Biomedical subjects
Publications and source records attributed to Marisa S Bartolomei.
Genomic imprinting ensures parent-of-origin gene expression and prevents uniparental development. In this issue of Cell Stem Cell, Li et al.1 extensively engineered androgenic haploid embryonic stem cells to overcome imprinting barriers, producing adult bi-paternal mice, albeit with low efficiency, and providing insights into roles of imprinted genes in development.
Transcriptional profiling by microarray hybridization has become a standard method to analyze global gene expression and has resulted in the availability of enormous amounts of experimental data. Given the number of different microarray platforms currently in use, it is critical to determine how reproducible results are from one platform to another. Additional variability may also arise from tissue collection and protocol differences among laboratories. In an effort to identify genes whose maternal mRNA pools are critical during preimplantation development, we compared published results of three independent studies of the mouse preimplantation embryo transcriptome, each performed in a different laboratory using different microarray platforms. We searched the combined data set for genes whose expression patterns were consistent among the three experiments. Querying for presence or absence at single developmental windows indicates that between 52% and 60% of genes are in agreement among the three experiments. Searching for expression patterns across three developmental windows (oocyte + 1-cell, 2- through 8-cell, and blastocyst stage) revealed approximately 33% agreement among the three experiments, although the majority of these genes were either always present or always absent. Using this approach, we identified 51 genes with a predicted expression pattern of maternal RNA only (not present during 2-cell through 8-cell or at the blastocyst stage). RT-PCR validation indicates 37 (72%) of these candidates have the microarray-predicted expression pattern and represent candidate maternal-effect genes. Based on our analysis, we conclude that data mining microarray experiments in this way greatly enhances candidate gene expression pattern accuracy.
Imprinting at the H19/Igf2 locus depends on a differentially methylated domain (DMD) acting as a maternal-specific, methylation-sensitive insulator and a paternal-specific locus of hypermethylation. Four repeats in the DMD bind CTCF on the maternal allele and have been proposed to recruit methylation on the paternal allele. We deleted the four repeats and assayed the effects of the mutation at the endogenous locus. The H19DMD-DeltaR allele can successfully acquire methylation during spermatogenesis and silence paternal H19, indicating that these paternal-specific functions are independent of the CTCF binding sites. Maternal inheritance of the mutations leads to biallelic Igf2 expression, consistent with the loss of a functional insulator. Additionally, we uncovered two previously undescribed roles for the CTCF binding sites. On the mutant allele, H19 RNA is barely detectable in 6.5 d.p.c. embryos and 9.5 d.p.c. placenta, for the first time identifying the repeats as the elements responsible for initiating H19 transcription. Furthermore, methylation is abruptly acquired on the mutant maternal allele after implantation, a time when the embryo is undergoing genome-wide de novo methylation. Together, these experiments show that in addition to being essential for a functional insulator, the CTCF repeats facilitate initiation of H19 expression in the early embryo and are required to maintain the hypomethylated state of the entire DMD.
DNA methylation is necessary for the silencing of endogenous retrotransposons and the maintenance of monoallelic gene expression at imprinted loci and on the X chromosome. Dynamic changes in DNA methylation occur during the initial stages of primordial germ cell development; however, all consequences of this epigenetic reprogramming are not understood. DNA demethylation in postmigratory primordial germ cells coincides with erasure of genomic imprints and reactivation of the inactive X chromosome, as well as ongoing germ cell differentiation events. To investigate a possible role for DNA methylation changes in germ cell differentiation, we have studied several marker genes that initiate expression at this time. Here, we show that the postmigratory germ cell-specific genes Mvh, Dazl and Scp3 are demethylated in germ cells, but not in somatic cells. Premature loss of genomic methylation in Dnmt1 mutant embryos leads to early expression of these genes as well as GCNA1, a widely used germ cell marker. In addition, GCNA1 is ectopically expressed by somatic cells in Dnmt1 mutants. These results provide in vivo evidence that postmigratory germ cell-specific genes are silenced by DNA methylation in both premigratory germ cells and somatic cells. This is the first example of ectopic gene activation in Dnmt1 mutant mice and suggests that dynamic changes in DNA methylation regulate tissue-specific gene expression of a set of primordial germ cell-specific genes.
