Structural heterochromatin and X-chromosome inactivation]

Our previous studies on the expression of the G6PD and alpha-GAL genes from the X chromosome of inter-specific hybrids of voles of the Microtus genus have demonstrated an unusual pattern of X-inactivation in the parents. The observed phenomenon was explained as the presumable result of nonrandom inactivation of the X chromosomes with a heterochromatin block in crosses involving Microtus arvalis whose X lacks a heterochromatin region and also of random X inactivation when both parents had heterochromatin blocks on the Xs. Based on known models, we discuss here the possible mechanisms of the effect of heterochromatin on X-inactivation; we give preference to the model postulating binding of nonhistone protein to the inactivation centre as the key event. The hypothesis we offer suggests change in chromatin conformation in the inactivation centre during packaging of heterochromatic region of a chromosome; the protein molecules diffusing along the chromosome towards the heterochromatin region by the "facilitated diffusion" mechanism may happen to be in the region of the X-inactivation centre, which, being in a favorable state, binds specifically to it; as a consequence, the binding probability of protein to heterochromatin increases as compared to chromosome without heterochromatin block.     

X chromosome inactivation pattern in elderly women over 70 years of age

In the present study we have analyzed X chromosome inactivation patterns in 40 women aged from 74 to 85 years (mean age 78 years). The control group was 36 women (mean age 30 years). The most common AR-assay was used to determine X-inactivation patterns (the study of methylation patterns of HpaII site in human androgen receptor gene (HUMARA) by quantative PCR). The age dependence of X-inactivation was not observed. We have detected skewed X-inactivation in three women among 40 (7.5%) elderly women comparing to two women among 36 (5.5%) women from control group. The difference was not found to be statistically significant. We made a suggestion that higher incidence of skewed X-inactivation in elderly women revealed by previous studies could occur due to some experimental ambiguities as heterogeneity of the group studied; inclusion of women having relatives with genetic abnormalities associated with skewed X-inactivation patterns; the difference of X chromosome inactivation skewing determination. We conclude that present study does not show X chromosome inactivation to be age dependent.     

Micromanipulating dosage compensation: understanding X-chromosome inactivation through nuclear transplantation

Nuclear transfer (NT) studies have provided insight into the functional importance of epigenetic alteration of the X chromosomes during X-inactivation. Uniparental embryos created by NT have been informative as to the time and location at which the imprint controlling extraembryonic X-inactivation is established. Experiments with female somatic cells, have demonstrated that the inactive X chromosome (Xi) is reactivated after NT, leading to random X-inactivation in the embryonic lineages of cloned embryos. However, in the extraembryonic lineages of clones, epigenetic information from the donor cell nucleus persists, leading to preferential inactivation of the donor cell's inactive X in the placenta of cloned animals. These results suggest epigenetic information established during embryonic X-inactivation is functionally equivalent to the gametic imprint.     

Commitment to X inactivation precedes the twinning event in monochorionic MZ twins.

To gain insight into the timing of twinning, we have examined a closely related event, X-chromosome inactivation, in female MZ twin pairs. X-inactivation patterns in peripheral blood and buccal mucosa were compared between monochorionic MZ (MC-MZ) and dichorionic MZ (DC-MZ) twins. Overall, the MC-MZ twins displayed highly similar X-inactivation patterns, whereas DC-MZ twins frequently differed in their X-inactivation patterns, when both tissues were tested. Previous experimental data suggest that commitment to X inactivation occurs when there are 10-20 cells in the embryo. Simulation of embryo splitting after commitment to X inactivation suggests that MC-MZ twinning occurs three or four rounds of replication after X inactivation, whereas a DC-MZ twinning event occurs earlier, before or around the time of X inactivation. Finally, the overall degree of skewing in the MZ twins was not significantly different from that observed in singletons. This indicates that X inactivation does not play a direct role in the twinning process, and it further suggests that extreme unequal splitting is not a common mechanism of twin formation.     

