Rsx is a metatherian RNA with Xist-like properties in X-chromosome inactivation

In female (XX) mammals, one of the two X chromosomes is inactivated to ensure an equal dose of X-linked genes with males (XY). X-chromosome inactivation in eutherian mammals is mediated by the non-coding RNA Xist. Xist is not found in metatherians (marsupials), and how X-chromosome inactivation is initiated in these mammals has been the subject of speculation for decades. Using the marsupial Monodelphis domestica, here we identify Rsx (RNA-on-the-silent X), an RNA that has properties consistent with a role in X-chromosome inactivation. Rsx is a large, repeat-rich RNA that is expressed only in females and is transcribed from, and coats, the inactive X chromosome. In female germ cells, in which both X chromosomes are active, Rsx is silenced, linking Rsx expression to X-chromosome inactivation and reactivation. Integration of an Rsx transgene on an autosome in mouse embryonic stem cells leads to gene silencing in cis. Our findings permit comparative studies of X-chromosome inactivation in mammals and pose questions about the mechanisms by which X-chromosome inactivation is achieved in eutherians.     

Conservation of position and exclusive expression of mouse Xist from the inactive X chromosome

X-chromosome inactivation in mammals is a regulatory phenomenon whereby one of the two X chromosomes in female cells is genetically inactivated, resulting in dosage compensation for X-linked genes between males and females. In both man and mouse, X-chromosome inactivation is thought to proceed from a single cis-acting switch region or inactivation centre (XIC/Xic). In the human, XIC has been mapped to band Xq13 (ref. 6) and in the mouse to band XD (ref. 7), and comparative mapping has shown that the XIC regions in the two species are syntenic. The recently described human XIST gene maps to the XIC region and seems to be expressed only from the inactive X chromosome. We report here that the mouse Xist gene maps to the Xic region of the mouse X chromosome and, using an interspecific Mus spretus/Mus musculus domesticus F1 hybrid mouse carrying the T(X;16)16H translocation, show that Xist is exclusively expressed from the inactive X chromosome. Conservation between man and mouse of chromosomal position and unique expression exclusively from the inactive X chromosome lends support to the hypothesis that XIST and its mouse homologue are involved in X-chromosome inactivation.     

Cloning of the breakpoint of an X;21 translocation associated with Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder which affects approximately 1 in 3,300 males, making it the most common of the neuromuscular dystrophies. The biochemical basis of the disease is unknown and as yet no effective treatment is available. A small number of females are also affected with the disease, and these have been found to carry X; autosome translocations involving variable autosomal sites but always with a breakpoint within band Xp21 of the X chromosome (implicated by other kinds of genetic evidence as the site of the DMD lesion). In these female patients the normal X chromosome is preferentially inactivated, which it is assumed silences their one normal DMD gene, leading to expression of the disease. In one such affected female the autosomal breakpoint lies in the middle of the short arm of chromosome 21, within a cluster of ribosomal RNA genes. Here we have used rRNA sequences as probes to clone the region spanning the translocation breakpoint. A sequence derived from the X-chromosomal portion of the clone detects a restriction fragment length polymorphism (RFLP) which is closely linked to the DMD gene and uncovers chromosomal deletions in some male DMD patients.     

Characterization of a murine gene expressed from the inactive X chromosome

In mammals, equal dosage of gene products encoded by the X chromosome in male and female cells is achieved by X inactivation. Although X-chromosome inactivation represents the most extensive example known of long range cis gene regulation, the mechanism by which thousands of genes on only one of a pair of identical chromosomes are turned off is poorly understood. We have recently identified a human gene (XIST) exclusively expressed from the inactive X chromosome. Here we report the isolation and characterization of its murine homologue (Xist) which localizes to the mouse X inactivation centre region and is the first murine gene found to be expressed from the inactive X chromosome. Nucleotide sequence analysis indicates that Xist may be associated with a protein product. The similar map positions and expression patterns for Xist in mouse and man suggest that this gene may have a role in X inactivation.     

