Disruption of imprinted gene expression and DNA methylation status in porcine parthenogenetic fetuses and placentas

Parthenogenetically activated oocytes cannot develop to term in mammals due to the lack of paternal gene expression and failed X chromosome inactivation (XCI). To further characterize porcine parthenogenesis, the expression of 18 imprinted genes was compared between parthenogenetic (PA) and normally fertilized embryos (Con) using quantitative real-time PCR (qRT-PCR). The results revealed that maternally expressed genes were over-expressed, whereas paternally expressed genes were significantly reduced in PA fetuses and placentas. The results of bisulfite sequencing PCR (BSP) demonstrated that PRE-1 and Satellite were hypermethylated in both Con and PA fetuses and placentas, while XIST DMRs were hypomethylated only in PA samples. Taken together, these results suggest that the aberrant methylation profile of XIST DMRs and abnormal imprinted gene expression may be responsible for developmental failure and impaired growth in porcine parthenogenesis.     

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.     

A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip

The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.     

Methylation dynamics of IG-DMR and Gtl2-DMR during murine embryonic and placental development

The Dlk1-Dio3 imprinted domain on mouse chromosome 12 contains IG-DMR and Gtl2-DMR, whose methylation patterns are established in the germline and after fertilization, respectively. In this study, we determine that acquisition of DNA methylation at the paternal allele of the Gtl2-DMR is initiated after the blastocyst stage and completed by embryonic day 6.5, and that Gtl2 (approved symbol: Meg3) is monoallelically expressed from the maternal allele as early as the blastocyst. Therefore, DNA methylation at the Gtl2-DMR is not a prerequisite for the imprinted expression of Gtl2, which may be involved in the control of proliferation and differentiation of cells during early gestation. We also reveal that a subregion of the IG-DMR exhibits tissue-specific differences in allelic methylation patterns. These results add to the growing body of knowledge elucidating the mechanism whereby parent-of-origin-dependent DNA methylation at the IG-DMR leads to the imprinted expression of the Dlk1-Dio3 cluster.     

CTR9/PAF1c regulates molecular lineage identity,histone H3K36 trimethylation and genomic imprinting during preimplantation development

Genome-wide epigenetic reprogramming is required for successful preimplantation development. Inappropriate or deficient chromatin regulation can result in defective lineage specification and loss of genomic imprinting, compromising normal development. Here we report that two members of the RNA polymerase II associated factor, homolog (Saccharomyces cerevisiae) complex (PAF1 complex) components, Ctr9 and Rtf1, are required during mammalian preimplantation development. We demonstrate that Ctr9-deficient embryos fail to correctly specify lineages at the blastocyst stage. Expression of some lineage specific factors is markedly reduced in Ctr9 knockdown embryos, including Eomes, Elf5 and Sox2, while others are inappropriately expressed (Oct4, Nanog, Gata6, Fgf4 and Sox17). We also show that several imprinted genes (Mest, Peg3, Snrpn and Meg3) are aberrantly expressed although allele specific DNA methylation is not altered. We document a loss of histone H3 lysine 36 trimethylation (H3K36me3) in Ctr9-deficient embryos and confirm that knockdown of either Setd2 or Rtf1 results in similar phenotypes. These findings show that the PAF1 complex is required for mammalian development, likely through regulation of H3K36me3, and indicate functional conservation of the PAF1 complex from yeast to mammals in vivo.     

Identification of the mouse paternally expressed imprinted gene Zdbf2 on chromosome 1 and its imprinted human homolog ZDBF2 on chromosome 2

In mammals, both the maternal and paternal genomes are necessary for normal embryogenesis due to parent-specific epigenetic modification of the genome during gametogenesis, which leads to non-equivalent expression of imprinted genes from the maternal and paternal alleles. In this study, we identified a paternally expressed imprinted gene, Zdbf2, by microarray-based screening using parthenogenetic and normal embryos. Expression analyses showed that Zdbf2 was paternally expressed in various embryonic and adult tissues, except for the placenta and adult testis, which showed biallelic expression of the gene. We also identified a differentially methylated region (DMR) at 10 kb upstream of exon 1 of the Zdbf2 gene and this differential methylation was derived from the germline. Furthermore, we also identified that the human homolog (ZDBF2) of the mouse Zdbf2 gene showed paternal allele-specific expression in human lymphocytes but not in the human placenta. Thus, our findings defined mouse chromosome 1 and human chromosome 2 as the loci for imprinted genes.     

Differential methylation persists at the mouse Rasgrf1 DMR in tissues displaying monoallelic and biallelic expression

A subset of mammalian genes exhibits genomic imprinting, whereby one parental allele is preferentially expressed. Differential DNA methylation at imprinted loci serves both to mark the parental origin of the alleles and to regulate their expression. In mouse, the imprinted gene Rasgrf1 is associated with a paternally methylated imprinting control region which functions as an enhancer blocker in its unmethylated state. Because Rasgrf1 is imprinted in a tissue-specific manner, we investigated the methylation pattern in monoallelic and biallelic tissues to determine if methylation of this region is required for both imprinted and non-imprinted expression. Our analysis indicates that DNA methylation is restricted to the paternal allele in both monoallelic and biallelic tissues of somatic and extraembryonic lineages. Therefore, methylation serves to mark the paternal Rasgrf1 allele throughout development, but additional factors are required for appropriate tissue-specific regulation of expression at this locus.     

