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1.  The role of X-chromosome inactivation in female predisposition to autoimmunity 
Arthritis Research  2000;2(5):399-406.
We propose that the phenomenon of X-chromosome inactivation in females may constitute a risk factor for loss of T-cell tolerance; specifically that skewed X-chromosome inactivation in the thymus may lead to inadequate thymic deletion. Using a DNA methylation assay, we have examined the X-chromosome inactivation patterns in peripheral blood from normal females (n = 30), female patients with a variety of autoimmune diseases (n = 167). No differences between patients and controls were observed. However, locally skewed X-chromsome inactivation may exist in the thymus, and therefore the underlying hypothesis remains to be disproved.
Introduction:
A reduction in the sex ratio (male : female) is characteristic of most autoimmune disorders. The increased prevalence in females ranges from a modest 2:1 for multiple sclerosis [1], to approximately 10:1 for systemic lupus erythematosus [2]. This tendency toward autoimmunity in females is often ascribed to hormonal differences, because in a number of experimental disease models estrogens exacerbated disease, and androgens can inhibit disease activity [3,4]. However, human studies have failed to demonstrate a clear-cut influence of hormonal environment on disease susceptibility to lupus or other autoimmune disorders. In addition, many childhood forms of autoimmunity, such as juvenile rheumatoid arthritis, exhibit female predominance [5]. Interestingly, juvenile (type 1) diabetes is an exception to this general trend, with a sex ratio close to 1 in most studies [6]. Therefore, it is reasonable to consider alternative explanations for the increased prevalence of autoimmune diseases in human females.
A unifying feature of autoimmune disorders appears to be the loss of immunologic tolerance to self-antigens, and in many of these diseases there is evidence that T-cell tolerance has been broken. The most profound form of T-cell tolerance involves deletion of potentially self-reactive T cells during thymic selection. Thus, lack of exposure to a self-antigen in the thymus may lead to the presence of autoreactive T cells and may increase the risk of autoimmunity. An elegant example of this has recently been reported [7].
The existence of X-chromosome inactivation in females offers a potential mechanism whereby X-linked self-antigens may escape presentation in the thymus or in other peripheral sites that are involved in tolerance induction. Early in female development, one of the two X chromosomes in each cell undergoes an ordered process of inactivation, with subsequent silencing of most genes on the inactive X chromosome [8]. This phenomenon occurs at a very early embryonic stage [9], and thus all females are mosaic and may occasionally exhibit extreme skewing towards one or the other parental X chromosome. In theory, this may result in a situation in which polymorphic self-antigens on one X chromosome may fail to be expressed at sufficiently high levels in a tolerizing compartment, such as the thymus, and yet may be expressed at a considerable frequency in the peripheral soma. Thus, females may be predisposed to a situation in which they can occasionally express X-linked autoantigens in the periphery to which they have been inefficiently tolerized. Stewart [10] has recently speculated that such a mechanism may play a role in the predisposition to systemic lupus.
This hypothesis predicts that females with autoimmunity may be particularly prone to this mechanism of `inadequate tolerization' by virtue of extremely skewed X-chromosome inactivation. We therefore performed a comprehensive analysis of X-chromosome inactivation patterns in populations of females with multiple sclerosis, systemic lupus erythematosus, juvenile rheumatoid arthritis, and type 1 (insulin-dependent) diabetes mellitus, and in female control individuals. The results do not provide support for a major role for skewed X-chromosome inactivation in female predisposition to autoimmunity; however, neither is the underlying hypothesis disproved by the present data.
Materials and method:
DNA was obtained from female patients from the following sources: 45 persons with juvenile diabetes seen at the Virginia Mason Research Center in Seattle, Washington; 58 multiple sclerosis patients seen at the New York Hospital Multiple Sclerosis Center; 46 patients with systemic lupus erythematosus seen at the Hospital for Special Surgery (New York); 18 patients with juvenile rheumatoid arthritis seen at the Children's Hospital Medical Center in Cleveland. In addition, 30 healthy age-matched females were studied as normal controls.
