Chromodomains are protein-RNA interaction modules

In Drosophila, compensation for the reduced dosage of genes located on the single male X chromosome involves doubling their expression in relation to their counterparts on female X chromosomes. Dosage compensation is an epigenetic process involving the specific acetylation of histone H4 at lysine 16 by the histone acetyltransferase MOF. Although MOF is expressed in both sexes, it only associates with the X chromosome in males. Its absence causes male-specific lethality. MOF is part of a chromosome-associated complex comprising male-specific lethal (MSL) proteins and at least one non-coding roX RNA. How MOF is integrated into the dosage compensation complex is unknown. Here we show that association of MOF with the male X chromosome depends on its interaction with RNA. MOF specifically binds through its chromodomain to roX2 RNA in vivo. In vitro analyses of the MOF and MSL-3 chromodomains indicate that these chromodomains may function as RNA interaction modules. Their interaction with non-coding RNA may target regulators to specific chromosomal sites.     

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.     

非整倍体胚胎发育中MSL复合体组分的RNA表达分析

MSL (male specific lethal) 复合体作为调节果蝇剂量补偿的关键元件，在非整倍体基因表达调控过程中发挥了重要作用．为深入探究非整倍体胚胎发育过程的分子调控机制，运用TSA-FISH (tyramide signal amplification- fluorescence in situ hybridization) 技术比较分析正常二倍体胚胎与常染色体三体胚胎中的MSL复合体组分基因表达模式，探究复合体重要组分在果蝇非整倍体胚胎不同时期的表达水平，以及亚细胞定位的变化．研究结果表明，在MSL复合体尚未组装的非整倍体胚胎发育早期，即母体表达阶段，多数复合体组分基因就已存在转录本水平上的差异，而这种表达差异主要源自于母体的非单倍体配子．随着发育的进行，染色体片段的增加导致基因组内剂量平衡发生变化，由此产生的反式剂量效应将引发MSL复合体各组分表达水平的差异变化，而这种差异将持续作用于后续非整倍体胚胎发育的各个时期．      

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.     

X-chromosome inactivation may explain the difference in viability of XO humans and mice

Only about 1% of human XO conceptuses survive to birth and these usually have the characteristics of Turner's syndrome, with a complex and variable phenotype including short stature, gonadal dysgenesis and anatomical defects. Both the embryonic lethality and Turner's syndrome are thought to be due to monosomy for a gene or genes common to the X and Y chromosomes. These genes would be expected to be expressed in females from both active and inactive X chromosomes to ensure correct dosage of gene product. Two genes with these properties are ZFX and RPS4X, both of which have been proposed to play a role in Turner's syndrome. In contrast to humans, mice that are XO are viable with no prenatal lethality (P. Burgoyne, personal communication) and are anatomically normal and fertile. We have devised a system to analyse whether specific genes on the mouse X chromosome are inactivated, and demonstrate that both Zfx and Rps4X undergo normal X-inactivation in mice. Thus the relative viability of XO mice compared to XO humans may be explained by differences between the two species in the way that dosage compensation of specific genes is achieved.     

The DNA sequence of the human X chromosome

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.     

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.     

Implications of fragile X expression in normal males for the nature of the mutation

The fragile site at Xq27, associated with a common form of X-linked mental retardation (XLMR), is expressed in a variable proportion of the peripheral lymphocytes of affected males when the cells are cultured under thymidylate stress (Td stress) produced by folate or thymidylate deprivation. Some clinically normal males--transmitting males--are known to carry and transmit the fragile X mutation and yet show no cytogenetic expression in lymphocytes. Normal males with no family history of X-linked mental retardation express the site only rarely. When the fragile X chromosome from affected males is isolated in a rodent genetic background by somatic cell hybridization, the level of expression is similar to that seen in lymphocytes under Td stress. Here we show that X chromosomes from two transmitting males and two normal control males, all of which were fragile X negative in lymphocytes or lymphoblasts, could be made to express the fragile site in hybrids, although at levels that were below those seen in hybrids from affected males. Furthermore, transmitting males could be differentiated from normal males by their significantly higher expression rates when hybrids were exposed to caffeine before cytogenetic harvest. One male chimpanzee also showed low level expression in hybrid cells. These data suggest that the hybrid system lowers the threshold for fragile X expression, a fragile site at Xq27 may be present on all human and chimpanzee X chromosomes and constitutes a previously unrecognized common fragile site and the hybrid system with caffeine post-treatment can distinguish between the common Xq27 fragile site of control males, the occult mutant fragile site of a transmitting male, and the fully expressed fragile site of an affected male with XLMR. Thus the mutation producing XLMR may represent a multi-step alteration of a naturally occurring DNA sequence producing a continuum of cytogenetic expression and a threshold for clinical manifestation.     

