Chromosome silencing mechanisms in X-chromosome inactivation: unknown unknowns

Fifty years ago, Mary Lyon hypothesised that one of the two X chromosomes in female mammalian cells is inactivated at random during early embryogenesis and that the inactive X is then stably maintained through all subsequent cell divisions. Although Lyon's hypothesis is now widely regarded as fact, we should not forget that her conceptual leap met with considerable resistance from the scientific establishment at the time - a common response to new ideas. Taking this point as a theme, I discuss our current understanding of the molecular mechanism of chromosome silencing in X-chromosome inactivation and focus on topics where new findings are challenging the prevailing view.     

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.     

X-chromosome inactivation: molecular mechanisms from the human perspective

X-chromosome inactivation is an epigenetic process whereby one X chromosome is silenced in mammalian female cells. Since it was first proposed by Lyon in 1961, mouse models have been valuable tools to uncover the molecular mechanisms underlying X inactivation. However, there are also inherent differences between mouse and human X inactivation, ranging from sequence content of the X inactivation center to the phenotypic outcomes of X-chromosome abnormalities. X-linked gene dosage in males, females, and individuals with X aneuploidies and X/autosome translocations has demonstrated that many human genes escape X inactivation, implicating cis-regulatory elements in the spread of silencing. We discuss the potential nature of these elements and also review the elements in the X inactivation center involved in the early events in X-chromosome inactivation.     

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.     

Weird mammals provide insights into the evolution of mammalian sex chromosomes and dosage compensation

The deep divergence of mammalian groups 166 and 190 million years ago (MYA) provide genetic variation to explore the evolution of DNA sequence, gene arrangement and regulation of gene expression in mammals. With encouragement from the founder of the field, Mary Lyon, techniques in cytogenetics and molecular biology were progressively adapted to characterize the sex chromosomes of kangaroos and other marsupials, platypus and echidna—and weird rodent species. Comparative gene mapping reveals the process of sex chromosome evolution from their inception 190 MYA (they are autosomal in platypus) to their inevitable end (the Y has disappeared in two rodent lineages). Our X and Y are relatively young, getting their start with the evolution of the sex-determining SRY gene, which triggered progressive degradation of the Y chromosome. Even more recently, sex chromosomes of placental mammals fused with an autosomal region which now makes up most of the Y. Exploration of gene activity patterns over four decades showed that dosage compensation via X-chromosome inactivation is unique to therian mammals, and that this whole chromosome control process is different in marsupials and absent in monotremes and reptiles, and birds. These differences can be exploited to deduce how mammalian sex chromosomes and epigenetic silencing evolved.     

Distribution of LINEs and other repetitive elements in the karyotype of the bat Carollia: implications for X-chromosome inactivation

The Lyon repeat hypothesis postulates that long interspersed elements (LINEs) play a role in X-chromosome inactivation. Evidence to support this hypothesis includes the observation that the degree of inactivation of autosomes translocated to the X chromosome is correlated with LINE density on that autosome. We examined the distribution of LINEs in the fruit bat Carollia brevicauda, which has an autosomal translocation to the X that occurred at least 7 million years ago. A quantitative analysis of LINE accumulation on multiple metaphase chromosome spreads revealed a significant accumulation on the original X relative to the attached autosome, the homolog of that autosome (Y(2)), and chromosome 1. Previous replication studies indicate that for the X and attached autosome, only the original X chromosome replicates late in Carollia females, and that the attached autosome replicates in the same timeframe as other autosomes. These data are compatible with the Lyon repeat hypothesis, and the possibility that LINEs act as booster elements for X inactivation remains a viable hypothesis. We address the procedures and limitations of quantitative analysis based on in situ hybridization.     

Mary Lyon and the hypothesis of random X chromosome inactivation

The 50th anniversary of Mary Lyon’s 1961 Nature paper, proposing random inactivation in early embryonic life of one of the two X chromosomes in the cells of mammalian females, provides an opportunity to remember and celebrate the work of those involved. While the hypothesis was initially put forward by Lyon based on findings in the mouse, it was founded on earlier studies, notably the work of Susumu Ohno; it was also suggested independently by Beutler and colleagues using experimental evidence from a human X-linked disorder, glucose-6-phosphate dehydrogenase deficiency, and has proved to be of as great importance for human and medical genetics as it has for general mammalian genetics. Alongside the hypothesis itself, previous cytological studies of mouse and human chromosomes, and the observations on X-linked mutants in both species deserve recognition for their essential role in underpinning the hypothesis of random X-inactivation, while subsequent research on the X-inactivation centre and the molecular mechanisms underlying the inactivation process represent some of the most outstanding contributions to human and wider mammalian genetics over the past 50 years.     

X chromosome inactivation in human and mouse pluripotent stem cells

Since the groundbreaking hypothesis of X chromosome inactivation (XCI) proposed by Mary Lyon over 50 years ago, a great amount of knowledge has been gained regarding this essential dosage compensation mechanism in female cells. For the mammalian system, most of the mechanistic studies of XCI have so far been investigated in the mouse model system, but recently, a number of interesting XCI studies have been extended to human pluripotent stem cells, including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Emerging data indicate that XCI in hESCs and hiPSCs is much more complicated than that of their mouse counterparts. XCI in human pluripotent stem cells is not as stable and is subject to environmental influences and epigenetic regulation in vitro. This mini-review highlights the key differences in XCI between mouse and human stem cells with a greater emphasis placed on the understanding of the epigenetic regulation of XCI in human stem cells.     