Employing a modification of previously described methods [11], we utilized a fluorescent Hpa II/PCR assay of the androgen receptor (AR) locus to assess X-chromosome inactivation patterns. The AR gene contains a polymorphic CAG repeat, which is flanked by Hpa II sites. These Hpa II sites are methylated on the inactive X chromosome, and are unmethylated on the active X chromosome. By performing PCR amplification across this region after cutting with the methylation-sensitive enzyme Hpa II, the relative amounts of the methylated AR alleles can be quantitatively determined with a high degree of accuracy; variance on repeated assays is approximately 4% [12].
Skewing of X-chromosome inactivation is expressed as percentage deviation from equal (50:50) inactivation of the upper and lower AR alleles. Therefore, the maximal possible deviation is 50%, in which case all of the X chromosomes bearing one of the AR alleles are inactivated.
Results:
We examined X-chromosome inactivation patterns in several different populations. The results are summarized in Fig. 1. A wide range of X-inactivation skewing was observed in all five groups. Approximately 5% (nine out of 197) of individuals exhibited extreme skewing (greater than 40% deviation from a 50:50 distribution). However, there was no difference between the groups, either in the overall mean skewing, or in the fraction of individuals with extreme skewing (>40%).
Although the present study was not initiated in order to examine allelic variation in the AR gene per se, the data provide an opportunity to address this question. Excessively long CAG repeats in the AR are a rare cause of spinal-bulbar muscular atrophy [13], and AR repeat length appears to have an influence on the biology of certain tumors [14,15]. In this context, it has been shown that transcription of AR correlates inversely with repeat length [16]. We therefore compared AR repeat length in control individuals and patients with autoimmunity. No differences were observed for mean repeat length, or for maximum and minimum repeat length, among the five groups.
Discussion:
The reason for the female predominance in most autoimmune diseases remains obscure. The present study was initiated in order to address the hypothesis that a nonhormonal mechanism related to X inactivation might be involved. The hypothesis rests on the idea that skewing of X inactivation might lead to a deficiency of tolerance induction in the thymus, particularly with respect to polymorphic X-linked autoantigens. The hypothesis predicts that skewed X inactivation would be more prevalent in females with autoimmune diseases than in female control individuals. This was not observed.
Nevertheless, these negative data do not rule out a role for X inactivation in female predisposition to loss of tolerance. A general model for how this mechanism might operate is shown in Fig. 2. Thymocytes undergo selection in the thymic parenchyma and, in the case of negative selection, the selecting elements appear to be derived from the bone marrow and consist mainly of thymic dendritic cells. If the thymic dendritic cell population exhibits random X inactivation, it is highly likely that differentiating thymocytes will contact dendritic cells that express self-antigens on both X chromosomes. This situation is outlined schematically on the left side of Fig. 2. However, if there is extremely skewed X inactivation in the thymic dendritic cell population, a particular thymocyte might not come into contact with dendritic cells that express one of the two X chormosomes. This would lead to a situation where T cells may undergo thymic maturation without having been negatively selected for antigens that are expressed on the predominantly inactive X chromosome. This situation is shown on the right side of Fig. 2.
In order for this mechanism to be physiologically relevant, some assumptions must be made. First, defective tolerance from skewed X inactivation should only be directed at X-linked antigens that are polymorphic, and for which the individual is heterozygous. Thus, this mechanism would not be expected to lead to lack of tolerance commonly, unless there are at least several highly polymorphic X-linked autoantigens in the population that are involved in thymic deletion events. Second, if this actually leads to autoimmunity, it also predicts that the initial break in tolerance that leads to disease should involve an X-linked autoantigen that is expressed in a peripheral nontolerizing site or circumstance.
A recent report [7] has elegantly demonstrated the importance of thymic deletion events in predisposition to autoimmune disease. The proteolipid protein (PLP) autoantigen is expressed in alternatively spliced forms, which exhibit tissue specific expression. A nonspliced variant is expressed in peripheral neural tissue. However, in the thymus a splice variant results in the lack of thymic expression of an immunodominant peptide. This results in loss of tolerace of T cells to this peptide, presumably on the basis of lack of thymic deletion of thymocytes that are reactive with this antigen. Interestingly, PLP is encoded on the X chromsome. However, there is no evidence that genetic polymorphisms control the level splicing of PLP within the thymus. Nevertheless, these data illustrate the potential importance of deficiencies in thymic deletion for autoimmune T-cell reactivity.
