Role of a 461-bp G-rich repetitive element in H19 transgene imprinting

 The molecular mechanism leading to the imprinted expression of genes is poorly understood. While no conserved cis-acting elements have been identified within the known loci, many imprinted genes are located near directly repetitive sequence elements, suggesting that such repeats might play a role in imprinted gene expression. The maternally expressed mouse H19 gene is located approximately 1.5 kb downstream from a 461-bp G-rich repetitive element. We have used a transgenic model to investigate whether this element is essential for H19 imprinting. Previous results demonstrated that a transgene, which contains 14 kb of H19 sequence, exhibits parent-of-origin specific expression and methylation analogous to the endogenous H19 imprinting pattern. Here, we have generated transgenes lacking the G-rich repeat. One transgene, containing a deletion of the G-rich repetitive element but which includes an additional 1.7 kb of 5’H19 sequence, is imprinted similarly to the endogenous H19 gene. To determine whether the G-rich repeat is conserved in other imprinted mammalian H19 homologues, additional 5’ flanking sequences were cloned from the rat and human. This element is conserved in the rat but not in human DNA. These results suggest that the 461-bp G-rich repetitive element is not essential for H19 imprinting. Received: 26 August 1998 / Accepted: 14 December 1998     

Genomic imprinting controls matrix attachment regions in the Igf2 gene

Genomic imprinting at the Igf2/H19 locus originates from allele-specific DNA methylation, which modifies the affinity of some proteins for their target sequences. Here, we show that AT-rich DNA sequences located in the vicinity of previously characterized differentially methylated regions (DMRs) of the imprinted Igf2 gene are conserved between mouse and human. These sequences have all the characteristics of matrix attachment regions (MARs), which are known as versatile regulatory elements involved in chromatin structure and gene expression. Combining allele-specific nuclear matrix binding assays and real-time PCR quantification, we show that retention of two of these Igf2 MARs (MAR0 and MAR2) in the nuclear matrix fraction depends on the tissue and is specific to the paternal allele. Furthermore, on this allele, the Igf2 MAR2 is functionally linked to the neighboring DMR2 while, on the maternal allele, it is controlled by the imprinting-control region. Our work clearly demonstrates that genomic imprinting controls matrix attachment regions in the Igf2 gene.     

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.     

Alterations of H19 Imprinting and IGF2 Replication Timing Are Infrequent in Beckwith–Wiedemann Syndrome

Beckwith–Wiedemann syndrome (BWS) is an overgrowth disorder resulting from dysregulation of multiple imprinted genes through a variety of distinct mechanisms. A frequent alteration in BWS involves changes in the imprinting status of the coordinately regulated IGF2 and H19 genes on 11p15. Patients have been categorized according to alterations in the imprinted expression, allele-specific methylation, and regional replication timing of these genes. In this work, IGF2/H19 expression, H19 DNA methylation, and IGF2 regional replication timing were studied in nine karyotypically normal BWS fibroblasts and two BWS patients with maternally inherited 11p15 chromosomal rearrangements. Informative patients (9/9) maintained normal monoallelic H19 expression/methylation, despite biallelic IGF2 expression in 6/9. Replication timing studies revealed no changes in the pattern of asynchronous replication timing for both a patient with biallelic IGF2 expression and a patient carrying an 11p15 inversion. In contrast, a patient with a chromosome 11;22 translocation and normal H19 expression/methylation exhibited partial loss of asynchrony and a shift toward earlier replication times. These results indicate that in BWS, (1) H19 imprinting alterations are less frequent than previously estimated, (2) IGF2 imprinting and H19 imprinting are not necessarily coordinated, and (3) alterations in regional replication timing are generally not correlated with either chromosomal rearrangements or the imprinting status of IGF2 and H19.     

Large-scale identification and mapping of nuclear matrix-attachment regions in the distal imprinted domain of mouse chromosome 7.

Mammalian imprinted genes, which are expressed from only one of the parental alleles, have a tendency to form clusters and are regulated by long-range mechanisms. Nuclear matrix-attachment regions (MARs), the anchor points of loop domains, are involved in coordination of gene expression and could play a role in regulation of imprinted domains. We have identified and mapped a total of 52 MARs in a 1-Mb imprinted domain on mouse distal chromosome 7 using our cosmid contigs and an in vitro MAR assay. We find two MAR clusters (comprising 20 and 19 MARs), one of which is mapped in the Th-Ins2 intergenic region, coincident with the boundary between the two imprinted subdomains. However, the imprinted/non-imprinted boundaries are not associated with a MAR. Based on the sequence information, we find that many of the MARs are rich in long interspersed nuclear elements. In addition, comparisons of the results obtained with several MAR-prediction software programs reveal good performance of ChrClass in terms of both sensitivity and specificity. This study presents the first large-scale mapping of MARs in an imprinted domain and provides a platform for understanding the roles of MARs in imprinting.     

