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.     

Low-copy-number human transgene is recognized as an X inactivation center in mouse ES cells, but fails to induce cis-inactivation in chimeric mice

X chromosome inactivation is initiated from a segment of the mammalian X chromosome called the X inactivation center. Transgenes from this region of the murine X chromosome are providing the means to identify the DNA needed for cis inactivation in mice. We recently showed that chimeric mice carrying transgenes from the human X inactivation center (XIC) region also provide a functional assay for human XIC activity; approximately 6 copies of a 480-kb human transgene (ES-10) were sufficient to initiate random X inactivation in cells of male chimeric mice (Migeon et al., 1999, Genomics, 59, 113-121). Now, we report studies of another human transgene (ES-5), which contains less than 300 kb of the human XIC region on Xq13.2 including an intact XIST locus and which has inserted in one or two copies into mouse chromosome 6. The ES-5 transgene is recognized as an X inactivation center in mouse embryonic stem cells, but is not sufficient to induce random X inactivation in somatic cells of highly chimeric mice. Human transgenes in chimeric mice provide a means to uncouple the key steps in this complex pathway and facilitate the search for essential components of the human XIC region.     

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.     

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.     

X inactivation in a mammal species with three sex chromosomes

X inactivation is a fundamental mechanism in eutherian mammals to restore a balance of X-linked gene products between XY males and XX females. However, it has never been extensively studied in a eutherian species with a sex determination system that deviates from the ubiquitous XX/XY. In this study, we explore the X inactivation process in the African pygmy mouse Mus minutoides, that harbours a polygenic sex determination with three sex chromosomes: Y, X, and a feminizing mutant X, named X*; females can thus be XX, XX*, or X*Y, and all males are XY. Using immunofluorescence, we investigated histone modification patterns between the two X chromosome types. We found that the X and X* chromosomes are randomly inactivated in XX* females, while no histone modifications were detected in X*Y females. Furthermore, in M. minutoides, X and X* chromosomes are fused to different autosomes, and we were able to show that the X inactivation never spreads into the autosomal segments. Evaluation of X inactivation by immunofluorescence is an excellent quantitative procedure, but it is only applicable when there is a structural difference between the two chromosomes that allows them to be distinguished.     

X chromosome choice occurs independently of asynchronous replication timing

In mammals, dosage compensation is achieved by X chromosome inactivation in female cells. Xist is required and sufficient for X inactivation, and Xist gene deletions result in completely skewed X inactivation. In this work, we analyzed skewing of X inactivation in mice with an Xist deletion encompassing sequence 5 KB upstream of the promoter through exon 3. We found that this mutation results in primary nonrandom X inactivation in which the wild-type X chromosome is always chosen for inactivation. To understand the molecular mechanisms that affect choice, we analyzed the role of replication timing in X inactivation choice. We found that the two Xist alleles and all regions tested on the X chromosome replicate asynchronously before the start of X inactivation. However, analysis of replication timing in cell lines with skewed X inactivation showed no preference for one of the two Xist alleles to replicate early in S-phase before the onset of X inactivation, indicating that asynchronous replication timing does not play a role in skewing of X inactivation.     

X inactivation in the mouse embryo deficient for Dnmt1: distinct effect of hypomethylation on imprinted and random X inactivation

It has been suggested that DNA methylation plays a crucial role in genomic imprinting and X inactivation. Using DNA methyltransferase 1 (Dnmt1)-deficient mouse embryos carrying X-linked lacZ transgenes, we studied the effects of genomic demethylation on X inactivation. Based on the expression pattern of lacZ, the imprinted X inactivation in the visceral endoderm, a derivative of the extraembryonic lineage, was unaffected in Dnmt1 mutant embryos at the time other imprinted genes showed aberrant expression. Random X inactivation in the embryonic lineage of Dnmt1 mutant embryos, however, was unstable as a result of hypomethylation, causing reactivation of, at least, one lacZ transgene that had initially been repressed. Our results suggest that maintenance of imprinted X inactivation in the extraembryonic lineage can tolerate extensive demethylation while normal levels of methylation are required for stable maintenance of X inactivation in the embryonic lineage.     

X chromosome regulation of autosomal gene expression in bovine blastocysts

Although X chromosome inactivation in female mammals evolved to balance the expression of X chromosome and autosomal genes in the two sexes, female embryos pass through developmental stages in which both X chromosomes are active in somatic cells. Bovine blastocysts show higher expression of many X genes in XX than XY embryos, suggesting that X inactivation is not complete. Here, we reanalyzed bovine blastocyst microarray expression data from a network perspective with a focus on interactions between X chromosome and autosomal genes. Whereas male-to-female ratios of expression of autosomal genes were distributed around a mean of 1, X chromosome genes were clearly shifted towards higher expression in females. We generated gene coexpression networks and identified a major module of genes with correlated gene expression that includes female-biased X genes and sexually dimorphic autosomal genes for which the sexual dimorphism is likely driven by the X genes. In this module, expression of X chromosome genes correlates with autosome genes, more than the expression of autosomal genes with each other. Our study identifies correlated patterns of autosomal and X-linked genes that are likely influenced by the sexual imbalance of X gene expression when X inactivation is inefficient.     

X chromosome inactivation patterns correlate with fetal-placental anatomy in monozygotic twin pairs: implications for immune relatedness and concordance for autoimmunity.

