Chromatin mechanisms in genomic imprinting

Mammalian imprinted genes are clustered in chromosomal domains. Their mono-allelic, parent-of-origin-specific expression is regulated by imprinting control regions (ICRs), which are essential sequence elements marked by DNA methylation on one of the two parental alleles. These methylation “imprints” are established during gametogenesis and, after fertilization, are somatically maintained throughout development. Nonhistone proteins and histone modifications contribute to this epigenetic process. The way ICRs mediate imprinted gene expression differs between domains. At some domains, for instance, ICRs produce long noncoding RNAs that mediate chromatin silencing. Lysine methylation on histone H3 is involved in this developmental process and is particularly important for imprinting in the placenta and brain. Together, the newly discovered chromatin mechanisms provide further clues for addressing imprinting-related pathologies in humans.     

The specification of imprints in mammals

At the heart of genomic imprinting in mammals are imprinting control regions (ICRs), which are the discrete genetic elements that confer imprinted monoallelic expression to several genes in imprinted gene clusters. A characteristic of the known ICRs is that they acquire different epigenetic states, exemplified by differences in DNA methylation, in the sperm and egg, and these imprint marks remain on the sperm- and oocyte-derived alleles into the next generation as a lifelong memory of parental origin. Although there has been much focus on gametic marking of ICRs as the point of imprint specification, recent mechanistic studies and genome-wide DNA methylation profiling do not support the existence of a specific imprinting machinery in germ cells. Rather, ICRs are part of more widespread methylation events that occur during gametogenesis. Instead, a decisive component in the specification of imprints is the choice of which sites of gamete-derived methylation to maintain in the zygote and preimplantation embryo at a time when much of the remainder of the genome is being demethylated. Among the factors involved in this selection, the zinc-finger protein Zfp57 can be regarded as an imprint-specific, sequence-specific DNA binding factor responsible for maintaining methylation at most ICRs. The recent insights into the balance of gametic and zygotic contributions to imprint specification should help understand mechanistic opportunities and constraints on the evolution of imprinting in mammals.     

Epigenetic deregulation of genomic imprinting in human disorders and following assisted reproduction

Imprinted genes play important roles in the regulation of growth and development, and several have been shown to influence behavior. Their allele-specific expression depends on inheritance from either the mother or the father, and is regulated by "imprinting control regions" (ICRs). ICRs are controlled by DNA methylation, which is present on one of the two parental alleles only. These allelic methylation marks are established in either the female or the male germline, following the erasure of preexisting DNA methylation in the primordial germ cells. After fertilization, the allelic DNA methylation at ICRs is maintained in all somatic cells of the developing embryo. This epigenetic "life cycle" of imprinting (germline erasure, germline establishment, and somatic maintenance) can be disrupted in several human diseases, including Beckwith-Wiedemann syndrome (BWS), Prader-Willi syndrome (PWS), Angelman syndrome and Hydatidiform mole. In the neurodevelopmental Rett syndrome, the way the ICR mediates imprinted expression is perturbed. Recent studies indicate that assisted reproduction technologies (ART) can sometimes affect the epigenetic cycle of imprinting as well, and that this gives rise to imprinting disease syndromes. This finding warrants careful monitoring of the epigenetic effects, and absolute risks, of currently used and novel reproduction technologies.     

Epigenetic instability at imprinting control regions in a KrasG12D-induced T-cell neoplasm

Although aberrant DNA methylation within imprinted domains has been reported in a variety of neoplastic diseases, it remains largely uncharacterized in the context of carcinogenesis. In this study, we induced T-cell lymphoma in mice by employing a breeding scheme involving mouse strains, LSL-Kras^G12D and MMTV-Cre. We then systematically surveyed imprinted domains for DNA methylation changes during tumor progression using combined bisulfite restriction analysis and NGS-based bisulfite sequencing. We detected hyper- or hypo-methylation at the imprinting control regions (ICRs) of the Dlk1, Peg10, Peg3, Grb10, and Gnas domains. These DNA methylation changes at ICRs were more prevalent and consistent than those observed at the promoter regions of well-known tumor suppressors, such as Mgmt, Fhit, and Mlh1. Thus, the changes observed at these imprinted domains are the outcome of isolated incidents affecting DNA methylation settings. Within imprinted domains, DNA methylation changes tend to be restricted to ICRs as nearby somatic differentially methylated regions and promoter regions experience no change. Furthermore, detailed analyses revealed that small cis-regulatory elements within ICRs tend to be resistant to DNA methylation changes, suggesting potential protection by unknown trans-factors. Overall, this study demonstrates that DNA methylation changes at ICRs are dynamic during carcinogenesis and advocates that detection of aberrant DNA methylation at ICRs may serve as a biomarker to enhance diagnostic procedures.     

