiple genes in cis. Here, we map a 250 kb region in mouse embryonic fibroblast cells to show that histone modifications associated with expressed and silent genes are mutually exclusive and localized to discrete regions. Expressed genes were modified at promoter regions by H3K4me3 + H3K4me2 + H3K9Ac and on putative regulatory elements flanking active promoters by H3K4me2 + H3K9Ac. Silent genes showed two types of nonoverlapping profile. One type spread over large domains of tissue-specific silent genes and contained H3K27me3 alone. A second type formed localized foci on silent imprinted gene promoters and a nonexpressed pseudogene and contained H3K9me3 + H4K20me3 +/- HP1. Thus, mammalian chromosome arms contain active chromatin interspersed with repressive chromatin resembling the type of heterochromatin previously considered a feature of centromeres, telomeres, and the inactive X chromosome.     

Epigenetic marks in somatic chromatin are remodelled to resemble pluripotent nuclei by amphibian oocyte extracts

Reprogramming pluripotency after nuclear transplantation shows that molecules in oocytes can remodel somatic chromatin to a stem cell state. Here we report on an ex-ovo system using axolotl oocyte extracts to remodel epigenetic marks of somatic chromatin. Molecules present in axolotl oocyte extracts induce the reduction of the overall levels of H3K9me3, HP1α, and DNA methylation of somatic cells, and they increase the levels of H3K9ac. The levels of signal intensity detected in treated differentiated cells resemble those detected in embryonic stem cells, which are, in contrast, unaffected by these extracts. Analysis of specific genome sequences shows that somatic cells exposed to oocyte extracts undergo demethylation of LINE-1 repeats but Major Satellite repeats and the imprinted gene H19 remain unchanged. In addition, they induce demethylation of the Oct-4 promoter. Finally, the kinetics of activation of Oct-4 and Nanog expression from MEF nuclei treated in extracts suggests that these genes are subject to different levels of epigenetic control. The results demonstrate that axolotl oocyte extracts are a useful tool for studying epigenetic remodelling of somatic cells to a stem cell configuration, and for elucidating oocyte specific mechanisms of nuclear reprogramming.     

Altered gene expression and methylation of the human chromosome 11 imprinted region in small for gestational age (SGA) placentae

Imprinted genes are known to be crucial for placental development and fetal growth in mammals, but no primary epigenetic abnormality in placenta has been documented to compromise human fetal growth. Imprinted genes demonstrate parent-of-origin-specific allelic expression that is epigenetically regulated i.e. extrinsic to the primary DNA sequence. To undertake an epigenetic analysis of poor fetal growth in placentae and cord blood tissues, we first established the tissue-specific patterns of methylation and imprinted gene expression for two imprinting clusters (KvDMR and H19 DMR) on chromosome 11p15 in placentae and neonatal blood for 20 control cases and 24 Small for Gestational Age (SGA) cases. We confirmed that, in normal human placenta, the H19 promoter is unmethylated. In contrast, most other human tissues show paternal methylation. In addition, we showed that the IGF2 DMR2, also paternally methylated in most human tissues, exhibits hypomethylation in placentae. However, in neonatal blood DNA, these two regions maintain the differential methylation status seen in most other tissues. Significantly, we have been able to demonstrate that placenta does maintain differential methylation at the imprinting control regions H19 DMR and KvDMR. Of note, in one SGA placenta, we found a methylation alteration at the H19 DMR and concomitant biallelic expression of the H19 gene, suggesting that loss of imprinting at H19 is one cause of poor fetal growth in humans. Of particular interest, we demonstrated also a decrease in IGF2 mRNA levels in all SGA placentae and showed that the decrease is, in most cases, independent of H19 regulation.