Preferential X-chromosome activity in human female placental tissues

Preferential inactivation of the paternally derived X chromosome in extraembryonic membranes of female rodents has been clearly demonstrated, but the mode of X-chromosome inactivation in the human placenta has not been so clearly defined. We examined A and B variants of the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) in 42 informative placentae to investigate whether the earliest differentiating human female embryonic cells exhibit preferential inactivation of the paternally inherited X. Contamination of villi with fetal blood was eliminated through culture of villous tissues, and maternal cell contamination was eliminated by careful dissection. Both fresh and cultured amnion and chorion, as well as cultured villi, revealed preferential maternal allele expression.     

The lesser known story of X-chromosome reactivation: A closer look into the reprogramming of the inactive X chromosome

X-chromosome inactivation (XCI) is an important mechanism employed by mammalian XX female cells to level X-linked gene expression with that of male XY cells. XCI occurs early in development as the pluripotent cells of the inner cell mass (ICM) in blastocysts successively differentiate into cells of all three germ layers. X-chromosome reactivation (XCR), the reversal of XCI, is critical for germ cell formation as a mechanism to diversify the X-chromosome gene pool. Here we review the characterization of XCR, and further explore its natural occurrence during development and the in vitro models of cellular reprogramming. We also review the key regulators involved in XCI for their role in suppressing the active histone marks and the genes in the active chromosome for their inhibition of X inactivation signals.Key words: X-chromosome reactivation, RNF12, reprogramming, primordial germ cells, iPS cellsX-chromosome inactivation (XCI) is an essential process occurring in female XX cells as a dosage compensation measure during development.¹ It ensures balanced X-chromosome-encoded proteins in male and female cells, and occurs randomly during early development, thus accounting for the mosaicism observed in female somatic cells. Once the cell has inactivated one of the X chromosomes, the pattern is maintained throughout the subsequent series of cell divisions. In mice, the paternal inactive X chromosome (Xi) is maintained throughout the early cleavage until the blastocyst stage, where cells of the inner cell mass (ICM) reactivate the inactive X chromosome.² At subsequent phases of early development, humans and mice share the pattern of XCI. Epiblast cells randomly inactivate one X chromosome, while the primordial germ cells (PGCs) reactivate the Xi during their migration to the genital ridges.^3–6 Interestingly, murine extra-embryonic trophoblast cells show non-random inactivation of the paternal X chromosome maintained in trophectoderm.^6,7 This pattern is, however, not conserved, as human trophectoderm cells randomly inactivate the paternal or maternal X chromosome. In addition to the PGCs and early developing embryo, cells cultured under defined conditions or undergoing reprogramming show X-chromosome reactivation (XCR).⁸ XCI has been extensively studied, while XCR is not well-understood, mainly due to the lack of easily accessible models. Here, we will review the developmental process of XCR and molecular mechanism involved in XCI and XCR.     

Cell lineage specific distribution of H3K27 trimethylation accumulation in an in vitro model for human implantation

Female mammals inactivate one of their two X-chromosomes to compensate for the difference in gene-dosage with males that have just one X-chromosome. X-chromosome inactivation is initiated by the expression of the non-coding RNA Xist, which coats the X-chromosome in cis and triggers gene silencing. In early mouse development the paternal X-chromosome is initially inactivated in all cells of cleavage stage embryos (imprinted X-inactivation) followed by reactivation of the inactivated paternal X-chromosome exclusively in the epiblast precursors of blastocysts, resulting temporarily in the presence of two active X-chromosomes in this specific lineage. Shortly thereafter, epiblast cells randomly inactivate either the maternal or the paternal X-chromosome. XCI is accompanied by the accumulation of histone 3 lysine 27 trimethylation (H3K27me3) marks on the condensed X-chromosome. It is still poorly understood how XCI is regulated during early human development. Here we have investigated lineage development and the distribution of H3K27me3 foci in human embryos derived from an in-vitro model for human implantation. In this system, embryos are co-cultured on decidualized endometrial stromal cells up to day 8, which allows the culture period to be extended for an additional two days. We demonstrate that after the co-culture period, the inner cell masses have relatively high cell numbers and that the GATA4-positive hypoblast lineage and OCT4-positive epiblast cell lineage in these embryos have segregated. H3K27me3 foci were observed in ～25% of the trophectoderm cells and in ～7.5% of the hypoblast cells, but not in epiblast cells. In contrast with day 8 embryos derived from the co-cultures, foci of H3K27me3 were not observed in embryos at day 5 of development derived from regular IVF-cultures. These findings indicate that the dynamics of H3K27me3 accumulation on the X-chromosome in human development is regulated in a lineage specific fashion.     