Sequence-specific methylation of the mouse H19 gene in embryonic cells deficient in the Dnmt-1 gene

We have used Dnmt^c/c ES cells that are homozygous for disruption of the DNA methyltransferase gene to address how de novo methylation is propagated and whether it is directed to specific sites in the early embryo. We examined the imprinted H19 gene and the specific-sequence region implicated as an “imprinting mark” to determine whether de novo methylation was occurring at a restricted set of sites. Since the “imprinting mark” was found to be methylated differentially at all stages of development, we reasoned that the sequence may still be a target for the de novo methylation activity found in the Dnmt^c/c cells, even though the loss of maintenance methylase activity renders the H19 promoter active. We used bisulfite genomic sequencing to determine the methylation state of the imprinted region of the H19 gene and found a low level of DNA methylation at specific single CpG sites in the upstream region of the imprinted H19 sequence in the Dnmt^c/c mutant ES cells. Moreover, these CpG sites appeared to be favoured targets for further de novo methylation of neighbouring CpG sites in rescued ES cells, which possess apparently normal maintenance activity. Our data provide further evidence for a separate methylating activity in ES cells and indicate that this activity displays sequence specificity. Dev. Genet. 22:111–121, 1998. © 1998 Wiley-Liss, Inc.     

Novel cis-regulatory function in ICR-mediated imprinted repression of H19

Expression of coregulated imprinted genes, H19 and Igf2, is monoallelic and parent-of-origin-dependent. Like most imprinted genes, H19 and Igf2 are regulated by a differentially methylated imprinting control region (ICR). CTCF binding sites and DNA methylation at the ICR have previously been identified as key cis-acting elements required for proper H19/Igf2 imprinting. Here, we use mouse models to elucidate further the mechanism of ICR-mediated gene regulation. We specifically address the question of whether sequences outside of CTCF sites at the ICR are required for paternal H19 repression. To this end, we generated two types of mutant ICRs in the mouse: (i) deletion of intervening sequence between CTCF sites (H19^ICR?IVS), which changes size and CpG content at the ICR; and (ii) CpG depletion outside of CTCF sites (H19^ICR-8nrCG), which only changes CpG content at the ICR. Individually, both mutant alleles (H19^ICR?IVS and H19^ICR-8nrCG) show loss of imprinted repression of paternal H19. Interestingly, this loss of repression does not coincide with a detectable change in methylation at the H19 ICR or promoter. Thus, neither intact CTCF sites nor hypermethylation at the ICR is sufficient for maintaining the fully repressed state of the paternal H19 allele. Our findings demonstrate, for the first time in vivo, that sequence outside of CTCF sites at the ICR is required in cis for ICR-mediated imprinted repression at the H19/Igf2 locus. In addition, these results strongly implicate a novel role of ICR size and CpG density in paternal H19 repression.     

Maintenance of paternal methylation and repression of the imprinted H19 gene requires MBD3

Paternal repression of the imprinted H19 gene is mediated by a differentially methylated domain (DMD) that is essential to imprinting of both H19 and the linked and oppositely imprinted Igf2 gene. The mechanisms by which paternal-specific methylation of the DMD survive the period of genome-wide demethylation in the early embryo and are subsequently used to govern imprinted expression are not known. Methyl-CpG binding (MBD) proteins are likely candidates to explain how these DMDs are recognized to silence the locus, because they preferentially bind methylated DNA and recruit repression complexes with histone deacetylase activity. MBD RNA and protein are found in preimplantation embryos, and chromatin immunoprecipitation shows that MBD3 is bound to the H19 DMD. To test a role for MBDs in imprinting, two independent RNAi-based strategies were used to deplete MBD3 in early mouse embryos, with the same results. In RNAi-treated blastocysts, paternal H19 expression was activated, supporting the hypothesis that MBD3, which is also a member of the Mi-2/NuRD complex, is required to repress the paternal H19 allele. RNAi-treated blastocysts also have reduced levels of the Mi-2/NuRD complex protein MTA-2, which suggests a role for the Mi-2/NuRD repressive complex in paternal-specific silencing at the H19 locus. Furthermore, DNA methylation was reduced at the H19 DMD when MBD3 protein was depleted. In contrast, expression and DNA methylation were not disrupted in preimplantation embryos for other imprinted genes. These results demonstrate new roles for MBD3 in maintaining imprinting control region DNA methylation and silencing the paternal H19 allele. Finally, MBD3-depleted preimplantation embryos have reduced cell numbers, suggesting a role for MBD3 in cell division.     

