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ABSTRACT: The ribonuclease protection assay (RPA) is a sensitive technique for the analysis of total cellular RNA. It involves generating
a specific antisense riboprobe, hybridizing the probe to total RNA, removing unprotected RNA by RNases, and finally isolating
and analyzing the protected RNA on a denaturing gel. Although the RPA is somewhat more labor-intensive than Northern analysis,
it has the advantage of being more sensitive (as little as 0.1 pg of target RNA can be detected with ideal hybridization conditions).
RPAs are also more tolerant of partially degraded RNA (provided the area that is protected is intact). Although RPAs are not
as sensitive as polymerase chain reaction (PCR)-based RNA analyses, the target RNA is analyzed directly; a reverse transcription
step is not required. Finally, the RPA is quantitative as long as the probe is in excess. More important for the study of
imprinted genes, the RPA can be designed to detect allele-specific expression of the target gene of interest.
02/2008: pages 133-144;
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ABSTRACT: In vitro culture of mouse embryos results in loss of imprinting. The aim of the present study was to examine how two of the techniques commonly used during assisted reproduction, namely embryo culture and embryo transfer, affect genomic imprinting after implantation in the mouse. F1 hybrid mouse embryos were subjected to three experimental conditions: control (unmanipulated), embryo transfer and in-vitro-culture followed by embryo transfer. Concepti were collected on d9.5 of development and allelic expression determination of ten imprinted genes (H19, Snrpn, Igf2, Kcnq1ot1, Cdkn1c, Kcnq1, Mknr3, Ascl2, Zim1, Peg3) was performed. Although control concepti had monoallelic imprinted gene expression in all tissues, both manipulated groups had aberrant expression of one or more imprinted genes in the yolk sac and placenta. Culture further exacerbated the effects of transfer by increasing the number of genes with aberrant allelic expression in extraembryonic, as well as embryonic tissues. Additionally, placentae of both groups of manipulated concepti exhibited reduced levels of Igf2 mRNA and increased levels of Ascl2 mRNA when compared with their unmanipulated counterparts. Furthermore, we show that biallelic expression of Kcnq1ot1 coincided with loss of methylation on the maternal allele of the KvDMR1 locus, a phenotype often associated with the human syndrome Beckwith-Wiedemann. In conclusion, our results show that even the most basic manipulation used during human-assisted reproduction, namely, embryo transfer, can lead to misexpression of several imprinted genes during post-implantation development. Additionally, our results serve as a cautionary tale for gene expression studies in which embryo transfer is used.
Human Molecular Genetics 02/2008; 17(1):1-14. · 7.64 Impact Factor
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ABSTRACT: Genomic imprinting governs allele-specific gene expression in an epigenetically heritable manner. The characterization of histone modifications at imprinted gene loci is incomplete, and whether specific histone marks determine transcription or are dependent on it is not understood. Using chromatin immunoprecipitations, we examined in multiple cell types and in an allele-specific manner the active and repressive histone marks of several imprinted loci, including H19, KvDMR1, Snrpn promoter/exon 1, and IG-DMR imprinting control regions. Expressed alleles are enriched for specific actively modified histones, including H3 di- and trimethylated at Lys4 and acetylated histones H3 and H4, while their silent counterparts are associated with repressive marks such as H3 trimethylated at Lys9 alone or in combination with H3 trimethylated at Lys27 and H4/H2A symmetrically dimethylated at Arg3. At H19, allele-specific histone modifications occur throughout the entire locus, including nontranscribed regions such as the differentially methylated domain (DMD) as well as sequences in the H19 gene body that are not differentially methylated. Significantly, the presence of active marks at H19 depends on transcriptional activity and occurs even in the absence of the DMD. These findings suggest that histone modifications are dependent on the transcriptional status of imprinted alleles and illuminate epigenetic mechanisms of genomic imprinting.