Differential histone modifications mark mouse imprinting control regions during spermatogenesis

Only some imprinting control regions (ICRs) acquire their DNA methylation in the male germ line. These imprints are protected against the global demethylation of the sperm genome following fertilisation, and are maintained throughout development. We find that in somatic cells and tissues, DNA methylation at these ICRs is associated with histone H4-lysine-20 and H3-lysine-9 trimethylation. The unmethylated allele, in contrast, has H3-lysine-4 dimethylation and H3 acetylation. These differential modifications are also detected at maternally methylated ICRs, and could be involved in the somatic maintenance of imprints. To explore whether the post-fertilisation protection of imprints relates to events during spermatogenesis, we assayed chromatin at stages preceding the global histone-to-protamine exchange. At these stages, H3-lysine-4 methylation and H3 acetylation are enriched at maternally methylated ICRs, but are absent at paternally methylated ICRs. H4 acetylation is enriched at all regions analysed. Thus, paternally and maternally methylated ICRs carry different histone modifications during the stages preceding the global histone-to-protamine exchange. These differences could influence the way ICRs are assembled into specific structures in late spermatogenesis, and may thus influence events after fertilisation.     

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.     

PR-SET7 and SUV4-20H regulate H4 lysine-20 methylation at imprinting control regions in the mouse

Imprinted genes are important in development and their allelic expression is mediated by imprinting control regions (ICRs). On their DNA-methylated allele, ICRs are marked by trimethylation at H3 Lys 9 (H3K9me3) and H4 Lys 20 (H4K20me3), similar to pericentric heterochromatin. Here, we investigate which histone methyltransferases control this methylation of histone at ICRs. We found that inactivation of SUV4-20H leads to the loss of H4K20me3 and increased levels of its substrate, H4K20me1. H4K20me1 is controlled by PR-SET7 and is detected on both parental alleles. The disruption of SUV4-20H or PR-SET7 does not affect methylation of DNA at ICRs but influences precipitation of H3K9me3, which is suggestive of a trans-histone change. Unlike at pericentric heterochromatin, however, H3K9me3 at ICRs does not depend on SUV39H. Our data show not only new similarities but also differences between ICRs and heterochromatin, both of which show constitutive maintenance of methylation of DNA in somatic cells.     

Partial Loss of Genomic Imprinting Reveals Important Roles for Kcnq1 and Peg10 Imprinted Domains in Placental Development

Mutations in imprinted genes or their imprint control regions (ICRs) produce changes in imprinted gene expression and distinct abnormalities in placental structure, indicating the importance of genomic imprinting to placental development. We have recently shown that a very broad spectrum of placental abnormalities associated with altered imprinted gene expression occurs in the absence of the oocyte–derived DNMT1o cytosine methyltransferase, which normally maintains parent-specific imprinted methylation during preimplantation. The absence of DNMT1o partially reduces inherited imprinted methylation while retaining the genetic integrity of imprinted genes and their ICRs. Using this novel system, we undertook a broad and inclusive approach to identifying key ICRs involved in placental development by correlating loss of imprinted DNA methylation with abnormal placental phenotypes in a mid-gestation window (E12.5-E15.5). To these ends we measured DNA CpG methylation at 15 imprinted gametic differentially methylated domains (gDMDs) that overlap known ICRs using EpiTYPER-mass array technology, and linked these epigenetic measurements to histomorphological defects. Methylation of some imprinted gDMDs, most notably Dlk1, was nearly normal in mid-gestation DNMT1o-deficient placentas, consistent with the notion that cells having lost methylation on these DMDs do not contribute significantly to placental development. Most imprinted gDMDs however showed a wide range of methylation loss among DNMT1o-deficient placentas. Two striking associations were observed. First, loss of DNA methylation at the Peg10 imprinted gDMD associated with decreased embryonic viability and decreased labyrinthine volume. Second, loss of methylation at the Kcnq1 imprinted gDMD was strongly associated with trophoblast giant cell (TGC) expansion. We conclude that the Peg10 and Kcnq1 ICRs are key regulators of mid-gestation placental function.     