G9a histone methyltransferase contributes to imprinting in the mouse placenta

Whereas DNA methylation is essential for genomic imprinting, the importance of histone methylation in the allelic expression of imprinted genes is unclear. Imprinting control regions (ICRs), however, are marked by histone H3-K9 methylation on their DNA-methylated allele. In the placenta, the paternal silencing along the Kcnq1 domain on distal chromosome 7 also correlates with the presence of H3-K9 methylation, but imprinted repression at these genes is maintained independently of DNA methylation. To explore which histone methyltransferase (HMT) could mediate the allelic H3-K9 methylation on distal chromosome 7, and at ICRs, we generated mouse conceptuses deficient for the SET domain protein G9a. We found that in the embryo and placenta, the differential DNA methylation at ICRs and imprinted genes is maintained in the absence of G9a. Accordingly, in embryos, imprinted gene expression was unchanged at the domains analyzed, in spite of a global loss of H3-K9 dimethylation (H3K9me2). In contrast, the placenta-specific imprinting of genes on distal chromosome 7 is impaired in the absence of G9a, and this correlates with reduced levels of H3K9me2 and H3K9me3. These findings provide the first evidence for the involvement of an HMT and suggest that histone methylation contributes to imprinted gene repression in the trophoblast.     

Ubiquitous expression and imprinting of Snrpn in the mouse

Snrpn is known to be abundantly expressed in rodent brain and heart, and in two separate studies with neonatal mouse brain it has been shown to be maternally imprinted, that is, the maternal allele is normally repressed. We now provide evidence on the expression profile and imprinting status of Snrpn throughout development. Using RT-PCR, we have established that Snrpn is further expressed at low levels in lung, liver, spleen, kidney, skeletal muscle, and gonads. Moreover, using mice with only maternal copies of Snrpn (maternal duplication for the chromosome region involved and parthenogenotes), we have shown that the gene is imprinted in all of these tissues and, generally, from the time the gene is first expressed at 7.5 days gestation. In contrast to the findings made with the imprinted genes, Igf2, Ins1, and Ins2, there is no evidence of tissue-specific imprinting in the embryo with Snrpn. Nor, as found with Igf2 and Igf2r, is there evidence of a window of biallelic expression between the germ line imprint and the time of gene repression. The absence of Snrpn expression in early embryos contrasts with the findings in ES cells.     

Genome-wide and single-base resolution DNA methylomes of the Pacific oyster Crassostrea gigas provide insight into the evolution of invertebrate CpG methylation

Background

Studies of DNA methylomes in a wide range of eukaryotes have revealed both conserved and divergent characteristics of DNA methylation among phylogenetic groups. However, data on invertebrates particularly molluscs are limited, which hinders our understanding of the evolution of DNA methylation in metazoa. The sequencing of the Pacific oyster Crassostrea gigas genome provides an opportunity for genome-wide profiling of DNA methylation in this model mollusc.

Results

Homologous searches against the C. gigas genome identified functional orthologs for key genes involved in DNA methylation: DNMT1, DNMT2, DNMT3, MBD2/3 and UHRF1. Whole-genome bisulfite sequencing (BS-seq) of the oyster’s mantle tissues revealed that more than 99% methylation modification was restricted to cytosines in CpG context and methylated CpGs accumulated in the bodies of genes that were moderately expressed. Young repeat elements were another major targets of CpG methylation in oysters. Comparison with other invertebrate methylomes suggested that the 5’-end bias of gene body methylation and the negative correlation between gene body methylation and gene length were the derived features probably limited to the insect lineage. Interestingly, phylostratigraphic analysis showed that CpG methylation preferentially targeted genes originating in the common ancestor of eukaryotes rather than the oldest genes originating in the common ancestor of cellular organisms.

Conclusions

Comparative analysis of the oyster DNA methylomes and that of other animal species revealed that the characteristics of DNA methylation were generally conserved during invertebrate evolution, while some unique features were derived in the insect lineage. The preference of methylation modification on genes originating in the eukaryotic ancestor rather than the oldest genes is unexpected, probably implying that the emergence of methylation regulation in these ''relatively young’ genes was critical for the origin and radiation of eukaryotes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1119) contains supplementary material, which is available to authorized users.     

哺乳动物印记域DLK1-DIO3的研究进展

DLK1-DIO3印记域定位于人14号染色体、小鼠12号染色体及绵羊18号染色体远端, 在真哺乳亚纲动物中印记保守。该印记域包含3个编码蛋白的父系表达基因Dlk1、Rtl1和Dio3以及若干大小不同的母系表达印记非编码RNA, 如miRNAs、snoRNAs 和大型非编码RNA Gtl2等。人和小鼠该印记域内印记基因剂量的改变将导致严重的表型异常甚至胚胎致死, 暗示正常的发育需要域内印记基因的正常表达。文章重点论述了哺乳动物DLK1-DIO3印记域的印记调控机制和域内印记基因及其功能的研究进展。