CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2

It is thought that the H19 imprinting control region (ICR) directs the silencing of the maternally inherited Igf2 allele through a CTCF-dependent chromatin insulator. The ICR has been shown to interact physically with a silencer region in Igf2, differentially methylated region (DMR)1, but the role of CTCF in this chromatin loop and whether it restricts the physical access of distal enhancers to Igf2 is not known. We performed systematic chromosome conformation capture analyses in the Igf2/H19 region over >160 kb, identifying sequences that interact physically with the distal enhancers and the ICR. We found that, on the paternal chromosome, enhancers interact with the Igf2 promoters but that, on the maternal allele, this is prevented by CTCF binding within the H19 ICR. CTCF binding in the maternal ICR regulates its interaction with matrix attachment region (MAR)3 and DMR1 at Igf2, thus forming a tight loop around the maternal Igf2 locus, which may contribute to its silencing. Mutation of CTCF binding sites in the H19 ICR leads to loss of CTCF binding and de novo methylation of a CTCF target site within Igf2 DMR1, showing that CTCF can coordinate regional epigenetic marks. This systematic chromosome conformation capture analysis of an imprinting cluster reveals that CTCF has a critical role in the epigenetic regulation of higher-order chromatin structure and gene silencing over considerable distances in the genome.     

Gain of imprinting at chromosome 11p15: A pathogenetic mechanism identified in human hepatocarcinomas

Genomic imprinting is a reversible condition that causes parental-specific silencing of maternally or paternally inherited genes. Analysis of DNA and RNA from 52 human hepatocarcinoma samples revealed abnormal imprinting of genes located at chromosome 11p15 in 51% of 37 informative samples. The most frequently detected abnormality was gain of imprinting, which led to loss of expression of genes present on the maternal chromosome. As compared with matched normal liver tissue, hepatocellular carcinomas showed extinction or significant reduction of expression of one of the alleles of the CDKN1C, SLC22A1L, and IGF2 genes. Loss of maternal-specific methylation at the KvDMR1 locus in hepatocarcinoma correlated with abnormal expression of CDKN1C and IGF2, suggesting a function for KvDMR1 as a long-range imprinting center active in adult tissues. These results point to the role of epigenetic mechanisms leading to loss of expression of imprinted genes at chromosome region 11p15 in human tumors.     

Epigenetic changes encompassing the IGF2/H19 locus associated with relaxation of IGF2 imprinting and silencing of H19 in Wilms tumor.

In most tissues IGF2 is expressed from the paternal allele while H19 is expressed from the maternal allele. We have previously shown that in some Wilms tumors the maternal IGF2 imprint is relaxed such that the gene is expressed biallelically. We have now investigated this subset of tumors further and found that biallelic expression of IGF2 was associated with undetectable or very low levels of H19 expression. The relaxation of IGF2 imprinting in Wilms tumors also involved a concomitant reversal in the patterns of DNA methylation of the maternally inherited IGF2 and H19 alleles. Furthermore, the only specific methylation changes that occurred in tumors with relaxation of IGF2 imprinting were solely restricted to the maternal IGF2 and H19 alleles. These data suggest that there has been an acquisition of a paternal epigenotype in these tumors as the result of a pathologic disruption in the normal imprinting of the IGF2 and H19 genes.     

Sexual dimorphism in parental imprint ontogeny and contribution to embryonic development

Genomic imprinting refers to the functional non-equivalence of parental genomes in mammals that results from the parent-of-origin allelic expression of a subset of genes. Parent-specific expression is dependent on the germ line acquisition of DNA methylation marks at imprinting control regions (ICRs), coordinated by the DNA-methyltransferase homolog DNMT3L. We discuss here how the gender-specific stages of DNMT3L expression may have influenced the various sexually dimorphic aspects of genomic imprinting: (1) the differential developmental timing of methylation establishment at paternally and maternally imprinted genes in each parental germ line, (2) the differential dependence on DNMT3L of parental methylation imprint establishment, (3) the unequal duration of paternal versus maternal methylation imprints during germ cell development, (4) the biased distribution of methylation-dependent ICRs towards the maternal genome, (5) the different genomic organization of paternal versus maternal ICRs, and finally (6) the overwhelming contribution of maternal germ line imprints to development compared to their paternal counterparts.     

