Maternal Xist Research Articles

Maternal H3K27me3-dependent autosomal and X chromosome imprinting.

Genomic imprinting and X-chromosome inactivation (XCI) are classic epigenetic phenomena that involve transcriptional silencing of one parental allele. Germline-derived differential DNA methylation is the best-studied epigenetic mark that initiates imprinting, but evidence indicates that other mechanisms exist. Recent studies have revealed that maternal trimethylation of H3 on lysine 27 (H3K27me3) mediates autosomal maternal allele-specific genesilencing and has an important role in imprinted XCI through repression of maternal Xist. Furthermore, loss of H3K27me3-mediated imprinting contributes to the developmental defects observed in cloned embryos. This novel maternal H3K27me3-mediated non-canonical imprinting mechanism further emphasizes the important role of parental chromatin in development and could provide the basis for improving the efficiency of embryo cloning.

Epigenetic signatures associated with imprinted paternally expressed genes in the Arabidopsis endosperm

BackgroundImprinted genes are epigenetically modified during gametogenesis and maintain the established epigenetic signatures after fertilization, causing parental-specific gene expression.ResultsIn this study, we show that imprinted paternally expressed genes (PEGs) in the Arabidopsis endosperm are marked by an epigenetic signature of Polycomb Repressive Complex2 (PRC2)-mediated H3K27me3 together with heterochromatic H3K9me2 and CHG methylation, which specifically mark the silenced maternal alleles of PEGs. The co-occurrence of H3K27me3 and H3K9me2 on defined loci in the endosperm drastically differs from the strict separation of both pathways in vegetative tissues, revealing tissue-specific employment of repressive epigenetic pathways in plants. Based on the presence of this epigenetic signature on maternal alleles, we are able to predict known PEGs at high accuracy and identify several new PEGs that we confirm using INTACT-based transcriptomes generated in this study.ConclusionsThe presence of the three repressive epigenetic marks, H3K27me3, H3K9me2, and CHG methylation on the maternal alleles in the endosperm serves as a specific epigenetic signature that allows prediction of genes with parental-specific gene expression. Our study reveals that there are substantially more PEGs than previously identified, indicating that paternal-specific gene expression is of higher functional relevance than currently estimated. The combined activity of PRC2-mediated H3K27me3 together with the heterochromatic H3K9me3 has also been reported to silence the maternal Xist locus in mammalian preimplantation embryos, suggesting convergent employment of both pathways during the evolution of genomic imprinting.

Manipulation of Xist Imprinting in Mouse Preimplantation Embryos.

X-chromosome inactivation (XCI) is established in preimplantation embryos in mice. Prior to the establishment of XCI, expression of the long noncoding RNA (lncRNA) Xist is essential. Xist expression in mouse preimplantation embryos is imprinted, and paternal Xist is predominantly expressed. Due to the manner of imprinting, maternal Xist is always silenced. However, the nature of imprinting to repress maternal Xist is variable. For example, parthenogenetic embryos that are composed of two maternal genomes exhibit maternal Xist derepression during the preimplantation phases. Maintenance of Xist imprinting to repress maternal Xist depends on the chromatin condensation states and the dosage of Rnf12, an essential Xist activator. Therefore, alterations of chromatin states and Rnf12 expression levels lead to maternal Xist derepression. In this chapter, we describe the method for derepressing maternal Xist by various approaches, such as mRNA injection, small molecule treatment, and nuclear transfer.

Genomic imprinting of Xist by maternal H3K27me3.

Maternal imprinting at the Xist gene is essential to achieve paternal allele-specific imprinted X-chromosome inactivation (XCI) in female mammals. However, the mechanism underlying Xist imprinting is unclear. Here we show that the Xist locus is coated with a broad H3K27me3 domain that is established during oocyte growth and persists through preimplantation development in mice. Loss of maternal H3K27me3 induces maternal Xist expression and maternal XCI in preimplantation embryos. Our study thus identifies maternal H3K27me3 as the imprinting mark of Xist.

Maintenance of Xist Imprinting Depends on Chromatin Condensation State and Rnf12 Dosage in Mice.