Embryological and molecular investigations of parental imprinting on mouse chromosome 7

Mouse embryos with duplications of whole maternal (parthenogenetic and gynogenetic) or paternal (androgenetic) genomes show reciprocal phenotypes and do not develop to term. Genetic complementation has identified the distal region of chromosome 7 (Chr 7) as one of the regions for which both a maternal and paternal chromosome copy are essential for normal development, presumably because of the presence of imprinted genes whose expression is dependent on their parental origin. Embryos with the maternal duplication and paternal deficiency of distal Chr 7 are growth retarded and die around day 16 of gestation; the reciprocal paternal duplication embryos die at an unidentified earlier stage. We report here the incorporation of cells with the paternal duplication into chimaeras, resulting in a striking growth enhancement of the embryos. One gene located on mouse distal Chr 7 (ref. 5) is the insulin-like growth factor 2 (Igf2) gene, an embryonic mitogen. In embryos with the maternal duplication of distal Chr 7, the two maternal alleles of the Igf2 gene are repressed. The presence of two paternal alleles of this gene in many cells is probably responsible for the growth enhancement observed in chimaeras. We propose that there are other imprinted genes in this Chr 7 region. We also compare the imprinting of this subgenomic region with phenotypes resulting from the duplication of the whole parental genome in parthenogenones and androgenones.     

X-inactivation profile reveals extensive variability in X-linked gene expression in females

In female mammals, most genes on one X chromosome are silenced as a result of X-chromosome inactivation. However, some genes escape X-inactivation and are expressed from both the active and inactive X chromosome. Such genes are potential contributors to sexually dimorphic traits, to phenotypic variability among females heterozygous for X-linked conditions, and to clinical abnormalities in patients with abnormal X chromosomes. Here, we present a comprehensive X-inactivation profile of the human X chromosome, representing an estimated 95% of assayable genes in fibroblast-based test systems. In total, about 15% of X-linked genes escape inactivation to some degree, and the proportion of genes escaping inactivation differs dramatically between different regions of the X chromosome, reflecting the evolutionary history of the sex chromosomes. An additional 10% of X-linked genes show variable patterns of inactivation and are expressed to different extents from some inactive X chromosomes. This suggests a remarkable and previously unsuspected degree of expression heterogeneity among females.     

Effect of ageing on reactivation of the human X-linked HPRT locus

In mammals, X-chromosome dosage compensation is achieved by inactivating one X chromosome in female cells. To test the hypothesis that genes on the silent X chromosome reactivate as a consequence of ageing, we examined the X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in 41 women who are heterozygous for mutations at this locus, leading to severe deficiency of the enzyme (Lesch-Nyhan syndrome). We find that heterozygotes who are more than 10 yr old have an excess of HPRT+ skin fibroblast clones (59% rather than the 50% expected as a consequence of random X inactivation) but this excess does not increase with age. Further studies of eight of these heterozygotes show that the silent locus does not detectably reactivate spontaneously in culture, but only in response to treatment with 5-aza-2-deoxycytidine, a potent inhibitor of methylation. There is no age difference in the frequency of this reactivation as assayed by HATr clones, and a more sensitive autoradiographic assay shows only a twofold difference between young and old heterozygotes. Thus, age-related reactivation is not a feature of all X-linked loci, and may have species, tissue and locus-specific determinants.     

The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus.

T-associated maternal effect (Tme) is the only known maternal-effect mutation in the mouse. The defect is nuclear-encoded and embryos that inherit a deletion of the Tme locus from their mother die at day 15 of gestation. There are many genomically imprinted regions known in the mouse genome but so far no imprinted genes have been cloned. The Tme locus is absent in two chromosome-17 deletion mutants, Thp and the tLub2, and its position has been localized using these deletions to a 1-cM region. We report here that the genes for insulin-like growth factor type-2 receptor (Igf2r) and mitochondrial superoxide dismutase-2 (Sod-2) are absent from both deletions. Probes for these genes and for plasminogen (Plg) and T-complex peptide 1 (Tcp-1) were used in pulsed-field gel mapping to show that Tme must lie within a region of 800-1,100 kb. We also demonstrate that embryos express Igf2r only from the maternal chromosome, and that Tcp-1, Plg and Sod-2 are expressed from both chromosomes. Therefore Igf2r is imprinted and closely linked or identical to Tme.     