The present results suggest that if skewed X inactivation is relevant to thymic tolerance induction, then the effect does not depend on global skewing of X-chromosome inactivation, at least in the hematopoietic compartment. In this study we examined X-inactivation patterns in peripheral blood mononuclear cells, and the results should reflect the state of X inactivation in all mesenchymal tissues, including dendritic cells. X inactivation occurs at a very early time point in development, and thus the results in one tissue should reflect the general situation in the rest of the body. However, there may be exceptions to this. We have occasionally observed differences in X-inactivation patterns between buccal mucosa (an ectodermally derived tissue) and peripheral blood in the same individiual (unpublished observations). This could be a chance event, or it may result from selection for certain X-linked alleles during embryonic development, as has been described in carriers of X-linked immunodeficiencies [17].
Another consideration is that certain tissue microenvironments may be derived from very small numbers of founder cells, and thus may exhibit skewed utilization of one or the other X chromosome, even if the tissue as a whole is not skewed. This situation could vary over time. Thus, there may be time points at which certain thymic microenvironments are populated by dendritic cells that, for stochastic reasons, all utilize the same X chromosome. This would create a `window of opportunity' in which a given thymocyte, in a given selecting location, could escape negative selection by antigens on the inactive X chromosome. The likelihood of this happening would obviously depend on the number of dendritic cells that are usually contacted by a thymocyte during thymic selection. There is limited information on this point, although Stewart [10] has theorized that this number may be as low as 15. If this is the case, then escape from thymic deletion may still occur in females who are heterozygous for a relevant X-linked antigen, even if the hematopoietic cells in general do not exhibit extreme skewing.
In conclusion, we suggest that X-chromosome inactivation needs to be considered as a potential factor in the predominance of females in most autoimmune diseases. Our inability to show an increase in X-chromosome skewing in females with autoimmunity does not eliminate this as an etiologic contributor to loss of immunologic tolerance. Future experiments must be directed at a detailed analysis of tissue patterns of X inactivation, as well as at a search for potential X-linked autoantigens.
PMCID: PMC17816  PMID: 11056674
autoimmunity; gender; immune tolerance; X chromosome
2.  Rapid De Novo Evolution of X Chromosome Dosage Compensation in Silene latifolia, a Plant with Young Sex Chromosomes 
PLoS Biology  2012;10(4):e1001308.
Evidence for dosage compensation in Silene latifolia, a plant with 10-million-year-old sex chromosomes, reveals that dosage compensation can evolve rapidly in young XY systems and is not an animal-specific phenomenon.
Silene latifolia is a dioecious plant with heteromorphic sex chromosomes that have originated only ∼10 million years ago and is a promising model organism to study sex chromosome evolution in plants. Previous work suggests that S. latifolia XY chromosomes have gradually stopped recombining and the Y chromosome is undergoing degeneration as in animal sex chromosomes. However, this work has been limited by the paucity of sex-linked genes available. Here, we used 35 Gb of RNA-seq data from multiple males (XY) and females (XX) of an S. latifolia inbred line to detect sex-linked SNPs and identified more than 1,700 sex-linked contigs (with X-linked and Y-linked alleles). Analyses using known sex-linked and autosomal genes, together with simulations indicate that these newly identified sex-linked contigs are reliable. Using read numbers, we then estimated expression levels of X-linked and Y-linked alleles in males and found an overall trend of reduced expression of Y-linked alleles, consistent with a widespread ongoing degeneration of the S. latifolia Y chromosome. By comparing expression intensities of X-linked alleles in males and females, we found that X-linked allele expression increases as Y-linked allele expression decreases in males, which makes expression of sex-linked contigs similar in both sexes. This phenomenon is known as dosage compensation and has so far only been observed in evolutionary old animal sex chromosome systems. Our results suggest that dosage compensation has evolved in plants and that it can quickly evolve de novo after the origin of sex chromosomes.