In female mammals, activation of Xist (X-inactive specific transcript) is essential for establishment of X chromosome inactivation. During early embryonic development in mice, paternal Xist is preferentially expressed whereas maternal Xist (Xm-Xist) is silenced. Unlike autosomal imprinted genes, Xist imprinting for Xm-Xist silencing was erased in cloned or parthenogenetic but not fertilized embryos. However, the molecular mechanism underlying the variable nature of Xm-Xist imprinting is poorly understood. Here, we revealed that Xm-Xist silencing depends on chromatin condensation states at the Xist/Tsix genomic region and on Rnf12 expression levels. In early preimplantation, chromatin decondensation via H3K9me3 loss and histone acetylation gain caused Xm-Xist derepression irrespective of embryo type. Although the presence of the paternal genome during pronuclear formation impeded Xm-Xist derepression, Xm-Xist was robustly derepressed when the maternal genome was decondensed before fertilization. Once Xm-Xist was derepressed by chromatin alterations, the derepression was stably maintained and rescued XmXpΔ lethality, indicating that loss of Xm-Xist imprinting was irreversible. In late preimplantation, Oct4 served as a chromatin opener to create transcriptional permissive states at Xm-Xist/Tsix genomic loci. In parthenogenetic embryos, Rnf12 overdose caused Xm-Xist derepression via Xm-Tsix repression; physiological Rnf12 levels were essential for Xm-Xist silencing maintenance in fertilized embryos. Thus, chromatin condensation and fine-tuning of Rnf12 dosage were crucial for Xist imprint maintenance by silencing Xm-Xist.

Round Spermatid Injection Rescues Female Lethality of a Paternally Inherited Xist Deletion in Mouse.

In mouse female preimplantation embryos, the paternal X chromosome (Xp) is silenced by imprinted X chromosome inactivation (iXCI). This requires production of the noncoding Xist RNA in cis, from the Xp. The Xist locus on the maternally inherited X chromosome (Xm) is refractory to activation due to the presence of an imprint. Paternal inheritance of an Xist deletion (XpΔXist) is embryonic lethal to female embryos, due to iXCI abolishment. Here, we circumvented the histone-to-protamine and protamine-to-histone transitions of the paternal genome, by fertilization of oocytes via injection of round spermatids (ROSI). This did not affect initiation of XCI in wild type female embryos. Surprisingly, ROSI using ΔXist round spermatids allowed survival of female embryos. This was accompanied by activation of the intact maternal Xist gene, initiated with delayed kinetics, around the morula stage, resulting in Xm silencing. Maternal Xist gene activation was not observed in ROSI-derived males. In addition, no Xist expression was detected in male and female morulas that developed from oocytes fertilized with mature ΔXist sperm. Finally, the expression of the X-encoded XCI-activator RNF12 was enhanced in both male (wild type) and female (wild type as well as XpΔXist) ROSI derived embryos, compared to in vivo fertilized embryos. Thus, high RNF12 levels may contribute to the specific activation of maternal Xist in XpΔXist female ROSI embryos, but upregulation of additional Xp derived factors and/or the specific epigenetic constitution of the round spermatid-derived Xp are expected to be more critical. These results illustrate the profound impact of a dysregulated paternal epigenome on embryo development, and we propose that mouse ROSI can be used as a model to study the effects of intergenerational inheritance of epigenetic marks.

Antagonist Xist and Tsix co-transcription during mouse oogenesis and maternal Xist expression during pre-implantation development calls into question the nature of the maternal imprint on the X chromosome

During the first divisions of the female mouse embryo, the paternal X-chromosome is coated by Xist non-coding RNA and gradually silenced. This imprinted X-inactivation principally results from the apposition, during oocyte growth, of an imprint on the X-inactivation master control region: the X-inactivation center (Xic). This maternal imprint of yet unknown nature is thought to prevent Xist upregulation from the maternal X (XM) during early female development. In order to provide further insight into the XM imprinting mechanism, we applied single-cell approaches to oocytes and pre-implantation embryos at different stages of development to analyze the expression of candidate genes within the Xic. We show that, unlike the situation pertaining in most other cellular contexts, in early-growing oocytes, Xist and Tsix sense and antisense transcription occur simultaneously from the same chromosome. Additionally, during early development, Xist appears to be transiently transcribed from the XM in some blastomeres of late 2-cell embryos concomitant with the general activation of the genome indicating that XM imprinting does not completely suppress maternal Xist transcription during embryo cleavage stages. These unexpected transcriptional regulations of the Xist locus call for a re-evaluation of the early functioning of the maternal imprint on the X-chromosome and suggest that Xist/Tsix antagonist transcriptional activities may participate in imprinting the maternal locus as described at other loci subject to parental imprinting.