BACKGROUND: Monozygotic (MZ) twinning is a poorly understood phenomenon that may result in subtle biologic differences between twins, despite their identical inheritance. These differences may in part account for discordant expression of disease in MZ twin pairs. Due to their stochastic nature, differences in X chromosome inactivation patterns are one source of such variation in female MZ twins. MATERIALS AND METHODS: We investigated X chromosome inactivation patterns in the blood of 41 MZ twin pairs based on methylation of the androgen receptor gene using a Hpa II-PCR assay. Twenty-six female MZ twin pairs with autoimmune disease (rheumatoid arthritis or multiple sclerosis) were studied. In addition, we studied 15 newborn female MZ twin pairs who were characterized at birth with respect to the anatomy of chorionic membranes (dichorionic versus monochorionic). RESULTS: We found a strong correlation between dichorionic fetal anatomy and differences in X chromosome inactivation patterns between members of an MZ twin pair. In contrast, all monochorionic twin pairs had closely correlated patterns of X chromosome inactivation. X chromosome inactivation patterns did not distinguish between MZ twin pairs who were concordant or discordant for autoimmune disease. CONCLUSIONS: The highly similar patterns of X chromosome inactivation among monochorionic twin pairs may result from their shared placental blood supply during intrauterine life. Alternatively, these patterns may indicate that X chromosome inactivation occurs before the twinning event in this anatomic subgroup of MZ twins. The data further suggest that these factors do not make a major contribution to the high discordance rates for autoimmune disease in MZ twin pairs.     

Sexual antagonism and X inactivation--the SAXI hypothesis

X inactivation has evolved in the soma of mammalian females so that both sexes have the same ratio of X:autosomal gene expression. The X chromosome in the germ cells of XY males is also precociously inactivated for reasons that remain unclear. Unlike X inactivation in the soma, this germline X inactivation is not restricted to mammals but has evolved independently in several animal phyla. Thus, germline X inactivation might have been the precursor of somatic X inactivation in mammals. We now propose a hypothesis for the evolution of germline X inactivation. The hypothesis predicts a redistribution of late spermatogenic genes from the X chromosome to the autosomes, leading eventually to germline X inactivation as the X chromosome becomes 'demasculinized'. Sexual antagonism could be the mechanism driving this redistribution. Recent expression and genetic studies in mammals, nematodes and Drosophila support this hypothesis, and expression data on taxa that have not evolved germline X inactivation, such as birds and butterflies, should shed further light on it.     

An N-ethyl-N-nitrosourea mutagenesis screen for epigenetic mutations in the mouse

The mammalian epigenetic phenomena of X inactivation and genomic imprinting are incompletely understood. X inactivation equalizes X-linked expression between males and females by silencing genes on one X chromosome during female embryogenesis. Genomic imprinting functionally distinguishes the parental genomes, resulting in parent-specific monoallelic expression of particular genes. N-ethyl-N-nitrosourea (ENU) mutagenesis was used in the mouse to screen for mutations in novel factors involved in X inactivation. Previously, we reported mutant pedigrees identified through this screen that segregate aberrant X-inactivation phenotypes and we mapped the mutation in one pedigree to chromosome 15. We now have mapped two additional mutations to the distal chromosome 5 and the proximal chromosome 10 in a second pedigree and show that each of the mutations is sufficient to induce the mutant phenotype. We further show that the roles of these factors are specific to embryonic X inactivation as neither genomic imprinting of multiple genes nor imprinted X inactivation is perturbed. Finally, we used mice bearing selected X-linked alleles that regulate X chromosome choice to demonstrate that the phenotypes of all three mutations are consistent with models in which the mutations have affected molecules involved specifically in the choice or the initiation of X inactivation.     

Trimethylation of Histone H3 Lysine 4 is an Epigenetic Mark at Regions Escaping Mammalian X Inactivation

It is now estimated that 150-200 genes clustered in several discrete regions escape X inactivation in somatic cells of human females by unknown mechanisms. Here, we show that although the human female inactive X chromosome is largely devoid of histone 3 lysine 4 trimethylation (H3K4me3), regions that are known to escape X inactivation, including the pseudoautosomal regions, are enriched with this modification. Also, H3K4me3, unlike H3K4me2 and H4 and H3 acetylation, is restricted to discrete regions on metaphase chromosomes. In contrast to humans, there are only a few genes that are known to escape X inactivation in the mouse. Therefore, we examined mouse female somatic cells with H3K4me3 to identify candidate regions with genes that escape X inactivation. We found the mouse female inactive X in somatic cells and the male inactive X in meiosis to have seven discrete regions that are enriched with H3K4me3. Furthermore, RNA polymerase II is largely excluded from the XY body at male pachytene except for several discrete regions on the X and Y suggesting the presence of regions that also escape sex chromosome inactivation during male meiosis.     

哺乳动物X染色体失活机制

哺乳动物X染色体连锁基因的剂量平衡,是通过雌性胚胎发育早期随机或印记失活一条X染色体来实现的,这是一个复杂的过程,包括：启动、计数、选择、维持等一系列的步骤。X染色体失活中心是X染色体失活的主控开关座位,调节X失活的早期事件,失活发生后,X染色体的失活状态可稳定地存在并传递给后代,这一过程涉及基因组印记的形成。此外,在雄性动物,精原细胞减数分裂早期也存在着短暂的X染色体失活现象。现对哺乳动物X染色体失活机制的最新进展进行综述。