Author Summary
The mammalian sex chromosomes originated from an ancestral pair of autosomes about 150 million years ago and the Y chromosome subsequently degenerated, losing most of its genes. During this process, a phenomenon called dosage compensation evolved to compensate for the gene loss on the Y chromosome and to equalize expression of X-linked genes in the two sexes. In humans, this is achieved by inactivating one of the two X chromosomes in females. Dosage compensation has also been reported in other animal XY systems such as fruit flies and worms, each 100 million years old or more. Here we studied dosage compensation in plants. We used high-throughput RNA sequencing in male and female Silene latifolia (white campion)—a dioecious plant whose XY chromosomes originated only about 10 million years ago—to identify hundreds of sex-linked genes. Analysis of their expression patterns in males and females revealed equal doses of sex-linked transcripts in both sexes, regardless of the degree of reduction of Y expression due to degeneration. Our results thus show that dosage compensation occurs in plants and is thus not an animal-specific phenomenon. They also reveal that proportionate dosage compensation can evolve rapidly de novo after the origin of sex chromosomes.
doi:10.1371/journal.pbio.1001308
PMCID: PMC3328428  PMID: 22529744
3.  X Chromosome Inactivation during Drosophila Spermatogenesis 
PLoS Biology  2007;5(10):e273.
Genes with male- and testis-enriched expression are under-represented on the Drosophila melanogaster X chromosome. There is also an excess of retrotransposed genes, many of which are expressed in testis, that have “escaped” the X chromosome and moved to the autosomes. It has been proposed that inactivation of the X chromosome during spermatogenesis contributes to these patterns: genes with a beneficial function late in spermatogenesis should be selectively favored to be autosomal in order to avoid inactivation. However, conclusive evidence for X inactivation in the male germline has been lacking. To test for such inactivation, we used a transgenic construct in which expression of a lacZ reporter gene was driven by the promoter sequence of the autosomal, testis-specific ocnus gene. Autosomal insertions of this transgene showed the expected pattern of male- and testis-specific expression. X-linked insertions, in contrast, showed only very low levels of reporter gene expression. Thus, we find that X linkage inhibits the activity of a testis-specific promoter. We obtained the same result using a vector in which the transgene was flanked by chromosomal insulator sequences. These results are consistent with global inactivation of the X chromosome in the male germline and support a selective explanation for X chromosome avoidance of genes with beneficial effects late in spermatogenesis.
Author Summary
During spermatogenesis, the X chromosome is inactivated in the male germline (sperm cells), thereby silencing, or inactivating, genes residing on the X chromosome. X chromosome silencing is thought to be common among species with XY sex determination and has important implications for genome evolution. For example, genes with increased expression in the male tend to be under-represented on the X chromosome, and many testes-specific genes have been “retrotransposed,” or moved, from the sex to autosomal chromosomes. However, compelling evidence for X chromosome inactivation in the fruit fly Drosophila has been lacking. Here, we used a transgenic technique to test for male germline X inactivation in this important model organism. We randomly inserted a “reporter gene” whose expression requires a regulatory element for an autosomal testis-specific gene into multiple autosomal and X-chromosomal locations. We found that autosomal insertions of the reporter gene have significantly higher expression in the male germline than X-linked insertions. This pattern holds for two different transgenes with nearly 50 independent insertions, providing strong evidence for X chromosome inactivation during spermatogenesis. The silencing of X-linked gene expression in the male germline may contribute to the observed paucity of male-expressed genes on the X chromosome and the excess of retrotransposed genes that have moved from the X chromosome to the autosomes.
Genes expressed in the Drosophila testes are under-represented on the X chromosome to avoid the global inactivation of the X chromosome that occurs in the male germ line.
doi:10.1371/journal.pbio.0050273
PMCID: PMC2001211  PMID: 17927450
4.  Evidence of Activity-Specific, Radial Organization of Mitotic Chromosomes in Drosophila 
PLoS Biology  2011;9(1):e1000574.