Loss of the maternal H19 gene induces changes in Igf2 methylation in both cis and trans

Recent investigations have shown that the maintenance of genomic imprinting of the murine insulin-like growth factor 2 (Igf2) gene involves at least two factors: the DNA (cytosine-5-)-methyltransferase activity, which is required to preserve the paternal specific expression of Igf2, and the H19 gene (lying 90 kb downstream of Igf2 gene), which upon inactivation leads to relaxation of the Igf2 imprint. It is not yet clear how these two factors are related to each other in the process of maintenance of Igf2 imprinting and, in particular, whether the latter is acting through cis elements or whether the H19 RNA itself is involved. By using Southern blots and the bisulfite genomic-sequencing technique, we have investigated the allelic methylation patterns (epigenotypes) of the Igf2 gene in two strains of mouse with distinct deletions of the H19 gene. The results show that maternal transmission of H19 gene deletions leads the maternal allele of Igf2 to adopt the epigenotype of the paternal allele and indicate that this phenomenon is influenced directly or indirectly by the H19 gene expression. More importantly, the bisulfite genomic-sequencing allowed us to show that the methylation pattern of the paternal allele of the Igf2 gene is affected in trans by deletions of the active maternal allele of the H19 gene. Selection during development for the appropriate expression of Igf2, dosage-dependent factors that bind to the Igf2 gene, or methylation transfer between the parental alleles could be involved in this trans effect.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:The structural H19 gene is required for transgene imprinting

The product of the H19 gene is an untranslated RNA that is expressed exclusively from the maternal chromosome during mammalian development. The H19 gene and its 5′-flanking sequence are required for the genomic imprinting of two paternally expressed genes, Ins-2 (encodes insulin-2) and Igf-2 (encodes insulin-like growth factor-2), that lie 90 and 115 kb 5′ to the H19 gene, respectively. In this report, the role of the H19 gene in its own imprinting is investigated by introducing a Mus spretus H19 gene into heterologous locations in the mouse genome. Multiple copies of the transgene were sufficient for its paternal silencing and DNA methylation. Replacing the H19 structural gene with a luciferase reporter gene resulted in loss of imprinting of the transgene. That is, high expression and low levels of DNA methylation were observed upon both paternal and maternal inheritance. The removal of 701 bp at the 5′ end of the structural gene resulted in a similar loss of paternal-specific DNA methylation, arguing that those sequences are required for both the establishment and maintenance of the sperm-specific gametic mark. The M. spretus H19 transgene could not rescue the loss of Igf-2 imprinting in trans in H19 deletion mice, implying a cis requirement for the H19 gene. In contrast to a previous report in which overexpression of a marked H19 gene was a prenatal lethal, expression of the M. spretus transgene had no deleterious effect, leading to the conclusion that the 20-base insertion in the marked gene created a neomorphic mutation.     

Paternally expressed genes predominate in the placenta

The discovery of genomic imprinting through studies of manipulated mouse embryos indicated that the paternal genome has a major influence on placental development. However, previous research has not demonstrated paternal bias in imprinted genes. We applied RNA sequencing to trophoblast tissue from reciprocal hybrids of horse and donkey, where genotypic differences allowed parent-of-origin identification of most expressed genes. Using this approach, we identified a core group of 15 ancient imprinted genes, of which 10 were paternally expressed. An additional 78 candidate imprinted genes identified by RNA sequencing also showed paternal bias. Pyrosequencing was used to confirm the imprinting status of six of the genes, including the insulin receptor (INSR), which may play a role in growth regulation with its reciprocally imprinted ligand, histone acetyltransferase-1 (HAT1), a gene involved in chromatin modification, and lymphocyte antigen 6 complex, locus G6C, a newly identified imprinted gene in the major histocompatibility complex. The 78 candidate imprinted genes displayed parent-of-origin expression bias in placenta but not fetus, and most showed less than 100% silencing of the imprinted allele. Some displayed variability in imprinting status among individuals. This variability results in a unique epigenetic signature for each placenta that contributes to variation in the intrauterine environment and thus presents the opportunity for natural selection to operate on parent-of-origin differential regulation. Taken together, these features highlight the plasticity of imprinting in mammals and the central importance of the placenta as a target tissue for genomic imprinting.     

Untargeted mutation of the maternally derived mouse hypervariable minisatellite allele in F1 mice born to irradiated spermatozoa

Length change mutation at the Ms6hm hypervariable mouse minisatellite locus was analyzed in C57BL/6N x C3H/HeN F(1) mice and the F(1) of the reciprocal cross born to irradiated male parents. Spontaneous mutant frequencies were 8.4% and 9.8% for the paternally derived and maternally derived C3H/HeN alleles, respectively. The mutant frequencies for the paternally derived allele increased to 22% and 19% when the male parents were irradiated with 6 Gy at the postmeiotic spermatozoa stage and the spermatogonia stage, respectively. These increases in the mutant frequency were at least 10 to 100 times higher than those expected from the frequency of hits to the 3- to 4-kb allele, suggesting that the length change mutation at this minisatellite locus was not a targeted event due directly to DNA damage in the region. Further analysis demonstrated that the mutant frequency increased also at the maternally derived C3H/HeN allele to 20% when the male parents were irradiated at the spermatozoa stage. This increase in the maternal allele mutation was not observed in F(1) born to irradiated spermatogonia. The present study suggests that introduction of DNA damage by irradiated sperm triggers genomic instability in zygotes and in embryos of subsequent developmental stages, and this genomic instability induces untargeted mutation in cis at the paternally derived minisatellite allele and in trans at the maternally derived unirradiated allele. Untargeted mutation revealed in the present study defines a previously unnoticed genetic hazard to the maternally derived genome by the paternally introduced DNA damage.     