X chromosome inactivation (XCI) is the phenomenon through which one of the two X chromosomes in female mammals is silenced to achieve dosage compensation with males. XCI is a highly complex, tightly controlled and developmentally regulated process. The mouse undergoes two forms of XCI: imprinted, which occurs in all cells of the preimplantation embryo and in the extraembryonic lineage, and random, which occurs in somatic cells after implantation. This review presents results and hypotheses that have recently been proposed concerning important aspects of both imprinted and random XCI in mice. We focus on how imprinted XCI occurs during preimplantation development, including a brief discussion of the debate as to when silencing initiates. We also discuss regulation of random XCI, focusing on the requirement for Tsix antisense transcription through the Xist locus, on the regulation of Xist chromatin structure by Tsix and on the effect of Tsix regulatory elements on choice and counting. Finally, we review exciting new data revealing that X chromosomes co-localize during random XCI. To conclude, we highlight other aspects of X-linked gene regulation that make it a suitable model for epigenetics at work.
In early mammalian development, one of the two X chromosomes is silenced in each female cell as a result of X chromosome inactivation, the mammalian dosage compensation mechanism. In the mouse epiblast, the choice of which chromosome is inactivated is essentially random, but can be biased by alleles at the X-linked X controlling element (Xce). Although this locus was first described nearly four decades ago, the identity and precise genomic localization of Xce remains elusive. Within the X inactivation center region of the X chromosome, previous linkage disequilibrium studies comparing strains of known Xce genotypes have suggested that Xce is physically distinct from Xist, although this has not yet been established by genetic mapping or progeny testing. In this report, we used quantitative trait locus (QTL) mapping strategies to define the minimal Xce candidate interval. Subsequent analysis of recombinant chromosomes allowed for the establishment of a maximum 1.85-Mb candidate region for the Xce locus. Finally, we use QTL approaches in an effort to identify additional modifiers of the X chromosome choice, as we have previously demonstrated that choice in Xce heterozygous females is significantly influenced by genetic variation present on autosomes (Chadwick and Willard 2005). We did not identify any autosomal loci with significant associations and thus show conclusively that Xce is the only major locus to influence X inactivation patterns in the crosses analyzed. This study provides a foundation for future analyses into the genetic control of X chromosome inactivation and defines a 1.85-Mb interval encompassing all the major elements of the Xce locus.
The differentially methylated domain (DMD) of the mouse H19 gene is a methylation-sensitive insulator that blocks access of the Igf2 gene to shared enhancers on the maternal allele and inactivates H19 expression on the methylated paternal allele. By analyzing H19 DMD deletion alleles H19DeltaDMD and H19Delta3.8kb-5'H19 in pre- and postimplantation embryos, we show that the DMD exhibits positive transcriptional activity and is required for H19 expression in blastocysts and full activation of H19 during subsequent development. We also show that the DMD is required to establish Igf2 imprinting by blocking access to shared enhancers when Igf2 monoallelic expression is initiated in postimplantation embryos and that the single remaining CTCF site of the H19DeltaDMD allele is unable to provide this function. Furthermore, our data demonstrate that sequence outside of the DMD can attract some paternal-allele-specific CpG methylation 5' of H19 in preimplantation embryos, although this methylation is not maintained during postimplantation in the absence of the DMD. Finally, we report a conditional allele floxing the 1.6-kb sequence deleted from the H19DeltaDMD allele and demonstrate that the DMD is required to maintain repression of the maternal Igf2 allele and the full activity of the paternal Igf2 allele in neonatal liver.