Isolation of a cDNA clone corresponding to an X-linked gene family (XLR) closely linked to the murine immunodeficiency disorder xid

The striking number of human and murine immunodeficiency disorders which map to the X chromosome suggests that genes localized on this chromosome must have important roles in lymphocyte development. At least seven distinct disorders in the human and two in the mouse disrupt lymphocyte maturation, particularly that of B cells, at characteristic stages. As functional genes mapping to the X chromosome in one mammal are found on the X chromosome in all other mammals, the same genes regulating lymphocyte development are expected to be found on the X chromosome in mouse and man. Investigations into the possible mechanisms of these X-linked disorders have been hampered by the lack of molecular probes for the genes or gene products affected; because of this, and the possibility of correlating one or more of the several hundred B- or T-cell-specific genes with a specific mutation, we surveyed 15 different B- and T-cell-specific cDNA clones for localization to the X chromosome. We report here the characterization of one of these murine cDNA clones, which hybridizes with a large, X-linked gene family, designated XLR (X-linked, lymphocyte-regulated). We show that the XLR gene family is closely linked to the X-linked immunodeficiency described in the CBA/N mouse strain (xid), by restriction fragment length polymorphism (RFLP) analysis of DNA from mice congeneic for xid. This finding, together with data on the expression of the XLR locus in B cells, indicates that this gene family either includes the locus defined by the xid mutation or is adjacent to it in a gene complex which may be important in lymphocyte differentiation.     

In the platypus a meiotic chain of ten sex chromosomes shares genes with the bird Z and mammal X chromosomes

Two centuries after the duck-billed platypus was discovered, monotreme chromosome systems remain deeply puzzling. Karyotypes of males, or of both sexes, were claimed to contain several unpaired chromosomes (including the X chromosome) that form a multi-chromosomal chain at meiosis. Such meiotic chains exist in plants and insects but are rare in vertebrates. How the platypus chromosome system works to determine sex and produce balanced gametes has been controversial for decades. Here we demonstrate that platypus have five male-specific chromosomes (Y chromosomes) and five chromosomes present in one copy in males and two copies in females (X chromosomes). These ten chromosomes form a multivalent chain at male meiosis, adopting an alternating pattern to segregate into XXXXX-bearing and YYYYY-bearing sperm. Which, if any, of these sex chromosomes bears one or more sex-determining genes remains unknown. The largest X chromosome, with homology to the human X chromosome, lies at one end of the chain, and a chromosome with homology to the bird Z chromosome lies near the other end. This suggests an evolutionary link between mammal and bird sex chromosome systems, which were previously thought to have evolved independently.     

Maternal Rnf12/RLIM is required for imprinted X-chromosome inactivation in mice

Two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Imprinted XCI begins with the detection of Xist RNA expression on the paternal X?chromosome (Xp) at about the four-cell stage of embryonic development. In the embryonic tissues of the inner cell mass, a random form of XCI occurs in blastocysts that inactivates either Xp or the maternal X?chromosome (Xm). Both forms of XCI require the non-coding Xist RNA that coats the inactive X?chromosome from which it is expressed. Xist has crucial functions in the silencing of X-linked genes, including Rnf12 (refs 3, 4) encoding the ubiquitin ligase RLIM (RING finger LIM-domain-interacting protein). Here we show, by targeting a conditional knockout of Rnf12 to oocytes where RLIM accumulates to high levels, that the maternal transmission of the mutant X?chromosome (Δm) leads to lethality in female embryos as a result of defective imprinted XCI. We provide evidence that in Δm female embryos the initial formation of Xist clouds and Xp silencing are inhibited. In contrast, embryonic stem cells lacking RLIM are able to form Xist clouds and silence at least some X-linked genes during random XCI. These results assign crucial functions to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.     

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.