Paternal RLIM/Rnf12 is a survival factor for milk-producing alveolar cells

In female mouse embryos, somatic cells undergo a random form of X chromosome inactivation (XCI), whereas extraembryonic trophoblast cells in the placenta undergo imprinted XCI, silencing exclusively the paternal X chromosome. Initiation of imprinted XCI requires a functional maternal allele of the X-linked gene Rnf12, which encodes the ubiquitin ligase Rnf12/RLIM. We find that knockout (KO) of Rnf12 in female mammary glands inhibits alveolar differentiation and milk production upon pregnancy, with alveolar cells that lack RLIM undergoing apoptosis as they begin to differentiate. Genetic analyses demonstrate that these functions are mediated primarily by the paternal Rnf12 allele due to nonrandom maternal XCI in mammary epithelial cells. These results identify paternal Rnf12/RLIM as a critical survival factor for milk-producing alveolar cells and, together with population models, reveal implications of transgenerational epigenetic inheritance.     

New lessons from random X-chromosome inactivation in the mouse

X-chromosome inactivation (XCI) ensures dosage compensation in mammals. Random XCI is a process where a single X chromosome is silenced in each cell of the epiblast of mouse female embryos. Operating at the level of an entire chromosome, XCI is a major paradigm for epigenetic processes. Here we review the most recent discoveries concerning the role of long noncoding RNAs, pluripotency factors, and chromosome structure in random XCI.     

De novo DNA methylation independent establishment of maternal imprint on X chromosome in mouse oocytes

In female mouse embryos, the paternal X chromosome (Xp) is preferentially inactivated during preimplantation development and trophoblast differentiation. This imprinted X-chromosome inactivation (XCI) is partly due to an activating imprint on the maternal X chromosome (Xm), which is set during oocyte growth. However, the nature of this imprint is unknown. DNA methylation is one candidate, and therefore we examined whether disruptions of the two de novo DNA methyltransferases in growing oocytes affect imprinted XCI. We found that accumulation of histone H3 lysine-27 trimethylation, a hallmark of XCI, occurs normally on the Xp, and not on the Xm, in female blastocysts developed from the mutant oocytes. Furthermore, the allelic expression patterns of X-linked genes including Xist and Tsix were unchanged in preimplantation embryos and also in the trophoblast. These results show that a maternal disruption of the DNA methyltransferases has no effect on imprinted XCI and argue that de novo DNA methylation is dispensable for Xm imprinting. This underscores the difference between imprinted XCI and autosomal imprinting.     

Self-imposed silence: parental antagonism and the evolution of X-chromosome inactivation

A model is proposed for the evolution of X-chromosome inactivation (XCI) in which natural selection initially favors the silencing of paternally derived alleles of X-linked demand inhibitors. The compensatory upregulation of maternally derived alleles establishes a requirement for monoallelic expression in females. For this reason, XCI is self-reinforcing once established. However, inactivation of a particular X chromosome is not. Random XCI (rXCI) is favored over paternal XCI because rXCI reduces the costs of functional hemizygosity in females. Once present, rXCI favors the evolution of locus-by-locus imprinting of X-linked loci, which creates an evolutionary dynamic in which different chromosomes compete to remain active.     