Insights into X chromosome inactivation from studies of species variation, DNA methylation and replication, and vice versa

I am indebted to Mary Lyon as her X-inactivation hypothesis stimulated my mentor, Barton Childs, and in turn, myself, to think about the consequences of X-inactivation in heterozygous females. I often reread her original papers setting forth the single active X hypothesis, and still marvel at the concise and compelling exposition of the hypothesis and the logical predictions which seemed prophetic at my first reading, and have survived the test of time. My contribution to this Festschrift reviews evidence derived from studies of DNA methylation, species variation and DNA replication that reveals an important role for methylated CpG islands and suggests a role for late DNA replication in propagating X inactivation from one cell to its progeny. These studies also show that X inactivation is a powerful research tool for identifying the factors which program and maintain developmental processes.     

Skewed X-chromosome inactivation is a common feature of X-linked mental retardation disorders

Some deleterious X-linked mutations may result in a growth disadvantage for those cells in which the mutation, when on the active X chromosome, affects cell proliferation or viability. To explore the relationship between skewed X-chromosome inactivation and X-linked mental retardation (XLMR) disorders, we used the androgen receptor X-inactivation assay to determine X-inactivation patterns in 155 female subjects from 24 families segregating 20 distinct XLMR disorders. Among XLMR carriers, ~50% demonstrate markedly skewed X inactivation (i.e., patterns 80:20), compared with only ~10% of female control subjects (P<.001). Thus, skewed X inactivation is a relatively common feature of XLMR disorders. Of the 20 distinct XLMR disorders, 4 demonstrate a strong association with skewed X inactivation, since all carriers of these mutations demonstrate X-inactivation patterns 80:20. The XLMR mutations are present on the preferentially inactive X chromosome in all 20 informative female subjects from these families, indicating that skewing is due to selection against those cells in which the XLMR mutation is on the active X chromosome.     

X-chromosome inactivation in XX androgenetic mouse embryos surviving implantation

Using genetic and cytogenetic markers, we assessed early development and X-chromosome inactivation (X-inactivation) in XX mouse androgenones produced by pronuclear transfer. Contrary to the current view, XX androgenones are capable of surviving to embryonic day 7.5, achieving basically random X-inactivation in all tissues including those derived from the trophectoderm and primitive endoderm that are characterized by paternal X-activation in fertilized embryos. This finding supports the hypothesis that in fertilized female embryos, the maternal X chromosome remains active until the blastocyst stage because of a rigid imprint that prevents inactivation, whereas the paternal X chromosome is preferentially inactivated in extra-embryonic tissues owing to lack of such imprint. In spite of random X-inactivation in XX androgenones, FISH analyses revealed expression of stable Xist RNA from every X chromosome in XX and XY androgenonetic embryos from the four-cell to morula stage. Although the occurrence of inappropriate X-inactivation was further suggested by the finding that Xist continues ectopic expression in a proportion of cells from XX and XY androgenones at the blastocyst and the early egg cylinder stage, a replication banding study failed to provide positive evidence for inappropriate X-inactivation at E6. 5.     

Pigmentary mosaicism in hypomelanosis of Ito

We report on a female with mental and motor retardation, facial dysmorphism, abnormal pigmentation reminiscent to hypomelanosis of Ito (HI), and karyotypic mosaicism involving a small supernumerary marker chromosome. The marker chromosome was defined by fluorescence in situ hybridisation (FISH) as a ring X chromosome with breakpoints in the juxtacentromeric region. FISH analysis showed that the ring does not include the XIST locus at the X-inactivation centre and, therefore, may not be subject to X inactivation. X-inactivation studies with the HUMARA (human androgen receptor) and FMR1 assay showed a skewed X-inactivation pattern (85:15) with preferential inactivation of the paternal X chromosome. These results are discussed with respect to the role of functional disomy of Xp in the pathogenesis of HI. Received: 16 February 1998 / Accepted: 17 July 1998     

Familial skewed X inactivation: a molecular trait associated with high spontaneous-abortion rate maps to Xq28.

