Autosomal location of genes from the conserved mammalian X in the platypus (<Emphasis Type="Italic">Ornithorhynchus anatinus</Emphasis>): implications for mammalian sex chromosome evolution

Mammalian sex chromosomes evolved from an ancient autosomal pair. Mapping of human X- and Y-borne genes in distantly related mammals and non-mammalian vertebrates has proved valuable to help deduce the evolution of this unique part of the genome. The platypus, a monotreme mammal distantly related to eutherians and marsupials, has an extraordinary sex chromosome system comprising five X and five Y chromosomes that form a translocation chain at male meiosis. The largest X chromosome (X₁), which lies at one end of the chain, has considerable homology to the human X. Using comparative mapping and the emerging chicken database, we demonstrate that part of the therian X chromosome, previously thought to be conserved across all mammals, was lost from the platypus X₁ to an autosome. This region included genes flanking the XIST locus, and also genes with Y-linked homologues that are important to male reproduction in therians. Since these genes lie on the X in marsupials and eutherians, and also on the homologous region of chicken chromosome 4, this represents a loss from the monotreme X rather than an additional evolutionary stratum of the human X.     

Molecular characterization and evolution of X and Y-borne <Emphasis Type="Italic">ATRX</Emphasis> homologues in American marsupials

In eutherians, the sex-reversing ATRX gene on the X has no homologue on the Y chromosome. However, testis-specific and ubiquitously expressed X-borne genes have been identified in Australian marsupials. We studied nucleotide sequence and chromosomal location of ATRX homologues in two American marsupials, the opossums Didelphis virginiana and Monodelphis domestica. A PCR fragment of M. domestica ATRX was used to probe Southern blots and to screen male genomic libraries. Southern analysis demonstrated ATRX homologues on both X and Y in D. virginiana, and two clones were isolated which hybridized to a single position on the Y chromosome in male-derived cells but to multiple sites of the X in female cells. In M. domestica, there was a single clone that mapped to the X but not to the Y, suggesting that it represents the M. domestica ATRX. However a male-specific band was detected in Southern blots probed with the D. virginiana ATRY and with a mouse ATRX clone, which implies that the Y copy in M. domestica has diverged further from other ATRX homologues. Thus there appears to be a Y-borne copy of ATRY in American, as well as Australian marsupials, although it has diverged in sequence, as have other Y genes that are testis-specific in both eutherian and marsupial lineages.     

Isolation,X location and activity of the marsupial homologue of <Emphasis Type="Italic">SLC16A2</Emphasis>, an <Emphasis Type="Italic">XIST</Emphasis>-flanking gene in eutherian mammals

X chromosome inactivation (XCI) achieves dosage compensation between males and females for most X-linked genes in eutherian mammals. It is a whole-chromosome effect under the control of the XIST locus, although some genes escape inactivation. Marsupial XCI differs from the eutherian process, implying fundamental changes in the XCI mechanism during the evolution of the two lineages. There is no direct evidence for the existence of a marsupial XIST homologue. XCI has been studied for only a handful of genes in any marsupial, and none in the model kangaroo Macropus eugenii (the tammar wallaby). We have therefore studied the sequence, location and activity of a gene SLC16A2 (solute carrier, family 16, class A, member 2) that flanks XIST on the human and mouse X chromosomes. A BAC clone containing the marsupial SLC16A2 was mapped to the end of the long arm of the tammar X chromosome and used in RNA FISH experiments to determine whether one or both loci are transcribed in female cells. In male and female cells, only a single signal was found, indicating that the marsupial SLC16A2 gene is silenced on the inactivated X.     

Lessons from comparative analysis of X-chromosome inactivation in mammals

In most mammals, X-chromosome inactivation is used as the strategy to achieve dosage compensation between XX females and XY males. This process is developmentally regulated, resulting in the differential treatment of the two X chromosomes in the same nucleus and mitotic heritability of the silent state. A lack of dosage compensation in an XX embryo is believed to result in early lethality, at least in eutherians. Given its fundamental importance, X-chromosome inactivation would be predicted to be a highly conserved process in mammals. However, recent studies have revealed major mechanistic differences in X inactivation between eutherians and marsupials, suggesting that the evolution of the X chromosome as well as developmental differences between mammals have led to diverse evolutionary strategies for dosage compensation.     

