Marisa S. Bartolomei

Differential Effects of Culture on Imprinted H19 Expression in the Preimplantation Mouse Embryo

Abstract The H19 gene is imprinted with preferential expression from the maternal allele. The putative imprinting control region for this locus is hypermethylated on the repressed paternal allele. Although maternal-specific expression of H19 is observed in mouse blastocysts that develop in vivo, biallelic expression has been documented in embryos and embryonic stem cells experimentally manipulated by in vitro culture conditions. In this study the effect of culture on imprinted H19 expression and methylation was determined. After culture of 2-cell embryos to the blastocyst stage in Whittens medium, the normally silent paternal H19 allele was aberrantly expressed, whereas little paternal expression was observed following culture in KSOM containing amino acids (KSOM+AA). Analysis of the methylation status of a CpG dinucleotide located in the upstream imprinting control region revealed a loss in methylation in embryos cultured in Whittens medium but not in embryos cultured in KSOM+AA. Thus, H19 expression and methylation were adversely affected by culture in Whittens medium, while the response of H19 to culture in KSOM+AA approximated more closely the in vivo situation. It is unlikely that biallelic expression of H19 following culture in Whittens medium is a generalized effect of lower methylation levels, since the amount of DNA methyltransferase activity and the spatial distribution of Dnmt1 protein were similar in in vivo-derived and cultured embryos. Moreover, imprinted expression of Snrpn was maintained following culture in either medium, indicating that not all imprinted genes are under the same stringent imprinting controls. The finding that culture conditions can dramatically, but selectively, affect the expression of imprinted genes provides a model system for further study of the linkage between DNA methylation and gene expression.

Thymine DNA Glycosylase Is Essential for Active DNA Demethylation by Linked Deamination-Base Excision Repair

DNA methylation is a major epigenetic mechanism for gene silencing. Whereas methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here, we show that either knockout or catalytic inactivation of the DNA repair enzyme thymine DNA glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific developmentally and hormonally regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.

X-Inactivation, Imprinting, and Long Noncoding RNAs in Health and Disease

X chromosome inactivation and genomic imprinting are classic epigenetic processes that cause disease when not appropriately regulated in mammals. Whereas X chromosome inactivation evolved to solve the problem of gene dosage, the purpose of genomic imprinting remains controversial. Nevertheless, the two phenomena are united by allelic control of large gene clusters, such that only one copy of a gene is expressed in every cell. Allelic regulation poses significant challenges because it requires coordinated long-range control in cis and stable propagation over time. Long noncoding RNAs have emerged as a common theme, and their contributions to diseases of imprinting and the X chromosome have become apparent. Here, we review recent advances in basic biology, the connections to disease, and preview potential therapeutic strategies for future development.

Selective loss of imprinting in the placenta following preimplantation development in culture.

Preimplantation development is a period of dynamic epigenetic change that begins with remodeling of egg and sperm genomes, and ends with implantation. During this time, parental-specific imprinting marks are maintained to direct appropriate imprinted gene expression. We previously demonstrated that H19 imprinting could be lost during preimplantation development under certain culture conditions. To define the lability of genomic imprints during this dynamic period and to determine whether loss of imprinting continues at later stages of development, imprinted gene expression and methylation were examined after in vitro preimplantation culture. Following culture in Whittens medium, the normally silent paternal H19 allele was aberrantly expressed and undermethylated. However, only a subset of individual cultured blastocysts (∼65%) exhibited biallelic expression, while others maintained imprinted H19 expression. Loss of H19 imprinting persisted in mid-gestation conceptuses. Placental tissues displayed activation of the normally silent allele for H19, Ascl2, Snrpn, Peg3 and Xist while in the embryo proper imprinted expression for the most part was preserved. Loss of imprinted expression was associated with a decrease in methylation at the H19 and Snrpn imprinting control regions. These results indicate that tissues of trophectoderm origin are unable to restore genomic imprints and suggest that mechanisms that safeguard imprinting might be more robust in the embryo than in the placenta.

Cohesins localize with CTCF at the KSHV latency control region and at cellular c-myc and H19/Igf2 insulators.

Cohesins, which mediate sister chromatin cohesion, and CTCF, which functions at chromatin boundaries, play key roles in the structural and functional organization of chromosomes. We examined the binding of these two factors on the Kaposis sarcoma‐associated herpesvirus (KSHV) episome during latent infection and found a striking colocalization within the control region of the major latency transcript responsible for expressing LANA (ORF73), vCyclin (ORF72), vFLIP (ORF71), and vmiRNAs. Deletion of the CTCF‐binding site from the viral genome disrupted cohesin binding, and crippled colony formation in 293 cells. Clonal instability correlated with elevated expression of lytic cycle gene products, notably the neighbouring promoter for K14 and vGPCR (ORF74). siRNA depletion of RAD21 from latently infected cells caused an increase in K14 and ORF74, and lytic inducers caused a rapid dissociation of RAD21 from the viral genome. RAD21 and SMC1 also associate with the cellular CTCF sites at mammalian c‐myc promoter and H19/Igf2 imprinting control region. We conclude that cohesin subunits associate with viral and cellular CTCF sites involved in complex gene regulation and chromatin organization.