The Polycomb group protein EED is dispensable for the initiation of random X-chromosome inactivation

The Polycomb group (PcG) proteins are thought to silence gene expression by modifying chromatin. The Polycomb repressive complex 2 (PRC2) plays an essential role in mammalian X-chromosome inactivation (XCI), a model system to investigate heritable gene silencing. In the mouse, two different forms of XCI occur. In the preimplantation embryo, all cells undergo imprinted inactivation of the paternal X-chromosome (Xp). During the peri-implantation period, cells destined to give rise to the embryo proper erase the imprint and randomly inactivate either the maternal X-chromosome or the Xp; extraembryonic cells, on the other hand, maintain imprinted XCI of the Xp. PRC2 proteins are enriched on the inactive-X during early stages of both imprinted and random XCI. It is therefore thought that PRC2 contributes to the initiation of XCI. Mouse embryos lacking the essential PRC2 component EED harbor defects in the maintenance of imprinted XCI in differentiating trophoblast cells. Assessment of PRC2 requirement in the initiation of XCI, however, has been hindered by the presence of maternally derived proteins in the early embryo. Here we show that Eed^−/− embryos initiate and maintain random XCI despite lacking any functional EED protein prior to the initiation of random XCI. Thus, despite being enriched on the inactive X-chromosome, PcGs appear to be dispensable for the initiation and maintenance of random XCI. These results highlight the lineage- and differentiation state–specific requirements for PcGs in XCI and argue against PcG function in the formation of the facultative heterochromatin of the inactive X-chromosome.     

A longitudinal twin study of skewed X chromosome-inactivation

X-chromosome inactivation (XCI) is a pivotal epigenetic mechanism involved in the dosage compensation of X-linked genes between males and females. In any given cell, the process of XCI in early female development is thought to be random across alleles and clonally maintained once established. Recent studies, however, suggest that XCI might not always be random and that skewed inactivation may become more prevalent with age. The factors influencing such XCI skewing and its changes over time are largely unknown. To elucidate the influence of stochastic, heritable and environmental factors in longitudinal changes in XCI, we examined X inactivation profiles in a sample of monozygotic (MZ) (n = 23) and dizygotic (DZ) (n = 22) female twin-pairs at ages 5 and 10 years. Compared to MZ twins who were highly concordant for allelic XCI ratios, DZ twins showed much lower levels of concordance. Whilst XCI patterns were moderately stable between ages 5 and 10 years, there was some drift over time with an increased prevalence of more extreme XCI skewing at age 10. To our knowledge, this study represents the earliest longitudinal assessment of skewed XCI patterns, and suggests that skewed XCI may already be established in early childhood. Our data also suggest a link between MZ twinning and the establishment of allelic XCI ratios, and demonstrate that acquired skewing in XCI after establishment is primarily mediated by stochastic mechanisms. These data have implications for our understanding about sex differences in complex disease, and the potential causes of phenotypic discordance between MZ female twins.     

Differential increase of steroid sulfatase activity in XX and XY trophoblast cells from human term placenta with syncytia formation in vitro

Steroid sulfatase (STS, EC 3.1.6.2) catalyzes the hydrolysis of the sulfate ester bonds of a variety of sulfated steroids, such as cholesterol, dehydroepiandrosterone, and estrone sulfate, a reaction influencing fertility and breast cancer in mammals. The activity of the enzyme is substantially elevated in placental syncytiotrophoblasts and is lower in other somatic cells. The polypeptide sequence of the enzyme is encoded by a gene located on the distal short arm of the human X chromosome. Prior studies have shown that the STS gene escapes X-chromosome inactivation. We studied the expression of the STS gene in primary cultures of cytotrophoblasts from human term placentae and compared it with the expression of autosomally encoded placental alkaline phosphatase (PALP) and X-linked glucose-6-phosphate dehydrogenase (G6PD). During 90 h in culture, the mononucleated cytotrophoblast cells did not proliferate, but differentiated into multinucleated, syncytiotrophoblast-like cells. STS activity in freshly isolated cytotrophoblasts was low (about 17%), compared to placental tis- sue, and about 1.7-fold higher in female than in male cells. During cultivation, STS activity increased 2- to 3-fold in female, but not in male, cells. PALP activity was very low in freshly isolated cytotrophoblasts (about 5% of placental tissue), and no significant difference between female and male cells was detectable. Within 90 h in culture, PALP activity increased in all preparations about 2- to 4-fold. G6PD activity in freshly isolated cytotrophoblasts showed great variation among preparations, and no significant difference between female and male cells was detectable. In both male and female cells the activity declined to about 50% of initial activity during cultivation. We conclude that human cytotrophoblasts in primary culture show a sex-specific regulation of STS activity, perhaps as a unique feature of the STS gene. The cytotrophoblast system may offer a new possibility to study the regulation of STS gene expression.     