In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions

The maintenance of H3K9 and DNA methylation at imprinting control regions (ICRs) during early embryogenesis is key to the regulation of imprinted genes. Here, we reveal that ZFP57, its cofactor KAP1, and associated effectors bind selectively to the H3K9me3-bearing, DNA-methylated allele of ICRs in ES cells. KAP1 deletion induces a loss of heterochromatin marks at ICRs, whereas deleting ZFP57 or DNMTs leads to ICR DNA demethylation. Accordingly, we find that ZFP57 and KAP1 associated with DNMTs and hemimethylated DNA-binding NP95. Finally, we identify the methylated TGCCGC hexanucleotide as the motif that is recognized by ZFP57 in all ICRs and in several tens of additional loci, several of which are at least ZFP57-dependently methylated in ES cells. These results significantly advance our understanding of imprinting and suggest a general mechanism for the protection of specific loci against the wave of DNA demethylation that affects the mammalian genome during early embryogenesis.     

Small regulatory RNAs controlled by genomic imprinting and their contribution to human disease

More than a hundred protein-coding genes are controlled by genomic imprinting in humans. These atypical genes are organized in chromosomal domains, each of which is controlled by a differentially methylated "imprinting control region" (ICR). How ICRs mediate the parental allele-specific expression of close-by genes is now becoming understood. At several imprinted domains, this epigenetic mechanism involves the action of long non-coding RNAs. It is less well appreciated that imprinted gene domains also transcribe hundreds of microRNA and small nucleolar RNA genes and that these represent the densest clusters of small RNA genes in mammalian genomes. The evolutionary reasons for this remarkable enrichment of small regulatory RNAs at imprinted domains remain unclear. However, recent studies show that imprinted small RNAs modulate specific functions in development and metabolism and also are frequently perturbed in cancer. Here, we review our current understanding of imprinted small RNAs in the human genome and discuss how perturbation of their expression contributes to disease.     

Epigenetic deregulation of imprinting in congenital diseases of aberrant growth

Human chromosome 11p15 comprises two imprinted domains important in the control of fetal and postnatal growth. Novel studies establish that imprinting at one of these, the IGF2-H19 domain, is epigenetically deregulated (with loss of DNA methylation) in Silver-Russell Syndrome (SRS), a congenital disease of growth retardation and asymmetry. Previously, the exact opposite epigenetic alteration (gain of DNA methylation) had been detected at the domain's 'imprinting control region' (ICR) in patients with Beckwith-Wiedemann Syndrome (BWS), a complex disorder of fetal overgrowth. However, more frequently, BWS is caused by loss of DNA methylation at the ICR that regulates the second imprinted domain at 11p15. Interestingly, a similar epigenetic alteration (with loss of methylation) at a putative ICR on human chromosome 6q24, is involved in transient neonatal diabetes mellitus (TNDM), a congenital disease with intrauterine growth retardation and a transient lack of insulin. Thus, fetal and postnatal growth is epigenetically controlled by different ICRs, at 11p15 and other chromosomal regions.     

Small regulatory RNAs controlled by genomic imprinting and their contribution to human disease

More than a hundred protein-coding genes are controlled by genomic imprinting in humans. These atypical genes are organized in chromosomal domains, each of which is controlled by a differentially methylated "imprinting control region" (ICR). How ICRs mediate the parental allele-specific expression of close-by genes is now becoming understood. At several imprinted domains, this epigenetic mechanism involves the action of long non-coding RNAs. It is less well appreciated that imprinted gene domains also transcribe hundreds of microRNA and small nucleolar RNA genes and that these represent the densest clusters of small RNA genes in mammalian genomes. The evolutionary reasons for this remarkable enrichment of small regulatory RNAs at imprinted domains remain unclear. However, recent studies show that imprinted small RNAs modulate specific functions in development and metabolism and also are frequently perturbed in cancer. Here, we review our current understanding of imprinted small RNAs in the human genome and discuss how perturbation of their expression contributes to disease.