Disruption of Imprinted Gene Methylation and Expression in Cloned Preimplantation Stage Mouse Embryos

Abstract Cloning by somatic cell nuclear transfer requires that epigenetic information possessed by the donor nucleus be reprogrammed to an embryonic state. Little is known, however, about this remodeling process, including when it occurs, its efficiency, and how well epigenetic markings characteristic of normal development are maintained. Examining the fate of epigenetic information associated with imprinted genes during clonal development offers one means of addressing these questions. We examined transcript abundance, allele specificity of imprinted gene expression, and parental allele-specific DNA methylation in cloned mouse blastocysts. Striking disruptions were seen in total transcript abundance and allele specificity of expression for five imprinted genes. Only 4% of clones recapitulated a blastocyst mode of expression for all five genes. Cloned embryos also exhibited extensive loss of allele-specific DNA methylation at the imprinting control regions of the H19 and Snprn genes. Thus, epigenetic errors arise very early in clonal development in the majority of embryos, indicating that reprogramming is inefficient and that some epigenetic information may be lost.

Mammalian Genomic Imprinting

Normal mammalian development requires a maternal and paternal contribution, which is attributed to imprinted genes, or genes that are expressed from a single parental allele. Approximately 100 imprinted genes have been reported in mammals thus far. Imprinted genes are controlled by cis-acting regulatory elements, termed imprinting control regions (ICRs), which have parental-specific epigenetic modifications, including DNA methylation. ICRs are methylated by de novo DNA methyltransferases during germline development; these parental-specific modifications must be maintained following fertilization when the genome is extensively reprogrammed. Many imprinted genes reside in ∼1-megabase clusters, with two major mechanisms of imprinting regulation currently recognized, CTCF-dependent insulators and long noncoding RNAs. Unclustered imprinted genes are generally regulated by germline-derived differential promoter methylation. Here, we describe the identification and functions of imprinted genes, cis-acting control sequences, trans-acting factors, and imprinting mechanisms in clusters. Finally, we define questions that require more extensive research.

GENOMIC IMPRINTING IN MAMMALS

Genomic imprinting affects a subset of genes in mammals and results in a monoallelic, parental-specific expression pattern. Most of these genes are located in clusters that are regulated through the use of insulators or long noncoding RNAs (lncRNAs). To distinguish the parental alleles, imprinted genes are epigenetically marked in gametes at imprinting control elements through the use of DNA methylation at the very least. Imprinted gene expression is subsequently conferred through lncRNAs, histone modifications, insulators, and higher-order chromatin structure. Such imprints are maintained after fertilization through these mechanisms despite extensive reprogramming of the mammalian genome. Genomic imprinting is an excellent model for understanding mammalian epigenetic regulation.

Genomic imprinting: employing and avoiding epigenetic processes

Genomic imprinting refers to an epigenetic mark that distinguishes parental alleles and results in a monoallelic, parental-specific expression pattern in mammals. Few phenomena in nature depend more on epigenetic mechanisms while at the same time evading them. The alleles of imprinted genes are marked epigenetically at discrete elements termed imprinting control regions (ICRs) with their parental origin in gametes through the use of DNA methylation, at the very least. Imprinted gene expression is subsequently maintained using noncoding RNAs, histone modifications, insulators, and higher-order chromatin structure. Avoidance is manifest when imprinted genes evade the genome-wide reprogramming that occurs after fertilization and remain marked with their parental origin. This review summarizes what is known about the establishment and maintenance of imprinting marks and discusses the mechanisms of imprinting in clusters. Additionally, the evolution of imprinted gene clusters is described. While considerable information regarding epigenetic control of imprinting has been obtained recently, much remains to be learned.

MECHANISMS OF GENOMIC IMPRINTING

A small number of mammalian genes undergo the process of genomic imprinting whereby the expression level of the alleles of a gene depends upon their parental origin. In the past year, attention has focused on the mechanisms that determine parental-specific expression patterns. Many imprinted genes are located in conserved clusters and, although it is apparent that imprinting of adjacent genes is jointly regulated, multiple mechanisms among and within clusters may operate. Recent developments have also refined the timing of the gametic imprints and further defined the mechanism by which DNA methyltransferases confer allelic methylation patterns.