A fluorescently labeled protein specifically binding to genes was reproducibly found at the periphery of condensed mitotic fruit fly chromosomes, illustrating preservation of a radial structure between consecutive divisions.
The organization and the mechanisms of condensation of mitotic chromosomes remain unsolved despite many decades of efforts. The lack of resolution, tight compaction, and the absence of function-specific chromatin labels have been the key technical obstacles. The correlation between DNA sequence composition and its contribution to the chromosome-scale structure has been suggested before; it is unclear though if all DNA sequences equally participate in intra- or inter-chromatin or DNA-protein interactions that lead to formation of mitotic chromosomes and if their mitotic positions are reproduced radially. Using high-resolution fluorescence microscopy of live or minimally perturbed, fixed chromosomes in Drosophila embryonic cultures or tissues expressing MSL3-GFP fusion protein, we studied positioning of specific MSL3-binding sites. Actively transcribed, dosage compensated Drosophila genes are distributed along the euchromatic arm of the male X chromosome. Several novel features of mitotic chromosomes have been observed. MSL3-GFP is always found at the periphery of mitotic chromosomes, suggesting that active, dosage compensated genes are also found at the periphery of mitotic chromosomes. Furthermore, radial distribution of chromatin loci on mitotic chromosomes was found to be correlated with their functional activity as judged by core histone modifications. Histone modifications specific to active chromatin were found peripheral with respect to silent chromatin. MSL3-GFP-labeled chromatin loci become peripheral starting in late prophase. In early prophase, dosage compensated chromatin regions traverse the entire width of chromosomes. These findings suggest large-scale internal rearrangements within chromosomes during the prophase condensation step, arguing against consecutive coiling models. Our results suggest that the organization of mitotic chromosomes is reproducible not only longitudinally, as demonstrated by chromosome-specific banding patterns, but also radially. Specific MSL3-binding sites, the majority of which have been demonstrated earlier to be dosage compensated DNA sequences, located on the X chromosomes, and actively transcribed in interphase, are positioned at the periphery of mitotic chromosomes. This potentially describes a connection between the DNA/protein content of chromatin loci and their contribution to mitotic chromosome structure. Live high-resolution observations of consecutive condensation states in MSL3-GFP expressing cells could provide additional details regarding the condensation mechanisms.
Author Summary
Mitotic chromosomes of eukaryotes are relatively large rod-like cellular organelles, about 1 µm in diameter and 10 µm long, of well-studied composition but unknown structure. The question of whether all DNA sequences equally contribute to the interactions leading to the formation of mitotic chromosomes has never been asked. To find an answer, we determined whether the radial positions of specific chromatin loci within mitotic chromosomes were reproduced at every cell cycle or were purely random. Based on fluorescence microscopy images of live or fixed chromosomes in cells from Drosophila embryos or Drosophila larval tissues expressing the MSL3-GFP fusion protein from a transgene, we report that the large-scale organization of mitotic chromosomes is reproduced not only longitudinally, as in the well-known chromosome banding phenomenon, but also radially. Actively transcribed, dosage-compensated genes of the Drosophila male X chromosome were always found at the periphery of mitotic chromosomes, starting from late prophase. Histone modifications specific to active chromatin were found to be more peripheral compared to silent chromatin that tended to be more central in the condensed chromosome. These findings are both exciting and significant for the field of cell and chromatin biology because they may help reconcile the old controversy between the existing models of chromosome structure that posit either radial loops of chromatin or consecutive coiling. In addition, we offer new insights into the mechanisms of mitotic condensation and suggest a link between structural and functional roles of different chromatin domains.
doi:10.1371/journal.pbio.1000574
PMCID: PMC3019107  PMID: 21264350
5.  The human Y chromosome. 
Journal of Medical Genetics  1985;22(5):329-344.