We previously reported that the genomes of gonadal germ cells at 11.5-19.5 days postcoitum (dpc) are incompetent to support full-term development of cloned mouse embryos. In this study, we performed nuclear transfer using primordial germ cells (PGCs) from earlier stages at 8.5-10.5 dpc. When PGC nuclei at 8.5, 9.5, and 10.5 dpc were transferred into enucleated oocytes, seven cloned embryos developed into full-term offspring. Of these, five, all derived from 8.5- or 9.5-dpc PGCs, developed into healthy adults with normal fertility. Of the remaining two offspring derived from 10.5-dpc PGCs, one died shortly after birth, and the other showed slight growth retardation but subsequently developed into a fertile adult. We examined allele-specific methylation at the imprinted H19 and Snrpn loci in 9.5- to 11.5-dpc PGCs. Although the beginning of methylation erasure was evident on the H19 paternal allele at 9.5 dpc, most PGCs did not demonstrate significant erasure of paternal allele-specific methylation until 10.5 dpc. Maternal allele-specific methylation was largely erased from Snrpn by 10.5 dpc. By 11.5 dpc, the majority of PGCs showed nearly complete or complete erasure of allele-specific methylation in both H19 and Snrpn. These results demonstrate that at least some genomic imprints remain largely intact in 8.5- to 9.5-dpc PGCs and then undergo erasure at approximately 10.5 dpc as the PGCs enter the genital ridges. Thus, migrating PGCs at 8.5-9.5 dpc can be successfully used as donors for nuclear transfer, whereas gonadal PGCs at 11.5 dpc and later are incompetent to support full-term development.
Somatic cell nuclear transfer embryos exhibit extensive epigenetic abnormalities, including aberrant methylation and abnormal imprinted gene expression. In this study, a thorough analysis of X chromosome inactivation (XCI) was performed in both preimplantation and postimplantation nuclear transfer embryos. Cloned blastocysts reactivated the inactive somatic X chromosome, possibly in a gradient fashion. Analysis of XCI by Xist RNA and Eed protein localization revealed heterogeneity within cloned embryos, with some cells successfully inactivating an X chromosome and others failing to do so. Additionally, a significant proportion of cells contained more than two X chromosomes, which correlated with an increased incidence of tetraploidy. Imprinted XCI, normally found in preimplantation embryos and extraembryonic tissues, was not observed in blastocysts or placentae from later stage clones, although fetuses recapitulated the Xce effect. We conclude that, although SCNT embryos can reactivate, count, and inactivate X chromosomes, they are not able to regulate XCI consistently. These results illustrate the heterogeneity of epigenetic changes found in cloned embryos.
Epigenetics generally refers to heritable changes in gene expression that are independent of nucleotide sequence. With complete genome sequences in hand, understanding the epigenetic control of genomes is the next step towards comprehending how the same DNA sequence gives rise to different cells, lineages and organs. Epigenetics also contributes to individual variation in normal biology and in disease states. The mouse provides a unique opportunity to understand how epigenetic differences contribute to both development and disease in a tractable mammalian system. Here we discuss current approaches and protocols used to study epigenetics in the mouse, including loss-of-function studies, mutagenesis screens, somatic cell nuclear transfer, genomics and proteomics.
Prader-Willi syndrome (PWS), most notably characterized by infantile hypotonia, short stature and morbid obesity, results from deficiencies in multiple genes that are subject to genomic imprinting. The usefulness of current mouse models of PWS has been limited by postnatal lethality in affected mice. Here, we report the survival of the PWS-imprinting center (IC) deletion mice on a variety of strain backgrounds. Expression analyses of the genes affected in the PWS region suggest that while there is low-level expression from both parental alleles in PWS-IC deletion pups, this expression does not explain their survival on certain strain backgrounds. Rather, the data provide evidence for strain-specific modifier genes that support the survival of PWS-IC deletion mice.
Imprinted expression at the H19-Igf2 locus depends on a differentially methylated domain (DMD) that acts both as a maternal-specific, methylation-sensitive insulator and as a paternal-specific site of hypermethylation. Four repeats in the DMD bind CCCTC-binding factor (CTCF) on the maternal allele and have been proposed to attract methylation on the paternal allele. We introduced point mutations into the DMD to deplete the repeats of CpGs while retaining CTCF-binding and enhancer-blocking activity. Maternal inheritance of the mutations left H19 expression and Igf2 imprinting intact, consistent with the idea that the DMD acts as an insulator. Conversely, paternal inheritance of these mutations disrupted maintenance of DMD methylation, resulting in biallelic H19 expression. Furthermore, an insulator was established on the paternally inherited mutated allele in vivo, reducing Igf2 expression and resulting in a 40% reduction in size of newborn offspring. Thus, the nine CpG mutations in the DMD showed that the two parental-specific roles of the H19 DMD, methylation maintenance and insulator assembly, are antagonistic.