Chromatin condensation of Xist genomic loci during oogenesis in mice

Repression of maternal Xist (Xm-Xist) during preimplantation in mouse embryos is essential for establishing imprinted X chromosome inactivation. Nuclear transplantation (NT) studies using nuclei derived from non-growing (ng) and full-grown (fg) oocytes have indicated that maternal-specific repressive modifications are imposed on Xm-Xist during oogenesis, as well as on autosomal imprinted genes. Recent studies have revealed that histone H3 lysine 9 trimethylation (H3K9me3) enrichments on Xm-Xist promoter regions are involved in silencing at the preimplantation stages. However, whether H3K9me3 is imposed on Xm-Xist during oogenesis is not known. Here, we dissected the chromatin states in ng and fg oocytes and early preimplantation stage embryos. Chromatin immunoprecipitation experiments against H3K9me3 revealed that there was no significant enrichment within the Xm-Xist region during oogenesis. However, NT embryos with ng nuclei (ngNT) showed extensive Xm-Xist derepression and H3K9me3 hypomethylation of the promoter region at the 4-cell stage, which corresponds to the onset of paternal Xist expression. We also found that the chromatin state at the Xist genomic locus became markedly condensed as oocyte growth proceeded. Although the condensed Xm-Xist genomic locus relaxed during early preimplantation phases, the extent of the relaxation across Xm-Xist loci derived from normally developed oocytes was significantly smaller than those of paternal-Xist and ngNT-Xist genomic loci. Furthermore, Xm-Xist from 2-cell metaphase nuclei became derepressed following NT. We propose that chromatin condensation is associated with imprinted Xist repression and that skipping of the condensation step by NT leads to Xist activation during the early preimplantation phase.

A new Xist allele driven by a constitutively active promoter is dominated by Xist locus environment and exhibits the parent-of-origin effects.

The dosage difference of X-linked genes between the sexes in mammals is compensated for by genetic inactivation of one of the X chromosomes in XX females. A noncoding RNA transcribed from the Xist gene at the onset of X chromosome inactivation coats the X chromosome in cis and induces chromosome-wide heterochromatinization. Here, we report a new Xist allele (Xist(CAG)) driven by a CAG promoter, which is known to be constitutively active in many types of cells. The paternal transmission of Xist(CAG) resulted in the preferential inactivation of the targeted paternal X (Xp) not only in the extra-embryonic but also the embryonic lineage, whereas maternal transmission ended with embryonic lethality at the early postimplantation stage with a phenotype that resembled mutant embryos carrying a maternal deficiency in Tsix, an antisense negative regulator of Xist, in both sexes. Interestingly, we found that the upregulation of Xist(CAG) in preimplantation embryos temporally differed depending on its parental origin: its expression started at the 4- to 8-cell stages when paternally inherited, and Xist(CAG) was upregulated at the blastocyst stage when maternally inherited. This might indicate that the Xist locus on Xp is permissive to transcription, but the Xist locus on the maternal X (Xm) is not. We extrapolated from these findings that the maternal Xist allele might manifest a chromatin structure inaccessible by transcription factors relative to the paternal allele. This might underlie the mechanism for the maternal repression of Xist at the early cleavage stage when Tsix expression has not yet occurred on Xm.

The role of maternal-specific H3K9me3 modification in establishing imprinted X-chromosome inactivation and embryogenesis in mice.