Despite its central role in sex determination, genetic analysis of the Y chromosome has been slow. This poor progress has been due to the paucity of available genetic markers. Whereas the X chromosome is known to include at least 100 functional genetic loci, only three or four loci have been ascribed to the Y chromosome and even the existence of several of these loci is controversial. Other factors limiting genetic analysis are the small size of the Y chromosome, which makes cytogenetic definition difficult, and the absence of extensive recombination. Based on cytogenetic observation and speculation, a working model of the Y chromosome has been proposed. In this classical model the Y chromosome is defined into subregions; an X-Y homologous meiotic pairing region encompassing most of the Y chromosome short arm and, perhaps, including a pseudoautosomal region of sex chromosome exchange; a pericentric region containing the sex determining gene or genes; and a long arm heterochromatic genetically inert region. The classical model has been supported by studies on the MIC2 loci, which encode a cell surface antigen defined by the monoclonal antibody 12E7. The X linked locus MIC2X, which escapes X inactivation, maps to the tip of the X chromosome short arm and the homologous locus MIC2Y maps to the Y chromosome short arm; in both cases, these loci are within the proposed meiotic pairing region. MIC2Y is the first biochemically defined, expressed locus to be found on the human Y chromosome. The proposed simplicity of the classical model has been challenged by recent molecular analysis of the Y chromosome. Using cloned probes, several groups have shown that a major part of the Y chromosome short arm is unlikely to be homologous to the X chromosome short arm. A substantial block of sequences of the short arm are homologous to sequences of the X chromosome long arm but well outside the pairing region. In addition, the short arm contains sequences shared with the Y chromosome long arm and sequences shared with autosomes. About two-thirds of XX males contain detectable Y derived sequences. As the amount of Y sequences present varies in different XX males, DNA from these subjects can be used to construct a map of the region around the sex determining gene. Assuming that XX males are usually caused by simple translocation, the sex determining genes cannot be located in the pericentric region. Although conventional genetic analysis of the Y chromosome is difficult, this chromosome is particularly suited to molecular analysis. Paradoxically, the Y chromosome may soon become the best defined human chromosome at the molecular level and may become the model for other chromosomes.
Images
PMCID: PMC1049475  PMID: 3908683
6.  Dosage Regulation of the Active X Chromosome in Human Triploid Cells 
PLoS Genetics  2009;5(12):e1000751.
In mammals, dosage compensation is achieved by doubling expression of X-linked genes in both sexes, together with X inactivation in females. Up-regulation of the active X chromosome may be controlled by DNA sequence–based and/or epigenetic mechanisms that double the X output potentially in response to autosomal factor(s). To determine whether X expression is adjusted depending on ploidy, we used expression arrays to compare X-linked and autosomal gene expression in human triploid cells. While the average X:autosome expression ratio was about 1 in normal diploid cells, this ratio was lower (0.81–0.84) in triploid cells with one active X and higher (1.32–1.4) in triploid cells with two active X's. Thus, overall X-linked gene expression in triploid cells does not strictly respond to an autosomal factor, nor is it adjusted to achieve a perfect balance. The unbalanced X:autosome expression ratios that we observed could contribute to the abnormal phenotypes associated with triploidy. Absolute autosomal expression levels per gene copy were similar in triploid versus diploid cells, indicating no apparent global effect on autosomal expression. In triploid cells with two active X's our data support a basic doubling of X-linked gene expression. However, in triploid cells with a single active X, X-linked gene expression is adjusted upward presumably by an epigenetic mechanism that senses the ratio between the number of active X chromosomes and autosomal sets. Such a mechanism may act on a subset of genes whose expression dosage in relation to autosomal expression may be critical. Indeed, we found that there was a range of individual X-linked gene expression in relation to ploidy and that a small subset (∼7%) of genes had expression levels apparently proportional to the number of autosomal sets.
Author Summary
Many organisms have a single X chromosome in males and two in females, leading to a chromosome imbalance between autosomes and sex chromosomes and between the sexes. In mammals, this dosage imbalance is adjusted by doubling expression of X-linked genes in both sexes and by silencing one X chromosome in females. We used expression array analyses of human triploid cultures to test X chromosome expression in the presence of three sets of autosomes and address the question of an autosomal counting factor. We found that overall X-linked gene expression is not tripled in the presence of three sets of autosomes. However, in triploid cells with a single active X chromosome, its expression is adjusted upward presumably by an epigenetic mechanism that senses the active X/autosome ratio. Based on the range of individual gene expression we identified a subset of dosage-sensitive genes whose expression is apparently proportional to the ploidy. Our findings are important for understanding the regulation of the X chromosome and the role of ploidy in the balance of gene expression and associated phenotypes.
doi:10.1371/journal.pgen.1000751
PMCID: PMC2777382  PMID: 19997486
7.  Accelerated Adaptive Evolution on a Newly Formed X Chromosome 
PLoS Biology  2009;7(4):e1000082.