X-chromosome inactivation in monkey embryos and pluripotent stem cells

Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.     

Developmental progression of Gpd expression from the inactive X chromosome of the virginia opossum

Metatherian (marsupial) mammals possess a non-random form of X-chromosome inactivation in which the paternally-derived X is always the one inactivated. To examine the progression of X-linked gene expression during metatherian development, we compared relative levels of the maternally and paternally encoded Gpd gene products in heterozygous female Virginia opossums (Didelphis virginiana) across a moior portion of the developmental period. Panels of tissues obtained from fetuses, newborns, and pouch young were examined via polyacrylamide gel electrophoresis of the G6PD protein. As in adults, G6PD phenotypes in these developmental stages were highly skewed in favor of the maternal allele product, but in some tissues there was a marked increase in paternal allele expression with advancing developmental age. However, even by 42 days of post-partum development, expression of the paternal Gpd allele had not attained the adult, tissue-specific activity pattern. Our findings indicate remarkable developmental changes in the activity of the paternal allele in several tissues/organs continuing well into mid pouch-life stages and beyond. Specifically we found that 1) a substantially repressed paternal Gpdgene is present in the cells of female stage 29 fetuses and later developmental stages, 2) the activity state of the paternal Gpd gene is not fixed during early embryonic development in this species, 3) maior changes in paternal Gpd expression occur in advanced developmental stages and comprise a maturation of the gene expression pattern during ontogeny, and 4) alterations of paternal Gpd allele activity during development occur in a tissue-specific manner. © 1995 Wiley-Liss, Inc.     

Sex chromosome inactivation in the male

Mammalian females have two X chromosomes, while males have only one X plus a Y chromosome. In order to balance X-linked gene dosage between the sexes, one X chromosome undergoes inactivation during development of female embryos. This process has been termed X-chromosome inactivation (XCI). Inactivation of the single X chromosome also occurs in the male, but is transient and is confined to the late stages of first meiotic prophase during spermatogenesis. This phenomenon has been termed meiotic sex chromosome inactivation (MSCI). A substantial portion (~15-25%) of X-linked mRNA-encoding genes escapes XCI in female somatic cells. While no mRNA genes are known to escape MSCI in males, ~80% of X-linked miRNA genes have been shown to escape this process. Recent results have led to the proposal that the RNA interference mechanism may be involved in regulating XCI in female cells. We suggest that some MSCI-escaping miRNAs may play a similar role in regulating MSCI in male germ cells.     

Heterochromatin as a factor influencing X-chromosome inactivation in hybrids of the common vole (Microtinae, Rodentia)]

Expression of X-linked genes for G6PD and alpha-GAL was studied in female interspecific hybrids of Microtus. The G6PD and alpha-GAL isozymes of Microtus arvalis were found to predominate in all cases when a species carrying a heterochromatin block on the X-chromosome served as one partner of hybridization and M. arvalis containing no heterochromatin block served as another. The proportions of G6PD and alpha-GAL parental forms were approx. equal in hybrid females when both species participating in hybridization contained heterochromatin blocks on X-chromosomes. Cytological analysis for revealing active and nonactive X-chromosomes on metaphase spreads of hybrid females supports the biochemical data. Non-random inactivation of X-chromosomes carrying the heterochromatin blocks in the interspecific hybrids with M. arvalis and a random one, when both parents contain heterochromatin blocks on the X-chromosomes are supposed to be the cause for the phenomenon observed. The study provided data supporting our previous hypothesis that heterochromatin affects the X-chromosome inactivation process in interspecific hybrid voles.     

