Spontaneous reactivation of the inactive X chromosome in mouse embryonal carcinoma cells

The mouse embryonal carcinoma cell line MC12 carries two X chromosomes, one of which replicates late in S phase and shares properties with the normal inactive X chromosome and, therefore, is considered to be inactivated. Since the hypoxanthine phosphoribosyl transferase (HPRT) gene on the active X chromosome is mutated (HPRT(NDASH;)), MC12 cells lack HPRT activity. After subjecting MC12 cells to selection in HAT medium, however, a number of HAT-resistant clones (HAT(R)) appeared. The high frequency of HAT resistance (3.18 x 10(-4)) suggested reactivation of HPRT(PLUS;) on the inactive X chromosome rather than reversion of HPRT(NDASH;). Consistent with this view, cytological analyses showed that the reactivation occurred over the length of the inactive X chromosome in 11 of 20 HAT(R) clones isolated. The remaining nine clones retained a normal heterochromatic inactive X chromosome. The spontaneous reactivation rate of the HPRT(PLUS;) on the inactive X chromosome was relatively high (1.34 x 10(-6)) and comparable to that observed for XIST-deleted somatic cells (Csankovszki et al., 2001), suggesting that the inactivated state is poorly maintained in MC12 cells.     

Polymorphic X-chromosome inactivation of the human TIMP1 gene.

X inactivation silences most but not all of the genes on one of the two X chromosomes in mammalian females. The human X chromosome preserves its activation status when isolated in rodent/human somatic-cell hybrids, and hybrids retaining either the active or inactive X chromosome have been used to assess the inactivation status of many X-linked genes. Surprisingly, the X-linked gene for human tissue inhibitor of metalloproteinases (TIMP1) is expressed in some but not all inactive X-containing somatic-cell hybrids, suggesting that this gene is either prone to reactivation or variable in its inactivation. Since many genes that escape X inactivation are clustered, we examined the expression of four genes (ARAF1, ELK1, ZNF41, and ZNF157) within approximately 100 kb of TIMP1. All four genes were expressed only from the active X chromosome, demonstrating that the factors allowing TIMP1 expression from the inactive X chromosome are specific to the TIMP1 gene. To determine if this variable inactivation of TIMP1 is a function of the hybrid-cell environment or also is observed in human cells, we developed an allele-specific assay to assess TIMP1 expression in human females. Expression of two alleles was detected in some female cells with previously demonstrated extreme skewing of X inactivation, indicating TIMP1 expression from the inactive chromosome. However, in other cells, no expression of TIMP1 was observed from the inactive X chromosome, suggesting that TIMP1 inactivation is polymorphic in human females.     

Stability of the "two active X" phenotype in triploid somatic cells.

We examined triploid cells of XXY karyotype heterozygous for glucose 6 phosphate dehydrogenase (G6PD) electrophoretic variants with regard to the stability of their X chromosome phenotype. Clonal populations of cells derived from these human fibroblasts maintained a precise 1:2:1 ratio of A:heteropolymer:B isozymes throughout their life span, indicating stability of the two active X chromosomes in these cells. To determine the influence of the autosomal complement on X chromosome expression, we attempted to perturb the relationship. Fusion of these triploid cells with human diploid fibroblasts carrying a novel G6PD variant (B') resulted in heterokaryons exprssing a novel heteropolymer, presumably indicating that all three parental X chromosomes were active. However, no derepression of the inactive X chromosome was observed. Analysis of interspecific hybrids derived from triploid cells and mouse fibroblasts confirmed that activity of parental X chromosomes is maintained. Some human mouse hybrid clones, however, expressed only a single human G6PD isozyme, probably attributable to segregation of the pertinent X chromosome, but elimination of a relevant autosome cannot be excluded. The triploid cells transformed by SV40 showed alterations in LDH pattern and an approximately 10-20% decrease in chromosome number, but maintained the precise G6PD phenotype of the untransformed cell. These studies provide evidence for the stability of the X chromosome phenotype in triploid cells.     

Reversal of X-inactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in vitro

A series of near-diploid embryonal carcinoma-like hybrid cells were obtained from polyethylene glycol mediated cell fusion between murine embryonal carcinoma cells (PSA-6TG1 or OTF9-63) having one X chromosome and thymocytes or bone marrow cells from female mice carrying Cattanach's or Searle's translocation. Prior to fusion with EC cells the somatic cells are presumed to contain only one active X chromosome. Following hybrid formation, the chronology of X chromosome replication and the expression of X-linked gene Pgk-1 indicated that all X chromosomes contributed by both parents were active in these hybrids. Experiments were performed to rule out the possibility that the hybrids were formed by fusion of EC cells with rare somatic cells in which both X chromosomes were active. Taken together the data indicate that within four days of fusion there is reactivation of the entire inactive X chromosome.     