Maintaining a single active X-chromosome by repressing Xist is crucial for embryonic development in mice. Although the Xist activator RNF12/RLIM is present as a maternal factor, maternal Xist (Xm-Xist) is repressed during preimplantation phases to establish imprinted X-chromosome inactivation (XCI). Here we show, using a highly reproducible chromatin immunoprecipitation method that facilitates chromatin analysis of preimplantation embryos, that H3K9me3 is enriched at the Xist promoter region, preventing Xm-Xist activation by RNF12. The high levels of H3K9me3 at the Xist promoter region are lost in embryonic stem (ES) cells, and ES-cloned embryos show RNF12-dependent Xist expression. Moreover, lack of Xm-XCI in the trophectoderm, rather than loss of paternally expressed imprinted genes, is the primary cause of embryonic lethality in 70–80% of parthenogenotes immediately after implantation. This study reveals that H3K9me3 is involved in the imprinting that silences Xm-Xist. Our findings highlight the role of maternal-specific H3K9me3 modification in embryo development.

Tsix-deficient X chromosome does not undergo inactivation in the embryonic lineage in males: implications for Tsix-independent silencing of Xist

Differential induction of the X-linked non-coding Xist gene is a key event in the process of X inactivation occurring in female mammalian embryos. Xist is negatively regulated in cis by its antisense gene Tsix through modification of the chromatin structure. The maternal Xist allele, which is normally silent in the extraembryonic lineages, is ectopically activated when Tsix is disrupted on the same chromosome, and subsequently the maternal X chromosome undergoes inactivation in the extraembryonic lineages even in males. However, it is still unknown whether the single Tsix-deficient X chromosome (X^Δ^Tsix) in males is also inactivated in the embryonic lineage. Here, we show that both male and female embryos carrying a maternally derived X^Δ^Tsix could survive if the extraembryonic tissues were complemented by wild-type tetraploid cells. In addition, Xist on the X^Δ^Tsix was properly silenced and methylated at CpG sites in adult male somatic cells. These results indicate that the embryonic lethality caused by the maternal X^Δ^Tsix is solely attributable to the defects in the extraembryonic lineages. X^Δ^Tsix does not seem to undergo inactivation in the embryonic lineage in males, suggesting the presence of a Tsix-independent silencing mechanism for Xist in the embryonic lineage.

35ANALYSIS OF EPIGENETIC MODIFICATIONS AND GENOMIC IMPRINTING IN NUCLEAR TRANSFER DERIVED BOS GAURUS×B. TAURUS CONCEPTI

Somatic cell nuclear transfer in cattle is an inefficient process hindered by low pregnancy rates and fetal placental abnormalities. Improper or incomplete epigenetic reprogramming of the donor genome has been implicated as a cause for these aberrations and has been investigated extensively in mice. Here we report the use of a bovine interspecies model (Bos gaurus×B. taurus) for the assessment and characterization of epigenetic modifications and genomic imprinting in 40-day-old female nuclear transfer (NT)-derived fetuses and placentas. Previously, we identified genomic imprinting at the IGF2, GTL2 and XIST loci in the Bos gaurus×B. taurus fetuses. These results indicated maternal and paternal imprinting of the IGF2 and GTL2 loci, respectively, in the chorion, allantois, liver, lung and brain, whereas the XIST locus was maternally imprinted solely in the chorion of females. We extended this analysis to 40-day-old NT fetuses derived from a hybrid lung fibroblast cell line (female). Analysis of the donor cell line indicated conservation of imprinting of the IGF2 and GTL2 loci and bialleic expression of the XIST locus, presumably from the random patterns of X-chromosome inactivation. Analysis of three NT and three control pregnancies indicated disruption of genomic imprinting at the XIST locus in the chorions of all three clones compared to controls. In contrast, proper allelic expression of the IGF2 and GTL2 loci was observed in all fetuses and placentas. Quantification of maternal and paternal XIST transcripts in the chorion of clones and controls demonstrated a significant skewing from preferential paternal expression in controls (95.0±0.882, mean±S.E.) to mixed paternal and maternal expression in clones (73.6±5.2), (t-test; P<0.05). In an attempt to determine the cause for the abnormal allelic expression of the XIST locus in the chorion of the clones, methylation analysis of the XIST Differentially Methylated Region (DMR) was performed. Methylation-sensitive restriction digests and subsequent PCR of the XIST DMR indicated patterns were not different between controls and clones. However, when genome-wide and promoter-specific methylation analysis (bisulfite sequencing) was extended to the satellite I repeat element and epidermal cytokeratin promoter, hypermethylation was observed in the chorion of clones. These results demonstrate disruption of genomic imprinting in XIST locus in the placenta of 40-day-old clones independent of DMR methylation. They also indicate that cloning is associated with increased levels of methylation in selected genomic regions in the chorion.