Sequences in the H19 ICR that are transcribed as small RNA in oocytes are dispensable for methylation imprinting in YAC transgenic mice

Allele-specific methylation of the endogenous H19 imprinting control region (ICR) is established in sperm. We previously showed that the paternal H19 ICR in yeast artificial chromosome (YAC) transgenic mice (TgM) was preferentially methylated in somatic cells, but not in germ cells, suggesting that differential methylation could be established after fertilization. In this report, we discovered small RNA molecules in growing oocytes, the nucleotide sequences of which mapped to the H19 ICR. To test if these small RNA sequences play a role in the establishment of differential methylation, we deleted the sequences from the H19 ICR DNA and generated YAC TgM. In somatic cells of these mice, methylation imprinting of the transgene was normally established. In addition, the mutant fragment was not methylated in sperm and eggs. These data demonstrate that sequences in the H19 ICR that correspond to the small RNA sequences are dispensable for methylation imprinting in YAC TgM.     

Imprinting of mammalian male gametes is gene specific and does not occur at a single stage of differentiation

Epigenetic modifications such as DNA methylation and alterations to chromatin structure have been proposed as hallmarks of imprinting in somatic cells after fertilization. In the germ cell line, gene imprinting needs to be reset in order to transmit the correct sex-specific imprinting pattern to the next generation. The precise timing of imprint erasure and re-establishment for many genes remains to be determined and precise molecular mechanisms of genomic imprinting have not yet been fully characterized. Here, we have analysed the methylation state and DNase-I sensitivity of two genes with reciprocal genomic imprinting (U2af1-rs1 and H19 genes) in a male mouse primordial germ cell (PGC) derived cell line (EG-1), isolated post-natal spermatogonia and mature sperm cells. Our results show that establishment of imprinting of the U2af1-rs1 and H19 genes during male germ cell differentiation occurs at different stages of differentiation. Furthermore, the presence of DNase-I hypersensitive sites may constitute a molecular marker to identify alleles and subsequently acquire the appropriate methylation imprint. We propose that this molecular identifier may be present or absent for a specific gene according to the sex of the gamete.     

Gender specific differences in levels of DNA methylation at selected loci from human total blood: a tendency toward higher methylation levels in males

Abnormal patterns of DNA methylation are observed in many diseases such as tumors and imprinting disorders. Little is known about inter-individual and gender specific variations. Here, we report on accurate and sensitive quantitative measurements of methylation in DNA from total blood in 96 healthy human males and 96 healthy human females. Global methylation was estimated by studying two repetitive DNA elements, namely Line-1 and Alu repeats, while single loci were investigated for three differentially methylated regions (DMRs) at PEG3, NESP55 and H19 imprinted genes and two additional loci at Xq28 (F8 gene) and at 19q13.4 (locus between PEG3 and ubiquitin specific protease 29). We observed inter-individual correlations in the degree of methylation between Alu and Line-1 repeats. Moreover, all studied CpGs showed slightly higher methylation in males (P < 0.0003–0.0381), with the exception of DMRs at imprinted genes (P = 0.0342–0.9616) which were almost equally methylated in both sexes with only a small tendency towards higher methylation in males. This observed difference could be due to the process of X chromosome inactivation or merely to the presence of an additional X chromosome in female cells or could be a result of downstream effects of sex determination. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hypomethylation trends in the intergenic region of the imprinted IGF2 and H19 genes in cloned cattle

Bos taurus is a good model for embryo biotechnologies such as nuclear transfer. However, animals produced from these technologies often suffer from large calf syndrome, suggesting fetal growth dysregulation. The imprinted fetal mitogen IGF2 is clustered with H19 and the two genes are co-regulated in humans and mice. Although the allelic expression pattern of IGF2/H19 has been elucidated in agricultural species such as sheep and cattle, the underlying mechanism of their imprinting regulation has not been characterized. Using bisulfite sequencing the methylation status of 44 CpG sites in a CpG rich intergenic region of IGF2/H19 in the liver, brain, lung, kidney and placenta of control calves (produced by conventional breeding). One fragment containing 16 CpG sites was differentially methylated region (DMR), and thus may be important in regulating IGF2/H19 allelic expression.The DMR in tissues from cloned term calves that either died immediately after birth or were sacrificed due to complications shortly thereafter were examined. There were significant variations in the methylation of this DMR in some of the cloned animals compared to the controls. Most of the observed variations tended toward hypomethylation. The hypomethylation of this DMR in the liver and placenta of clones correlates with the previous observation of abnormal, biallelic expression of the H19 allele in those clones [Zhang, S., Kubota, C., Yang, L., Zhang, Y., Page, R., O’Neill, M., Yang, X., Tian, X.C., 2004. Genomic imprinting of H19 in naturally reproduced and cloned cattle. Biol. Reprod.] but not with allelic expression of IGF2 (as determined in this study). These data suggest that this DMR is involved in H19 allelic expression, but that other mechanisms probably regulate the expression of IGF2/H19. Contrary to global hypermethylation observed in cloned embryos, putative imprinting control regions can display hypomethylation trends in specific organs of cloned calves.