Sex chromosomes originated from ordinary autosomes, and their evolution is characterized by continuous gene loss from the ancestral Y chromosome. Here, we document a new feature of sex chromosome evolution: bursts of adaptive fixations on a newly formed X chromosome. Taking advantage of the recently formed neo-X chromosome of Drosophila miranda, we compare patterns of DNA sequence variation at genes located on the neo-X to genes on the ancestral X chromosome. This contrast allows us to draw inferences of selection on a newly formed X chromosome relative to background levels of adaptation in the genome while controlling for demographic effects. Chromosome-wide synonymous diversity on the neo-X is reduced 2-fold relative to the ancestral X, as expected under recent and recurrent directional selection. Several statistical tests employing various features of the data consistently identify 10%–15% of neo-X genes as targets of recent adaptive evolution but only 1%–3% of genes on the ancestral X. In addition, both the rate of adaptation and the fitness effects of adaptive substitutions are estimated to be roughly an order of magnitude higher for neo-X genes relative to genes on the ancestral X. Thus, newly formed X chromosomes are not passive players in the evolutionary process of sex chromosome differentiation, but respond adaptively to both their sex-biased transmission and to Y chromosome degeneration, possibly through demasculinization of their gene content and the evolution of dosage compensation.
Author Summary
Sex chromosomes have evolved independently many times in both animals and plants from ordinary chromosomes. Much research on sex chromosome evolution has focused on the degeneration and loss of genes from the Y chromosome. Here, we describe another principle of sex chromosome evolution: bursts of adaptive fixations on a newly formed X chromosome. By employing a comparative population genomics approach and taking advantage of the recently formed sex chromosomes in the fruit fly Drosophila miranda, we show that rates of adaptation are increased about 10-fold on a newly formed X chromosome relative to background levels of selection in the genome. This suggests that a young X chromosome responds adaptively to both its female-biased transmission and to Y chromosome degeneration. Thus, contrary to the traditional view of being passive players, the X chromosome has a very active role in the evolutionary process of sex chromosome differentiation.
Research on sex chromosome molecular evolution has focused on the degeneration of the Y chromosome, but new evidence highlights that important changes occur on the evolving X chromosome in the form of rapid bursts of adaptive evolution.
doi:10.1371/journal.pbio.1000082
PMCID: PMC2672600  PMID: 19402745
8.  Chromosomal Redistribution of Male-Biased Genes in Mammalian Evolution with Two Bursts of Gene Gain on the X Chromosome 
PLoS Biology  2010;8(10):e1000494.
Two bursts of gene gains occurred on the mammalian X chromosome contribute to an age-dependent chromosomal distribution of male-biased genes.
Mammalian X chromosomes evolved under various mechanisms including sexual antagonism, the faster-X process, and meiotic sex chromosome inactivation (MSCI). These forces may contribute to nonrandom chromosomal distribution of sex-biased genes. In order to understand the evolution of gene content on the X chromosome and autosome under these forces, we dated human and mouse protein-coding genes and miRNA genes on the vertebrate phylogenetic tree. We found that the X chromosome recently acquired a burst of young male-biased genes, which is consistent with fixation of recessive male-beneficial alleles by sexual antagonism. For genes originating earlier, however, this pattern diminishes and finally reverses with an overrepresentation of the oldest male-biased genes on autosomes. MSCI contributes to this dynamic since it silences X-linked old genes but not X-linked young genes. This demasculinization process seems to be associated with feminization of the X chromosome with more X-linked old genes expressed in ovaries. Moreover, we detected another burst of gene originations after the split of eutherian mammals and opossum, and these genes were quickly incorporated into transcriptional networks of multiple tissues. Preexisting X-linked genes also show significantly higher protein-level evolution during this period compared to autosomal genes, suggesting positive selection accompanied the early evolution of mammalian X chromosomes. These two findings cast new light on the evolutionary history of the mammalian X chromosome in terms of gene gain, sequence, and expressional evolution.