Molecular and cellular biology 02/2008; 28(1):71-82. · 6.06 Impact Factor
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ABSTRACT: Genomic imprinting is an epigenetic mechanism of transcriptional regulation through which expression of a subset of mammalian genes is restricted to one parental allele. An intriguing characteristic of imprinted genes is that they often cluster in megabase-sized chromosomal domains, indicating that domain-specific mechanisms regulate imprinting. Detailed study of the known imprinted domains has revealed a number of common characteristics. First, all clusters have an imprinting control region (ICR) that is typically 1-5 kb in size and differentially methylated, and that regulates imprinting across the entire domain. Second, the clusters have at least one noncoding RNA (ncRNA) that is usually expressed from the maternal allele and multiple paternally expressed protein-coding genes. Finally, the clusters are likely regulated by one of two mechanisms, transcription of a long ncRNA that silences expression of protein-coding genes bidirectionally in cis and blocking of shared enhancer elements by CCCTC binding factor (CTCF) binding insulators. More recent experiments may even suggest that both mechanisms operate at some clusters. In this chapter, we will describe what is known about imprinting at five well-studied imprinted loci and highlight some of the critical experiments that are required before a full understanding of imprinting mechanisms is achieved.
Advances in genetics 02/2008; 61:207-23. · 3.39 Impact Factor
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Cell 10/2007; 130(5):958. · 32.40 Impact Factor
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ABSTRACT: Paternal repression of the imprinted H19 gene is mediated by a differentially methylated domain (DMD) that is essential to imprinting of both H19 and the linked and oppositely imprinted Igf2 gene. The mechanisms by which paternal-specific methylation of the DMD survive the period of genome-wide demethylation in the early embryo and are subsequently used to govern imprinted expression are not known. Methyl-CpG binding (MBD) proteins are likely candidates to explain how these DMDs are recognized to silence the locus, because they preferentially bind methylated DNA and recruit repression complexes with histone deacetylase activity. MBD RNA and protein are found in preimplantation embryos, and chromatin immunoprecipitation shows that MBD3 is bound to the H19 DMD. To test a role for MBDs in imprinting, two independent RNAi-based strategies were used to deplete MBD3 in early mouse embryos, with the same results. In RNAi-treated blastocysts, paternal H19 expression was activated, supporting the hypothesis that MBD3, which is also a member of the Mi-2/NuRD complex, is required to repress the paternal H19 allele. RNAi-treated blastocysts also have reduced levels of the Mi-2/NuRD complex protein MTA-2, which suggests a role for the Mi-2/NuRD repressive complex in paternal-specific silencing at the H19 locus. Furthermore, DNA methylation was reduced at the H19 DMD when MBD3 protein was depleted. In contrast, expression and DNA methylation were not disrupted in preimplantation embryos for other imprinted genes. These results demonstrate new roles for MBD3 in maintaining imprinting control region DNA methylation and silencing the paternal H19 allele. Finally, MBD3-depleted preimplantation embryos have reduced cell numbers, suggesting a role for MBD3 in cell division.
PLoS Genetics 09/2007; 3(8):e137. · 8.69 Impact Factor
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ABSTRACT: 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.
Developmental Biology 11/2006; 298(2):344-53. · 4.07 Impact Factor
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ABSTRACT: 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.
Human Molecular Genetics 11/2006; 15(19):2945-54. · 7.64 Impact Factor
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ABSTRACT: 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.
Development 10/2006; 133(17):3411-8. · 6.60 Impact Factor
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ABSTRACT: 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.
Mammalian Genome 10/2006; 17(9):941-9. · 2.89 Impact Factor
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ABSTRACT: 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.
Genetics 09/2006; 173(4):2103-10. · 4.01 Impact Factor
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ABSTRACT: 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.
Molecular and Cellular Biology 03/2006; 26(4):1245-58. · 5.53 Impact Factor
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ABSTRACT: 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.
Nature Genetics 12/2005; 37(11):1194-200. · 35.53 Impact Factor
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ABSTRACT: 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.
Proceedings of the National Academy of Sciences 09/2005; 102(32):11361-6. · 9.68 Impact Factor
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ABSTRACT: 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.
Developmental Biology 04/2005; 279(2):525-40. · 4.07 Impact Factor
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ABSTRACT: 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.
Human Molecular Genetics 01/2005; 13(23):2971-7. · 7.64 Impact Factor
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ABSTRACT: 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.
Development 09/2004; 131(15):3727-35. · 6.60 Impact Factor
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ABSTRACT: 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.
Nature Genetics 09/2004; 36(8):883-8. · 35.53 Impact Factor
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ABSTRACT: 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.
Genomics 08/2004; 84(1):59-68. · 3.02 Impact Factor
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ABSTRACT: 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.
Human Molecular Genetics 05/2004; 13(8):839-49. · 7.64 Impact Factor