We report a family ascertained for molecular diagnosis of muscular dystrophy in a young girl, in which preferential activation (> or = 95% of cells) of the paternal X chromosome was seen in both the proband and her mother. To determine the molecular basis for skewed X inactivation, we studied X-inactivation patterns in peripheral blood and/or oral mucosal cells from 50 members of this family and from a cohort of normal females. We found excellent concordance between X-inactivation patterns in blood and oral mucosal cell nuclei in all females. Of the 50 female pedigree members studied, 16 showed preferential use (> or = 95% cells) of the paternal X chromosome; none of 62 randomly selected females showed similarly skewed X inactivation was maternally inherited in this family. A linkage study using the molecular trait of skewed X inactivation as the scored phenotype localized this trait to Xq28 (DXS1108; maximum LOD score [Zmax] = 4.34, recombination fraction [theta] = 0). Both genotyping of additional markers and FISH of a YAC probe in Xq28 showed a deletion spanning from intron 22 of the factor VIII gene to DXS115-3. This deletion completely cosegregated with the trait (Zmax = 6.92, theta = 0). Comparison of clinical findings between affected and unaffected females in the 50-member pedigree showed a statistically significant increase in spontaneous-abortion rate in the females carrying the trait (P < .02). To our knowledge, this is the first gene-mapping study of abnormalities of X-inactivation patterns and is the first association of a specific locus for recurrent spontaneous abortion in a cytogenetically normal family. The involvement of this locus in cell lethality, cell-growth disadvantage, developmental abnormalities, or the X-inactivation process is discussed.     

Parental source of chromosome imprinting and its relevance for X chromosome inactivation

Abstract. In imprinting, homologous chromosomes behave differently during development according to their parental origin. Typically, paternally derived chromosomes are preferentially inactivated or eliminated. Examples of such phenomena include inactivation of the mammalian X chromosome, inactivation or elimination of one haploid chromosome set in male coccids, and elimination of paternal X chromosomes in the fly Sciara . It has generally been thought that the paternal chromosomes bear an imprint leading to their inactivation or elimination. However, alteration of the parental origin of chromosomes, as in the study of parthenogenotes in mammals and coccids, shows that passage of chromosomes through a male germ cell or fertilization is not essential for inactivation or elimination. It appears that neither chromosome set is programmed to resist or undergo inactivation. Instead the two sets differ in relative sensitivity, and the question is whether the maternal set have an imprint for resistance, or the paternal set one for susceptibility. Very early in development of mammals both X chromosomes are active. This makes it simpler to envisage the maternal X bearing an imprint for resistance to inactivation, which persists through the early developmental period. Similar considerations also apply in coccids and Sciara . Thus, imprinting should be regarded as a phenomenon conferred on the maternal chromosomes in the oocyte. This permits simpler models for the mechanism of X-inactivation, and weakens the case for evolution of X-inactivation from an earlier form of inactivation during male gametogenesis. One may speculate whether imprinting affects timing of gene action in development.     

The search for the mouse X-chromosome inactivation centre

The phenomenon of X-chromosome inactivation in female mammals, whereby one of the two X chromosome present in each cell of the female embryo is inactivated early in development, was first described by Mary Lyon in 1961. Nearly 30 years later, the mechanism of X-chromosome inactivation remains unknown. Strong evidence has accumulated over the years, however, for the involvement of a major switch or inactivation centre on the mouse X chromosome. Identification of the inactivation centre at the molecular level would be an important step in understanding the mechanism of X-inactivation. In this paper we review the evidence for the existence and location of the X-inactivation centre on the mouse X-chromosome, present data on the molecular genetic mapping of this region, and describe ongoing strategies we are using to attempt to identify the inactivation centre at the molecular level.     

A practical metaphase marker of the inactive X chromosome.

It is paradoxical that the inactivated X is the only chromosome that can be identified in the interphase nucleus, yet in metaphase, it is indistinguishable from its genetically active homolog unless special culture and staining procedures are employed. A specific inactivation-associated fold in proximal Xq resolves that paradox. We describe here how the fold in the proximal long arm can be used as a simple and reliable marker to identify the inactivated X in G-, Q-, or R-banded preparations. Several examples are given, including localization of the inactivation center to band Xq13 or q21.1, identification of nonrandom inactivation in X-chromosome rearrangements, identification of multiple active X chromosomes in tumor cell lines, analysis of X-inactivation patterns in female carriers of the fragile site at Xq27, and comparison of X-inactivation patterns among primate species.