The late-replicating X chromosome in digynous mouse triploid embryos

Using BrdU-labeling and acridine orange staining, the behavior of X-chromosome replication was studied in 28 XXX and 19 XXY digynous mouse triploids. In some of these the paternal and maternal X chromosome could by cytologically distinguished. Such embryos were obtained by mating chromosomally normal females with males carrying Cattanach's X chromosome which contains an autosomal insertion that substantially increases the length of this chromosome. In the XXX triploids there were two distinct cell lines, one with two late-replicating X chromosomes, and the other with only one late-replicating X. The XXY triploids were also composed of two cell populations, one with a single late-replicating X and the other with no late replicating X chromosome. Assuming that the late-replicating X is genetically inactive, in both XXX and XXY triploids, cells from the embryonic region tended to have only one active X chromosome, whereas those from the extra-embryonic membranes tended to have two active X chromosomes. The single active X chromosome was either paternal or maternal in origin, but two active X chromosomes were overwhelmingly maternal in origin, suggesting paternal X-inactivation in extra-embryonic tissues.     

DNase I sensitivity of Microtus agrestis active,inactive and reactivated X chromosomes in mouse-Microtus cell hybrids

We isolated Microtus agrestis-mouse somatic cell hybrid clones which had retained either the active or the inactive M. agrestis X chromosome. In both hybrid clones the X chromosomes retained their original chromatin conformation as studied by the in situ nick translation technique — the active X chromosome retained its high sensitivity to DNase I while the inactive one remained insensitive. A clone in which the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene had been spontaneously reactivated was isolated from the hybrid containing the inactive X chromosome. The in situ nick translation technique was used to study possible DNA conformation changes in the euchromatin of the inactive X chromosome with special reference to the reactivated HPRT locus. We found that the euchromatin in this X chromosome exhibited the same low sensitivity to DNase I as is characteristic of the inactive X chromosome.Professor Marcus passed away on 2 January 1987     

Frequency of reactivation and variability in expression of X-linked enzyme loci.

A mouse-human cell hybrid clone retaining an inactive human X chromosome was treated with 5-azacytidine. Following treatment, expression of the X-linked enzyme markers, hypoxanthine-guanine phosphoribosyltransferase (HPRT), glucose-6-phosphate dehydrogenase (G6PD), phosphoglycerate kinase (PGK), and alpha-galactosidase A (GLA) was examined. Results presented here show that 45 of the 62 clones positive for human HPRT expressed human GLA, while only four of 68 clones negative for human HPRT expressed human GLA. These results strongly suggest that there is coordinate reactivation of GLA and HPRT. Reactivated expression of G6PD was studied in detail. The studies show that 5-azacytidine can induce heritable changes in the inactive human X chromosome resulting in the expression of G6PD activity at a level lower than that from an active human X chromosome.     

Variable X chromosome inactivation patterns in near-tetraploid murine EC × somatic cell hybrid cells differentiatedin vitro

For the cytogenetic study of X chromosome inactivation as an X chromosome dosage compensation mechanism, we isolated a number of XXXX, XXX, and XXY near-tetraploid mouse hybrid cell clones by fusing XX or XO embryonal carcinoma cells with lymphocytes carrying a structurally altered X chromosome(s). The inactive X chromosome from the female lymphocyte was reactivated in these hybrid clones which retained embryonal carcinoma morphology so far as they were cultured on the collagen-coated plastic surface in the medium supplemented with leukemia inhibitory factor (LIF) and betamercaptoethanol (BME). Some of these clones developed balloon-like cystic embryoid bodies when they were allowed to form cell aggregates in medium without LIF and BME in bacteriological petri dishes to which they do not adhere. X chromosome inactivation occurring during this process detected by the incorporation of 5-bromodeoxyuridine did not conform to the expected pattern leaving two X chromosomes active in every tetraploid cells. This may suggest either that the X-inactivation mechanism evolved primarily, for the diploid cell is unable to deal with tetraploid conditions efficiently, or that the present system ofin vitro differentiation represents an anomalous situation never encounteredin vivo.     