Genome-wide scan for body composition in pigs reveals important role of imprinting

The role of imprinting in body composition was investigated in an experimental cross between Chinese Meishan pigs and commercial Dutch pigs. A whole-genome scan revealed significant evidence for five quantitative trait loci (QTL) affecting body composition, of which four were imprinted. Imprinting was tested with a statistical model that separated the expression of paternally and maternally inherited alleles. For back fat thickness, a paternally expressed QTL was found on Sus scrofa chromosome 2 (SSC2), and a Mendelian-expressed QTL was found on SSC7. In the same region of SSC7, a maternally expressed QTL affecting muscle depth was found. Chromosome 6 harbored a maternally expressed QTL on the short arm and a paternally expressed QTL on the long arm, both affecting intramuscular fat content. The individual QTL explained from 2% up to 10% of the phenotypic variance. The known homologies to human and mouse did not reveal positional candidate genes. This study demonstrates that testing for imprinting should become a standard procedure to unravel the genetic control of multifactorial traits.     

No loss of genomic imprinting of IGF-II and H19 in placentas of diabetic pregnancies with fetal macrosomia.

OBJECTIVES: Fetal macrosomia is a common complication of maternal diabetes mellitus and is associated with substantial morbidity, but the precise cellular and molecular mechanisms that induce fetal macrosomia are not well understood. The imprinted genes IGF-II and H19 are crucial for placental development and fetal growth. The term placentas from diabetic pregnancies express more insulin-like growth factor II (IGF-II) than those from normal pregnancies. Deregulation of their imprinting status is observed in the macrosomia-associated syndrome, the Beckwith-Wiedemann syndrome. The aim of this study was to determine whether loss of imprinting hence biallelic expression was also a hallmark of macrosomia in diabetic pregnancies. DESIGN AND METHODS: IGF-II and H19 maternal and paternal expressions were studied in placentas from two groups of type 1 diabetic mothers: one with macrosomic babies and the other with babies of normal weight. Maternal or paternal allele specific expressions were defined by using DNA polymorphic markers of the IGF-II and H19 genes. RFLP analysis was performed on PCR products from genomic DNA of the father, the mother and the child, and on RT-PCR products from placental mRNA. RESULTS: RFLP analysis showed that the IGF-II gene remains paternally expressed and the H19 gene remains maternally expressed in all placentas examined, independently of the birth weight status. CONCLUSIONS: These results suggest that, in contrast with Beckwith-Wiedemann syndrome-associated macrosomia, loss of imprinting for IGF-II or H19 is not a common feature of diabetic pregnancies associated with macrosomia.     

Paternal and maternal genomes confer opposite effects on proliferation, cell-cycle length, senescence, and tumor formation

Loss of imprinting is the silencing of active imprinted genes or the activation of silent imprinted genes, and it is one of the most common epigenetic changes associated with the development of a wide variety of tumors. Here, we have analyzed the effects that global imprinted gene expression has on cell proliferation and transformation. Primary mouse embryonic fibroblasts (MEFs), whose entire genome is either exclusively paternal (androgenetic) or maternal (parthenogenetic), exhibit dramatically contrasting patterns of growth. In comparison with biparental MEFs, andro-genetic proliferation is characterized by a shorter cell cycle, increased saturation density, spontaneous transformation, and formation of tumors at low passage number. Parthenogenetic MEFs reach a lower saturation density, senesce, and die. The maternally expressed imprinted genes p57kip2 and M6P/Igf2r retard proliferation and reduce the long-term growth of MEFs. In contrast, the paternally expressed growth factor Igf2 is essential for the long-term proliferation of all genotypes. Increased Igf2 expression in primary MEFs not only stimulates proliferation, but also results in their rapid conversion to malignancy with tumor formation of short latency. Our results reveal that paternally expressed imprinted genes, in the absence of maternal imprinted genes, predispose fibroblasts to rapid transformation. A potent factor in their transformation is IGF2, which on increased expression results in the rapid conversion of primary cells to malignancy. These results reveal a route by which malignant choriocarcinoma may arise from molar pregnancies. They also suggest that the derivation of stem cells from parthenogenetic embryos, for the purposes of therapeutic cloning, may be ineffective.