Specific patterns of histone marks accompany X chromosome inactivation in a marsupial

The inactivation of one of the two X chromosomes in female placental mammals represents a remarkable example of epigenetic silencing. X inactivation occurs also in marsupial mammals, but is phenotypically different, being incomplete, tissue-specific and paternal. Paternal X inactivation occurs also in the extraembryonic cells of rodents, suggesting that imprinted X inactivation represents a simpler ancestral mechanism. This evolved into a complex and random process in placental mammals under the control of the XIST gene, involving notably variant and modified histones. Molecular mechanisms of X inactivation in marsupials are poorly known, but occur in the absence of an XIST homologue. We analysed the specific pattern of histone modifications using immunofluorescence on metaphasic chromosomes of a model kangaroo, the tammar wallaby. We found that all active marks are excluded from the inactive X in marsupials, as in placental mammals, so this represents a common feature of X inactivation throughout mammals. However, we were unable to demonstrate the accumulation of inactive histone marks, suggesting some fundamental differences in the molecular mechanism of X inactivation between marsupial and placental mammals. A better understanding of the epigenetic mechanisms underlying X inactivation in marsupials will provide important insights into the evolution of this complex process. Edda Koina and Julie Chaumeil contributed equally to this work.     

Comparative mapping of X chromosomes in vole species of the genus Microtus

Comparative mapping of X-linked genes has progressed rapidly since Ohno's prediction that genes on the X chromosome should be conserved as a syntenic group in all mammals. Although several conserved blocks of homology between human and mouse have been discovered, rearrangements within the X chromosome have also been characterized. More recently, some exceptions to Ohno's law have been reported. We have used fluorescence in situ hybridization (FISH) to map five genes, Gla, G6pd, Hprt, Pgk1 and Xist, to two of the largest conserved segments of X material in five members of the genus Microtus (grey vole) and show that vole X chromosomes demonstrate greater homology to human than to mouse. Cytogenetic analysis indicates a relatively high frequency of rearrangement during vole evolution, although certain blocks of homology appear to be highly conserved in all species studied to date. On this basis we were able to predict the probable location of the rat X inactivation centre (Xic) based solely on high-resolution G-banding. Our prediction was then confirmed by mapping the rat Xist gene by FISH. The possible significance of conserving long-range chromosome structure in the vicinity of the Xic is discussed with respect to the mechanism of X inactivation.     

Transgenic mice carrying an Xist-containing YAC

The initiation of X-chromosome inactivation in female mammals is controlled by a key locus, the X-inactivation centre (Xic). The Xist gene, which maps to the candidate region for Xic and is expressed exclusively from the inactive X chromosome, is thought to be an essential component of the Xic. To test whether sequences spanning several hundred kilobases and including Xist from the Xic region are capable of initiating inactivation, we have created a series of transgenic mice using a 460 kb yeast artificial chromosome (YAC). Analysis in these mice of the expression of Xist, of a LacZ reporter gene and of two genes in the region that are normally silent on the inactive X chromosome, suggests that essential sequences for Xist expression and X-inactivation may be absent in these transgenic animals.      

Human postmeiotic sex chromatin and its impact on sex chromosome evolution

Sex chromosome inactivation is essential epigenetic programming in male germ cells. However, it remains largely unclear how epigenetic silencing of sex chromosomes impacts the evolution of the mammalian genome. Here we demonstrate that male sex chromosome inactivation is highly conserved between humans and mice and has an impact on the genetic evolution of human sex chromosomes. We show that, in humans, sex chromosome inactivation established during meiosis is maintained into spermatids with the silent compartment postmeiotic sex chromatin (PMSC). Human PMSC is illuminated with epigenetic modifications such as trimethylated lysine 9 of histone H3 and heterochromatin proteins CBX1 and CBX3, which implicate a conserved mechanism underlying the maintenance of sex chromosome inactivation in mammals. Furthermore, our analyses suggest that male sex chromosome inactivation has impacted multiple aspects of the evolutionary history of mammalian sex chromosomes: amplification of copy number, retrotranspositions, acquisition of de novo genes, and acquisition of different expression profiles. Most strikingly, profiles of escape genes from postmeiotic silencing diverge significantly between humans and mice. Escape genes exhibit higher rates of amino acid changes compared with non-escape genes, suggesting that they are beneficial for reproductive fitness and may allow mammals to cope with conserved postmeiotic silencing during the evolutionary past. Taken together, we propose that the epigenetic silencing mechanism impacts the genetic evolution of sex chromosomes and contributed to speciation and reproductive diversity in mammals.     