Correlation between X-chromosome inactivation and cell differentiation in female preimplantation mouse embryos

By means of a cytological method involving BrdU incorporation and acridine orange fluorescence staining in combination with embryo manipulation, we studied X-chromosome activity in female preimplantation mouse embryos with special reference to the correlation between X-chromosome inactivation and cell differentiation. There was no sign of asynchronous replication between the two X chromosomes from the one-cell to intermediate blastocyst stage. The allocyclic X chromosome, first detected in late blastocysts, was paternal in origin, mostly replicating early in the S phase and limited to the trophectoderm. Subsequent X-chromosome inactivation occurring in the primary endoderm was also characterized by the involvement of the paternal X and early replication. Both X chromosomes continued to replicate synchronously in the embryonic ectoderm or epiblast at this stage. It was evident that overt cell differentiation preceded the appearance of the asynchronously replicating X chromosome in the trophectoderm and primary endoderm. This finding seems to support the view that cell differentiation is an important correlate of X-chromosome inactivation.     

X-inactivation and X-reactivation: epigenetic hallmarks of mammalian reproduction and pluripotent stem cells

X-chromosome inactivation is an epigenetic hallmark of mammalian development. Chromosome-wide regulation of the X-chromosome is essential in embryonic and germ cell development. In the male germline, the X-chromosome goes through meiotic sex chromosome inactivation, and the chromosome-wide silencing is maintained from meiosis into spermatids before the transmission to female embryos. In early female mouse embryos, X-inactivation is imprinted to occur on the paternal X-chromosome, representing the epigenetic programs acquired in both parental germlines. Recent advances revealed that the inactive X-chromosome in both females and males can be dissected into two elements: repeat elements versus unique coding genes. The inactive paternal X in female preimplantation embryos is reactivated in the inner cell mass of blastocysts in order to subsequently allow the random form of X-inactivation in the female embryo, by which both Xs have an equal chance of being inactivated. X-chromosome reactivation is regulated by pluripotency factors and also occurs in early female germ cells and in pluripotent stem cells, where X-reactivation is a stringent marker of naive ground state pluripotency. Here we summarize recent progress in the study of X-inactivation and X-reactivation during mammalian reproduction and development as well as in pluripotent stem cells.     

X-linked glucose-6-phosphate dehydrogenase (G6PD) and autosomal 6-phosphogluconate dehydrogenase (6PGD) polymorphisms in baboons

Electrophoretic polymorphisms of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were examined in captive colonies of five subspecies of baboons (Papio hamadryas). Phenotype frequencies and family data verified the X-linked inheritance of the G6PD polymorphism. Insufficient family data were available to confirm autosomal inheritance of the 6PGD polymorphism, but the electrophoretic patterns of variant types (putative heterozygotes) suggested the codominant expression of alleles at an autosomal locus. Implications of the G6PD polymorphism are discussed with regard to its utility as a marker system for research on X-chromosome inactivation during baboon development and for studies of clonal cell proliferation and/or cell selection during the development of atherosclerotic lesions in the baboon model.     

Histochemical differences in expression of X-linked glucose-6-phosphate dehydrogenase between ectoderm- and endoderm-derived embryonic and extra-embryonic tissues

We examined the activity of X-linked glucose-6-phosphate dehydrogenase (G6PD) in concepti of the enzyme-deficient mutant and wild-type C3H mice. By using different crosses between the G6PD-deficient homozygous, heterozygous, or wild-type females and hemizygous or wild-type males, we confirmed the inactivation of one of the two X chromosomes in female concepti by a histochemical method. With this technique, a dual (G6PD + or -) cell population could be observed in the tissue sections. We demonstrate that the paternal X chromosome is inactivated in the endoderm of parietal and visceral yolk sac and in the trophoblast, whereas in the embryo and in the yolk sac mesoderm this inactivation is random. Our results confirm biochemical observations showing that only the maternal X chromosome is expressed in all derivatives of trophectoderm and primitive endoderm, whereas derivatives of the primitive ectoderm show random X chromosome expression.