Studies on the activity of the X chromosomes in female teratocarcinoma cells in culture

Embryonal carcinoma cells derived from murine teratocarcinomas are able to differentiate into the same variety of tissue types as early embryonic cells. Because embryonal carcinoma cells resemble those of the embryo at a stage before X chromosome inactivation has occurred in females embyronal carcinoma cells containing two X chromosomes were examined to determine whether both are genetically active. The specific activities of X-linked enzymes were measured in embryonal carcinoma cells containing either one or two X chromosomes. The activities in both cell types were similar, suggesting that only one X chromosome was active in the female cells. Further support for this conclusion came from experiments in which azaguanine-resistant mutants were recovered with similar frequencies from embryonal carcinoma cell lines containing one and two X chromosomes. Late replication of an X chromosome DNA was detected in one embryonal carcinoma cell line with two X chromosomes but not in another. This suggests that cells of these two lines were arrested at different developmental stages, and that late DNA replication may not be a necessary adjunct of X inactivation. Evidence is presented which suggests that X chromosome reactivation does not occur during differentiation of the cells in vitro.     

Manifestation of the fragile site Xq27 in fibroblasts

Summary Fibroblasts from a heterozygous carrier for the Martin-Bell syndrome, who manifests the fragile site Xq27, were cloned to separate the population carrying the primary defect on the active X chromosome from the population with this defect on the inactive X. Clones with this defect on the active X manifest the fra(X)(q27) whereas clones from the other population are fra(X)-negative (Steinbach et al. 1983b). In this project, the replication status of the X chromosome manifesting the fra(X)(q27) was studied in seven clones with this defect on the active X.The results obtained on the clones were very similar to the results obtained from uncloned fibroblasts and lymphocytes. In the clones the fragile site was found manifested on the early replicating X in 73 cells and on the late replicating X in four cells.Since the defect is located on the active X chromosome of these cells the manifestation of the fragile site on the late replicating X suggests that the defect and the fragile site cannot be identical. It is concluded that there is no obligate synteny of this defect and the manifested fragile site.     

Human X chromosomes: synchrony of DNA replication in diploid and triploid fibroblasts with multiple active or inactive X chromosomes

We have analyzed patterns of DNA replication in X chromosomes from diploid cultured human fibroblasts and from three triploid 69,XXY fibroblast strains, using BrdU--33258 Hoechst--Giemsa techniques. Both X chromosomes in each of these Barr body-negative triploid strains were early-replicating. The results of gene dosage studies using (1) a histochemical stain to measure X-linked glucose-6-phosphate dehydrogenase (G6PD) activity in single cells and (2) cellulose acetate electrophoresis of G6PD activity in cell extracts also indicated that both Xs in these strains were genetically active. When we compared the synchrony of X chromosome DNA replication kinetics both between cells and within cells containing multiple inactive Xs, a marked variability and asynchrony was observed for late-replicating X chromosomes. In a culture of 47,XXX fibroblasts administered an 8-h terminal pulse of dT after growth in BrdU-containing medium, asynchrony was detected between the two late-replicating Xs in approximately 70% of cells examined. No such asynchrony was observed between the two early-replicating Xs in similarly cultured 69,XXY cells; in the triploid strains, the two Xs were distinguished by asynchronous replication in only approximately 15% of cells. The striking variability in late X chromosome replication kinetics appears, then, to be a property unique to inactive Xs and is not inherent to all X chromosomes.     

X chromosomes alternate between two states prior to random X-inactivation

Early in the development of female mammals, one of the two X chromosomes is silenced in half of cells and the other X chromosome is silenced in the remaining half. The basis of this apparent randomness is not understood. We show that before X-inactivation, the two X chromosomes appear to exist in distinct states that correspond to their fates as the active and inactive X chromosomes. Xist and Tsix, noncoding RNAs that control X chromosome fates upon X-inactivation, also determine the states of the X chromosomes prior to X-inactivation. In wild-type ES cells, X chromosomes switch between states; among the progeny of a single cell, a given X chromosome exhibits each state with equal frequency. We propose a model in which the concerted switching of homologous X chromosomes between mutually exclusive future active and future inactive states provides the basis for the apparently random silencing of one X chromosome in female cells.     