The search for a marsupial XIC reveals a break with vertebrate synteny

X-chromosome inactivation (XCI) evolved in mammals to deal with X-chromosome dosage imbalance between the XX female and the XY male. In eutherian mammals, random XCI of the soma requires a master regulatory locus known as the ‘X-inactivation center’ (XIC/Xic), wherein lies the noncoding XIST/Xist silencer RNA and its regulatory antisense Tsix gene. By contrast, marsupial XCI is imprinted to occur on the paternal X chromosome. To determine whether marsupials and eutherians share the XIC-driven mechanism, we search for the sequence equivalents in the genome of the South American opossum, Monodelphis domestica. Positional cloning and bioinformatic analysis reveal several interesting findings. First, protein-coding genes that flank the eutherian XIC are well-conserved in M. domestica, as well as in chicken, frog, and pufferfish. However, in M. domestica we fail to identify any recognizable XIST or TSIX equivalents. Moreover, cytogenetic mapping shows a surprising break in synteny with eutherian mammals and other vertebrates. Therefore, during the evolution of the marsupial X chromosome, one or more rearrangements broke up an otherwise evolutionarily conserved block of vertebrate genes. The failure to find XIST/TSIX in M. domestica may suggest that the ancestral XIC is too divergent to allow for detection by current methods. Alternatively, the XIC may have arisen relatively late in mammalian evolution, possibly in eutherians with the emergence of random XCI. The latter argues that marsupial XCI does not require XIST and opens the search for alternative mechanisms of dosage compensation.     

Clcn4-2 genomic structure differs between the X locus in Mus spretus and the autosomal locus in Mus musculus: AT motif enrichment on the X

In Mus spretus, the chloride channel 4 gene Clcn4-2 is X-linked and dosage compensated by X up-regulation and X inactivation, while in the closely related mouse species Mus musculus, Clcn4-2 has been translocated to chromosome 7. We sequenced Clcn4-2 in M. spretus and identified the breakpoints of the evolutionary translocation in the Mus lineage. Genetic and epigenetic differences were observed between the 5'ends of the autosomal and X-linked loci. Remarkably, Clcn4-2 introns have been truncated on chromosome 7 in M. musculus as compared with the X-linked loci from seven other eutherian mammals. Intron sequences specifically preserved in the X-linked loci were significantly enriched in AT-rich oligomers. Genome-wide analyses showed an overall enrichment in AT motifs unique to the eutherian X (except for genes that escape X inactivation), suggesting a role for these motifs in regulation of the X chromosome.     

Dosage compensation in mammals: fine-tuning the expression of the X chromosome

Mammalian females have two X chromosomes and males have only one. This has led to the evolution of special mechanisms of dosage compensation. The inactivation of one X chromosome in females equalizes gene expression between the sexes. This process of X-chromosome inactivation (XCI) is a remarkable example of long-range, monoallelic gene silencing and facultative heterochromatin formation, and the questions surrounding it have fascinated biologists for decades. How does the inactivation of more than a thousand genes on one X chromosome take place while the other X chromosome, present in the same nucleus, remains genetically active? What are the underlying mechanisms that trigger the initial differential treatment of the two X chromosomes? How is this differential treatment maintained once it has been established, and how are some genes able to escape the process? Does the mechanism of X inactivation vary between species and even between lineages? In this review, X inactivation is considered in evolutionary terms, and we discuss recent insights into the epigenetic changes and developmental timing of this process. We also review the discovery and possible implications of a second form of dosage compensation in mammals that deals with the unique, potentially haploinsufficient, status of the X chromosome with respect to autosomal gene expression.     

The region homologous to the X-chromosome inactivation centre has been disrupted in marsupial and monotreme mammals

Summary Marsupial, as well as eutherian, mammals are subject to X chromosome inactivation in the somatic cells of females, although the phenotype and the molecular mechanism differ in important respects. Monotreme mammals appear to subscribe at least to a form of dosage compensation of X-borne genes. An important question is whether inactivation in these non-eutherian mammals involves co-ordination by a control locus homologous to the XIST gene and neighbouring genes, which play a key regulatory role in human and mouse X inactivation. We mapped BACs containing several orthologues of protein-coding genes that flank human and mouse XIST and genes that lie in the homologous region in chicken and frog. We found that these genes map to two distant locations on the opossum X, and also to different locations on a platypus autosome. We failed to find any trace of an XIST orthologue in any marsupial or monotreme or on any flanking BAC, confirming the conclusion from recent work that non-eutherian mammals lack XIST. We propose the region homologous to the human and mouse X-inactivation centre expanded in early mammals, and this unstable region was disrupted independently in marsupial and monotreme lineages. In the eutherian lineage, inserted and existing sequences provided the starting material for the non-translated RNAs of the X-inactivation centre, including XIST. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.