Species Differences in TSIX/Tsix Reveal the Roles of These Genes in X-Chromosome Inactivation

Transcriptional silencing of the human inactive X chromosome is induced by the XIST gene within the human X-inactivation center. The XIST allele must be turned off on one X chromosome to maintain its activity in cells of both sexes. In the mouse placenta, where X inactivation is imprinted (the paternal X chromosome is always inactive), the maternal Xist allele is repressed by a cis-acting antisense transcript, encoded by the Tsix gene. However, it remains to be seen whether this antisense transcript protects the future active X chromosome during random inactivation in the embryo proper. We recently identified the human TSIX gene and showed that it lacks key regulatory elements needed for the imprinting function of murine Tsix. Now, using RNA FISH for cellular localization of transcripts in human fetal cells, we show that human TSIX antisense transcripts are unable to repress XIST. In fact, TSIX is transcribed only from the inactive X chromosome and is coexpressed with XIST. Also, TSIX is not maternally imprinted in placental tissues, and its transcription persists in placental and fetal tissues, throughout embryogenesis. Therefore, the repression of Xist by mouse Tsix has no counterpart in humans, and TSIX is not the gene that protects the active X chromosome from random inactivation. Because human TSIX cannot imprint X inactivation in the placenta, it serves as a mutant for mouse Tsix, providing insights into features responsible for antisense activity in imprinted X inactivation.

Control of Xist expression for imprinted and random X chromosome inactivation in mice.

Applying RNA fluorescence in situ hybridization to parthenogenetic embryos with two maternally derived X (X(M)) chromosomes and embryos with X chromosome aneuploidy such as X(P)0 (X(P), paternally derived X chromosome), X(M)X(M)X(P) and X(M)X(M)Y, we studied the control of Xist/Tsix expression for silencing the entire X chromosome in mice. The data show that the paternally derived Xist allele is highly expressed in every cell of the embryo from the 4-cell stage onward, irrespective of the number of X chromosomes in a diploid cell. The high level of Xist transcription is maintained in non-epiblast cells culminating in X(P)-inactivation, whereas in X(P)0 embryos it is terminated by the blastocyst stage, probably as a result of counting the number of X chromosomes in a cell occurring at the morula/blastocyst stage. Xist is also down-regulated in epiblast cells of X(M)X(P) and X(M)X(M)X(P) embryos to make X-inactivation random. In epiblast cells, Xist seems to be up-regulated after counting and random choice of the future inactive X chromosome(s). Although the maternal Xist allele is never activated in fertilized embryos before implantation, some parthenogenetic embryos show Xist up-regulation in a proportion of cells. These and other data reported earlier suggest that imprinted X-inactivation in non-epiblast tissues of rodents had been derived from the random X-inactivation system.

X chromosome imprinting and inactivation in the early mammalian embryo

Quantitative differences in X-linked gene expression between androgenetic (two paternal genomes), gynogenetic (two maternal genomes) and normal embryos provide clues into the roles of genomic imprinting and the X:autosome ratio in controlling X chromosome function during development. These data and many others can be accounted for by a new model of X-chromosome-inactivation (XCI). Expression of the Xist RNA from all paternal X chromosomes during development preimplantation leads to repression of genes near the X-chromosome-inactivation center (Xic). Other genes are repressed as a result of spreading of the inactivation, but only in embryos with at least two X chromosomes. XY androgenones are only deficient in expression of genes near the Xic and can form blastocysts, whereas XX androgenones completely inactivate both X chromosomes and die before the blastocyst stage. The X:autosome ratio regulates XCI solely by promoting the spread of inactivation away from the Xic on chromosomes that express Xist. Methylation of the maternal Xist gene is retained in extraembryonic tissues, so that gynogenones and parthenogenones cannot express Xist, do not undergo XCI in those tissues, and so have extraembryonic defects. This model should be relevant to understanding how aberrant X chromosome regulation might occur and how this might contribute to distortion of the X-chromosome-transmission ratio, sex ratio distortion, and disease.