Repressive but not activating epigenetic modifications are aberrant on the inactive X chromosome in live cloned cattle

X inactivation is the process of a chromosome-wide silencing of the majority of genes on the X chromosome during early mammalian development. This process may be aberrant in cloned animals. Here we show that repressive modifications, such as methylation of DNA, and the presence of methylated histones, H3K9me2 and H3K27me3, exhibit distinct aberrance on the inactive X chromosome in live clones. In contrast, H3K4me3, an active gene marker, is obviously missing from the inactive X chromosome in all cattle studied. This suggests that the disappearance of active histone modifications (H3K4me3) seems to be more important for X inactivation than deposition of marks associated with heterochromatin (DNA methylation, H3K27me3 and H3K9me2). It also implies that even apparently normal clones may have subtle abnormalities in repressive, but not activating epigenetic modifications on the inactive X when they survive to term. We also found that the histone H3 methylations were enriched and co-localized at q21-31 of the active X chromosome, which may be associated with an abundance of LINE1 repeat elements.     

Targeted mutagenesis of Tsix leads to nonrandom X inactivation.

During X inactivation, mammalian female cells make the selection of one active and one inactive X chromosome. X chromosome choice occurs randomly and results in Xist upregulation on the inactive X. We have hypothesized that the antisense gene, Tsix, controls Xist expression. Here, we create a targeted deletion of Tsix in female and male mouse cells. Despite a deficiency of Tsix RNA, X chromosome counting remains intact: female cells still inactivate one X, while male cells block X inactivation. However, heterozygous female cells show skewed Xist expression and primary nonrandom inactivation of the mutant X. The ability of the mutant X to block Xist accumulation is compromised. We conclude that Tsix regulates Xist in cis and determines X chromosome choice without affecting silencing. Therefore, counting, choice, and silencing are genetically separable. Contrasting effects in XX and XY cells argue that negative and positive factors are involved in choosing active and inactive Xs.     

Histone H4 acetylation analyses in patients with polysomy X: implications for the mechanism of X inactivation

In humans, it is thought that the X-inactivation phenomenon occurs no matter how many X chromosomes are present, and that only one of them remains active. Nevertheless, individuals who have an abnormal number of X chromosomes show a wide spectrum of abnormalities, which increase with the number of X chromosomes present in a given individual. It has been shown that the inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, and that this could be used as an accessible marker for distinguishing between Xi and Xa in spreads of metaphase chromosomes. We studied three X-polysomic patients for the presence of active chromatin by analysis of histone H4 acetylation on unfixed metaphase spreads. Using antisera to H4 acetylated at lysines 16, 8 and 5, respectively, we observed frequencies different from those expected from cells with only one underacetylated X chromosome. In particular, when antiserum to H4 acetylated at lysine 16 was used about 90% of the cells showed acetylation of all X chromosomes. This suggests a possible disturbance in the deacetylation process, probably due to the presence of multiple Xs. Received: 25 April 1997 / Accepted: 15 March 1998     

The single active X in human cells: evolutionary tinkering personified

All mammals compensate for sex differences in numbers of X chromosomes by transcribing only a single X chromosome in cells of both sexes; however, they differ from one another in the details of the compensatory mechanisms. These species variations result from chance mutations, species differences in the staging of developmental events, and interactions between events that occur concurrently. Such variations, which have only recently been appreciated, do not interfere with the strategy of establishing a single active X, but they influence how it is carried out. In an overview of X dosage compensation in human cells, I point out the evolutionary variations. I also argue that it is the single active X that is chosen, rather than inactive ones. Further, I suggest that the initial events in the process—those that precede silencing of future inactive X chromosomes—include randomly choosing the future active X, most likely by repressing its XIST locus.     

Methylation profile of genes on the human X chromosome

X chromosome inactivation occurs in female mammals for the purpose of equalisation of dosage of X linked genes between the two sexes. In eutherian mammals, one of the two copies of the X chromosome present in female individuals is silenced. Epigenetic modifications of both DNA and histones have been implicated to play a crucial role in this inactivation phenomenon. In this work, we have employed a novel method published earlier by us, to assess the DNA methylation levels of genes on the inactive X chromosome in the human system. We have used genomic DNA from cells with the following karyotype namely, 47,XXX and 45,X to compare methylation levels from the active and inactive X. We report differential methylation of genes from the active and the inactive X chromosome with higher number of methylated genes being present on the inactive X chromosome. Our work has also led to identification of motifs that show a significant similarity to microRNA sequences which are enriched in methylated regions specific to the inactive X.