Nuclear transplantation experiments in mice demonstrated that both a maternal and paternal genome are required for normal mammalian development. This failure of uniparental development suggested the existence of genes expressed exclusively from a single parental allele (imprinted genes). While many advances have contributed to our understanding of parental-specific transcriptional regulation, the nature of the mark that designates which parental allele is to be transcribed and the mechanisms that translate this mark into monoallelic expression remain incompletely understood. My laboratory studies the mechanisms that govern the monoallelic expression of imprinted genes and X-linked genes in females. We have pursued the epigenetic modifications, the cis-acting sequences, and the trans-acting factors that establish and maintain monoallelic expression patterns in the mouse.
The H19/Igf2-Imprinting Control Region
Genomic imprinting is defined as the unequal expression of the maternal and paternal alleles of a gene. Two of the first three identified imprinted genes are the linked and oppositely imprinted H19 and Igf2 genes. The imprinting of these genes, which is conserved in mammals, depends on shared regulatory elements. Our initial work focused on understanding how parental-specific expression is achieved for H19 and how reciprocal imprinting of H19 and Igf2 is mediated. We previously demonstrated that a 2-kb region, designated the differentially methylated domain (DMD), located upstream from the start of H19 transcription, is hypermethylated on the repressed paternal allele throughout development and is a prime candidate for conferring locus-wide parental identity. This same region exhibits an open chromatin conformation on the expressed maternal allele in somatic tissues.
To address the role of the DMD in imprinted gene regulation, we used gene targeting to generate deletions at the endogenous locus. We demonstrated that this element is required for the reciprocal imprinting of the H19 and Igf2 genes; the DMD is necessary to repress H19 expression from the paternal allele and confer exclusive H19 expression from the maternal allele. Based on the DMD deletions and the demonstration that the insulator protein CTCF (CCCTC-binding factor) binds to short conserved repeats within the DMD, we and others have proposed that the 2-kb element acts as an insulator on the maternal allele.
The following model has emerged for H19/Igf2 imprinting: On the maternal chromosome, CTCF binds to the four conserved repeats within the DMD and assembles an insulator, allowing H19 exclusive access to the enhancers. On the paternal chromosome, the methylation-sensitive CTCF protein cannot bind to the DMD. Furthermore, since the hypermethylated DMD and H19 promoter are in an inactive chromatin conformation, only Igf2 can access the enhancers.
The above experiments demonstrate that the DMD mediates two opposing functions: paternal-specific DNA hypermethylation resulting in H19 repression and maternal-specific insulation causing H19 activation and Igf2 repression. While attempting to determine if these activities are separable, we found that they are antagonistic. Point mutations were introduced into the endogenous H19 locus that deplete the CTCF-binding sites of CpGs but still allow CTCF to bind on the mutant maternal allele. Although the paternal allele acquires DNA methylation in the male germline, this methylation is lost in the embryo and an insulator is subsequently assembled on the mutant paternal allele. We postulate that mutation of CpG dinucleotides in the CTCF sites allows CTCF to bind in the preimplantation embryo, causing a further reduction in DNA methylation, assembly of an insulator, and activation of the normally repressed paternal H19 allele. Thus, the two parental-specific roles of the H19 DMD—maintenance of DNA methylation and insulator assembly—are antagonistic. (This work is supported in part by a grant from the National Institutes of Health.)
Essential Function for Maternal CTCF in H19 Gene Imprinting
Most imprinted genes examined to date harbor maternal-specific hypermethylated imprinting control regions. In contrast, the H19 DMD possesses one of the few maternal hypomethylation marks, implying that mechanisms may exist that not only confer germline methylation but also protect it from such modifications. Since CTCF binds to the maternal allele in somatic cells, it is possible that CTCF is responsible for this protective role in the female germline. Using a transgenic RNA interference (RNAi)-based approach to generate oocytes with reduced amounts of CTCF protein, we found increased methylation of the H19 DMD and decreased developmental competence of CTCF-deficient oocytes. These results suggest that CTCF protects the H19 DMD from de novo methylation during oocyte growth and is required for normal preimplantation development. Experiments are in progress to determine if CTCF has a wider role in the protection of CTCF-binding sites from DNA methylation and to examine the cause of developmental failure of CTCF-depleted embryos. (This work is supported in part by a grant from the National Institutes of Health.)
Effect of Suboptimal Culture on Imprinted Gene Expression
The mammalian embryo undergoes a critical set of events during preimplantation development that lead to extensive epigenetic reprogramming of its genome. Proper reprogramming is required for the successful development of the postimplantation embryo. A variety of insults, including in vitro culture of preimplantation embryos, can lead to epigenetic dysregulation, suggesting that these embryos are acutely sensitive to environmental perturbations. For example, there is extensive documentation on abnormalities that arise in sheep and cattle (large-offspring syndrome) as a consequence of embryo culture. More recently a significant increase in the frequency of imprinting disorders was reported in babies derived by assisted reproductive technology (ART). The molecular defects associated with these disorders (Beckwith-Wiedemann syndrome and Angelman syndrome) likely occur during embryo culture.
To understand the etiology of these defects, we have developed a mouse embryo culture model for loss of imprinting. When mouse embryos were cultured under standard conditions, H19 was hypomethylated and biallelically expressed. Appropriate imprinting, however, was maintained when culture conditions were optimized. We have since determined that loss of imprinting is confined to genes expressed in extraembryonic derivatives. When embryos cultured under suboptimal conditions were transferred to recipient females and harvested at 9.5 dpc, multiple imprinted genes were biallelically expressed in placental tissues. These genes were appropriately imprinted in embryonic tissues and in all tissues from embryos propagated under optimal culture conditions. Loss of imprinting observed under suboptimal culture conditions could explain at least some of the problems associated with somatic cell cloning, large-offspring syndrome, and ART. We propose that trophectodermal cells are particularly sensitive to in vitro culture. This system also allows further dissection of imprinting mechanisms. (This work is supported in part by a grant from the National Institutes of Health.)
Screen for X-Inactivation Mutations in Mice
X chromosome inactivation (XCI) is the silencing mechanism used by mammals to inactivate one X chromosome in females, thereby equalizing expression of X-linked genes in males and females. The most elusive step in the X-inactivation pathway is the initial choice of which X chromosome to inactivate. While all factors known to be involved in XCI map to the X chromosome, it is probable that unidentified autosomal factors are essential to the process. To isolate such factors, we used N-ethyl-N-nitrosourea (ENU) mutagenesis in the mouse to select for mutations that affect XCI. In collaboration with Huntington Willard (Duke University), we have recovered three independent autosomal-dominant mutations that perturb XCI patterns. Affected heterozygous females exhibit alterations in the proportion of cells expressing a given X chromosome. The observation that 6.5-day embryos are affected by the mutations suggests that we have disrupted autosomal factors that act early in the X-inactivation pathway. Such factors may regulate the choice process of XCI. These results are the first evidence of an autosomal mutation affecting any component of the XCI pathway. We have mapped mutations to chromosomes 5, 10, and 15 and are refining the locations of these mutations and characterizing the phenotypes of the mutant animals.