Role of paternal and maternal genomes in mouse development

There has been much speculation on whether mammalian eggs with two male pronuclei can develop normally. Eggs with two female pronuclei can sometimes develop as far as the 25-somite stage but with only very meagre extraembryonic tissues. We suggested that the genome undergoes specific imprinting during gametogenesis and that some paternal genes may be necessary for normal development of the extraembryonic tissues, in which only the maternal X chromosome remains active. However, the need for the maternal genome for development to term is not yet unequivocally established. The detailed study described here demonstrates that while between 40 and 50% of heterozygous reconstituted eggs with a male and a female pronucleus develop to term, none of the eggs with two male pronuclei does so. Furthermore, embryos in the latter case are very retarded, even though the trophoblast develops relatively well compared with embryos having two female pronuclei. Our combined results indicate that while the paternal genome is essential for the normal development of extraembryonic tissues, the maternal genome may be essential for some stages of embryogenesis.     

Exchange of terminal portions of X- and Y-chromosomal short arms in human XX males

In most human 'XX males', DNA sequences normally found on Yp, the short arm of the Y chromosome, are present on Xp, the short arm of the X chromosome. To establish whether this transfer involves a terminal portion of Yp, and whether a terminal portion of Xp is lost in the process, we followed the inheritance of pseudoautosomal restriction fragment length polymorphisms in two XX-male families. One XX male apparently inherited the entire pseudoautosomal region of his father's Y chromosome and no part of the pseudoautosomal region of his father's X chromosome. The second XX male also inherited the entire pseudoautosomal region of his father's Y, but in addition inherited a proximal portion of the pseudoautosomal region of his father's X. These findings argue that XX males result from the transfer of a terminal portion of Yp onto Xp in exchange for a terminal portion of Xp (ref. 7). This implies that the testis-determining factor gene (TDF) maps distally in the strictly sex-linked portion of Yp, near the pseudoautosomal domain. The XX males described here appear to result from single (and, at least in the second case, unequal) crossovers proximal to the pseudoautosomal region on Yp and proximal to or within the pseudoautosomal region on Xp.     

Isolation of the dorsal locus of Drosophila

The establishment of embryonic polarity is a crucial step in pattern formation and morphogenesis. In the fruitfly Drosophila melanogaster, embryonic polarity depends primarily on genes expressed in the female during oogenesis. Mutations in these 'maternal effect' genes can lead to major disruptions in normal pattern formation. Two classes of maternal genes essential for the establishment of polarity in the embryo have been identified. Lesions in one class, the 'bicaudal' genes, disrupt the anterior-posterior axis; lesions in the other class disrupt dorsal-ventral polarity, and in the most extreme cases embryos fail to form any ventral or lateral structures. Genetic studies suggest that the anterior-posterior and dorsal-ventral axes may be independent as the defects observed in mutants from each class seem to be restricted to one axis only. The dorsal (dl) locus is one of the maternal effect genes involved in the establishment of dorsal-ventral polarity. Homozygous dl females produce embryos exhibiting the mutant phenotype--complete lack of dorsal-ventral polarity in the strongest alleles--irrespective of the genotype of the father. Although dl is a maternal effect locus and must be expressed during oogenesis, the gene product, or a substance depending on the normal function of the dl gene, seems to be active early in embryogenesis, as the dl phenotype can be partially rescued by injection of cytoplasm from wild-type cleavage-stage embryos. Here we report the molecular cloning of the dorsal locus and a study of its expression.