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 Whitten's medium, the normally silent paternal H19 allele was aberrantly expressed and undermethylated. However, only a subset of individual cultured blastocysts (approximately 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.
Imprinted genes are differentially marked during germ cell development to allow for their eventual parent-of-origin specific expression. A subset of imprinted genes becomes methylated during oocyte growth in both mouse and human. However the timing and mechanisms of methylation acquisition are unknown. Here, we examined the methylation of the Snrpn, Igf2r, Peg1 and Peg3 differentially methylated regions in postnatal growing mouse oocytes. Our findings indicate that methylation was acquired asynchronously at these different genes. Further analysis of Snrpn DMR1 revealed that parental alleles retain an epigenetic memory of their origin as the two alleles were recognized in a parental-specific manner in the absence of DNA methylation. In addition, we show that methylation acquisition was probably related to oocyte diameter and coincided with the accumulation of Dnmt3a, Dnmt3b and Dnmt3L transcripts. Methylation of the repetitive retroviral-like intracisternal A particle also occurred during this same window of oocyte growth. These findings contribute to our understanding of the epigenetic mechanisms underlying imprint acquisition during female germ cell development and have implications for the practice of assisted reproductive technologies.
The imprinted regulation of H19 and Insulin-like growth factor 2 expression involves binding of the vertebrate insulator protein, CCCTC binding factor (CTCF), to the maternally hypomethylated differentially methylated domain (DMD). How this hypomethylated state is maintained during oogenesis and the role of CTCF, if any, in this process are not understood. With the use of 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. Our results suggest that CTCF protects the H19 DMD from de novo methylation during oocyte growth and is required for normal preimplantation development.
The regulation of H19 and Igf2 imprinting and expression depends on common elements. Using comparative analysis between human and mouse, we identified conserved regions 3' of the H19 transcription unit, including the H19/Igf2 endodermal enhancers and elements within a 4.2-kb domain between the H19 transcription unit and the enhancers. Transgene experiments implicate these elements in imprinting regulation. To establish whether they are required at the endogenous locus, first we replaced the endodermal enhancers with the alpha-fetoprotein endodermal enhancers (H19Afp). Second, we deleted the 4.2-kb region (H19delta4.2). Our analysis revealed that H19 and Igf2 imprinting and tissue-specific expression were maintained for both mutations, except for a slight reduction in paternal Igf2 expression from the H19Afp allele in liver. These results demonstrate that the H19 insulator can interact with heterologous enhancers to imprint Igf2. Furthermore, for H19, chromatin context or additional sequences possibly compensate for loss of conserved 3' elements.
Germ cells undergo epigenetic modifications as they develop, which suggests that they may be ideal donors for nuclear transfer (cloning). In this study, nuclei from confirmed embryonic germ cells were used as donors to determine whether they are competent for cloning and at which stage they are most competent. Embryos cloned from migrating 10.5-days-postcoitum (dpc) primordial germ cells (PGCs) showed normal morphological development to midgestation but died shortly thereafter. In contrast, embryos cloned from later-stage germ cells were developmentally delayed at midgestation. Thus, donor germ cell age inversely correlated with the developmental stage attained by cloned embryos. The methylation status of the H19- and Snrpn-imprinting control regions in germ cell clones paralleled that of the donors, and revealed that demethylation, or erasure of imprints, was already initiated in PGCs at 10.5 dpc and was complete by 13.5 dpc. Similarly, clones derived from male 15.5-dpc germ cells showed increased methylation correlating with the initiation of de novo methylation that resets imprints at this stage, and clones from neonatal germ cells showed nearly complete methylation in the H19 imprinting control region. These results indicate that the epigenetic state of the donor nucleus is retained in cloned embryos, and that germ cells are therefore inadequate nuclear donors for cloning because they are either erasing or resetting epigenetic patterns.
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.