The 48,XXXX syndrome: study of psychomotor development from birth to 11 years of age and review of the literature

A child with four X chromosomes is described. This case and the literature review allow to underline the mental retardation and some other "major" but inconstant signs that are extremely helpful for the early clinical diagnosis. They are hypertelorism, epicanthal fold and genital anomalies. The mental evolution is assessed on an eleven year period. The bad results concern particularly the child's use of language and the complicated works. They become worse with time. The additional X chromosomes Mary Lyon inactivation, perhaps incomplete, is discussed because its determinism. Enzymatic measuring out is our approach to this problem.     

X-chromosome inactivation and the search for chromosome-wide silencers

X-chromosome inactivation leads to divergent fates for two homologous chromosomes. Whether one X remains active or becomes silenced depends on the activity of Xist, a gene expressed only from the inactive X and whose RNA product 'paints' the X in cis. Recent work argues that Xist RNA itself is the acting agent for initiating the silencing step. Xist RNA contains separable domains for RNA localization and chromosome silencing. While no Xist RNA-interacting factors have been identified, a growing collection of chromatin alterations have been identified on the inactive X, including variant histone H2A composition and histone H3 methylation. Some or all of these changes may be critical for chromosome-wide silencing. As none of the silencing proteins identified so far is unique to X chromosome inactivation, the specificity must partly reside in Xist RNA whose spread along the X orchestrates general silencing factors for this specific task.     

Escape from X inactivation

Although the process of X inactivation in mammalian cells silences the majority of genes on the inactivated X chromosome, some genes escape this chromosome-wide silencing. Genes that escape X inactivation present a unique opportunity to study the process of silencing and the mechanisms that protect some genes from being turned off. In this review, we will discuss evolutionary aspects of escape from X inactivation, in relation to the divergence of the sex chromosomes. Molecular characteristics, expression, and epigenetic modifications of genes that escape will be presented, including their developmental regulation and the implications of chromatin domains along the X chromosome in modeling the escape process.     

Hematopoietic precursor cells transiently reestablish permissiveness for X inactivation

Xist is the trigger for X inactivation in female mammals. The long noncoding Xist RNA localizes along one of the two female X chromosomes and initiates chromosome-wide silencing in the early embryo. In differentiated cells, Xist becomes dispensable for the maintenance of the inactive X, and its function for initiation of silencing is lost. How Xist mediates gene repression remains an open question. Here, we use an inducible Xist allele in adult mice to identify cells in which Xist can cause chromosome-wide silencing. We show that Xist has the ability to initiate silencing in immature hematopoietic precursor cells. In contrast, hematopoietic stem cells and mature blood cells are unable to initiate ectopic X inactivation. This indicates that pathways critical for silencing are transiently activated in hematopoietic differentiation. Xist-responsive cell types in normal female mice show a change of chromatin marks on the inactive X. However, dosage compensation is maintained throughout hematopoiesis. Therefore, Xist can initiate silencing in precursors with concomitant maintenance of dosage compensation. This suggests that Xist function is restricted in development by the limited activity of epigenetic pathways rather than by a change in the responsiveness of chromatin between embryonic and differentiated cell types.     

A cross-species comparison of X-chromosome inactivation in Eutheria

Mammalian X-chromosome inactivation achieves dosage compensation between the sexes by the silencing of one X chromosome in females. In Eutheria, X inactivation is initiated by the large noncoding RNA Xist; however, it is unknown how this RNA results in silencing of the chromosome or why, at least in humans, many genes escape silencing in somatic cells. We have sequenced the coast mole Xist gene and compared the Xist RNA sequence among seven eutherians to provide insight into the structure of the RNA and origins of the gene. Using DNA methylation of promoter sequences to assess whether genes are silenced in females we report the inactivation status of seven X-linked genes in humans and mice as well as two additional eutherians, the mole and the cow, providing evidence that escape from inactivation is common among Eutheria.     

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.     

Fifty years of X-inactivation research

The third X-inactivation meeting 'Fifty years of X-inactivation research', which celebrated the fiftieth anniversary of Mary Lyon's formulation of the X-inactivation hypothesis, was an EMBO workshop held in Oxford, UK, in July 2011. This conference brought together the usual suspects from the field, as well as younger researchers, to discuss recent advances in X-inactivation research. Here, we review the results presented at the meeting and highlight some of the exciting progress that has been made. We also discuss the future challenges for the field, which aim to further our understanding of the developmental regulation of X inactivation, the randomness (or skewing) of X inactivation, and the diverse strategies used by mammalian species to mediate X inactivation.     

X inactivation: Tsix and Xist as yin and yang

A new study shows that expression of Tsix, an antisense Xist gene, can be controlled by imprinting, and that high Tsix activity during X inactivation can protect the future active X chromosome from silencing by Xist. Tsix and Xist seem to have a yin and yang relationship, with opposite effects on X inactivation.     

RNAi in X inactivation: contrasting findings on the role of interference

X inactivation is the process that brings about the dosage equivalence of X‐linked genes in females to that of males. This complex process initiated at a very early stage of female embryonic development is orchestrated by long non‐coding RNAs transcribed in both sense and antisense orientation. Recent studies present contradicting evidence for the role of small RNAs and RNase III enzyme Dicer in the X inactivation process. In this review, I discuss these results in the overall perspective of X inactivation and gene silencing.     

X-chromosome inactivation: closing in on proteins that bind Xist RNA

X inactivation is the developmentally regulated silencing of a single X chromosome in XX female mammals. In recent years, the Xist gene has been revealed as the master regulatory switch controlling this process. Parental imprinting and/or counting mechanisms ensure that Xist is expressed only on the inactive X chromosome. Chromosome silencing then results from the accumulation of the Xist RNA silencing signal, in cis, over the entire length of the X chromosome. A key issue has been to identify the factors that interact with Xist RNA to initiate heritable gene silencing. This review discusses recent progress that has put this goal in sight.