Author Summary
Some evolutionary theories predict that the X chromosome will be enriched for genes with male functions. However, recent studies showed there had been gene traffic in which autosomal male-biased genes were retroposed from X-linked parental genes. A question remains about whether this pattern also holds for all types of new genes. Herein, using comparative genomic analysis, we dated all human and mouse genes to the vertebrate phylogenetic tree. We found that the X chromosome evolved with two bursts of gene origination events. The recent burst includes mainly male-biased genes in contrast to older X-linked genes that are often female-biased in expression. Meiotic sex chromosome inactivation contributes to this dynamic since it silences the older but not the younger X-linked genes. The older burst was after the split of eutherian mammals and the marsupial opossum, and the genes from this burst were quickly incorporated into transcriptional networks of multiple tissues, especially in the brain. The transcriptional expansion, together with the rapid protein evolution of the preexisting old X-linked genes, suggests that positive selection was acting in the early evolution of the mammalian X chromosome. These two lines of findings revealed extensive gene evolution in the mammalian X chromosome.
doi:10.1371/journal.pbio.1000494
PMCID: PMC2950125  PMID: 20957185
9.  Novel Gene Acquisition on Carnivore Y Chromosomes 
PLoS Genetics  2006;2(3):e43.
Despite its importance in harboring genes critical for spermatogenesis and male-specific functions, the Y chromosome has been largely excluded as a priority in recent mammalian genome sequencing projects. Only the human and chimpanzee Y chromosomes have been well characterized at the sequence level. This is primarily due to the presumed low overall gene content and highly repetitive nature of the Y chromosome and the ensuing difficulties using a shotgun sequence approach for assembly. Here we used direct cDNA selection to isolate and evaluate the extent of novel Y chromosome gene acquisition in the genome of the domestic cat, a species from a different mammalian superorder than human, chimpanzee, and mouse (currently being sequenced). We discovered four novel Y chromosome genes that do not have functional copies in the finished human male-specific region of the Y or on other mammalian Y chromosomes explored thus far. Two genes are derived from putative autosomal progenitors, and the other two have X chromosome homologs from different evolutionary strata. All four genes were shown to be multicopy and expressed predominantly or exclusively in testes, suggesting that their duplication and specialization for testis function were selected for because they enhance spermatogenesis. Two of these genes have testis-expressed, Y-borne copies in the dog genome as well. The absence of the four newly described genes on other characterized mammalian Y chromosomes demonstrates the gene novelty on this chromosome between mammalian orders, suggesting it harbors many lineage-specific genes that may go undetected by traditional comparative genomic approaches. Specific plans to identify the male-specific genes encoded in the Y chromosome of mammals should be a priority.
Synopsis
Y chromosomes are typically gene poor and enriched with repetitive elements, making them difficult to sequence by standard methods. Hence, the Y chromosome gene repertoire in mammalian species other than human has not been explored until very recently. Here the authors used a directed approach to isolate Y chromosome genes of the domestic cat, an evolutionary divergent species from human and mouse. They found that the feline Y chromosome harbors its own unique set of genes that are expressed specifically in the testes, presumably where they play an important role in spermatogenesis. Paralleling the discoveries seen from the full human Y chromosome sequence, the feline Y chromosome has acquired and remodeled some genes from autosomes, while other genes have a shared ancestry with the X chromosome. However, none of the four new genes are found on the Y chromosomes of human or mouse, although two are shared with the canine Y chromosome. This work highlights the Y chromosome as a source of potential gene novelty in different species and suggests that more directed efforts at characterizing this hitherto understudied chromosome will further enrich our understanding of the types of genes found there and the roles they may play in mammalian spermatogenesis.
doi:10.1371/journal.pgen.0020043
PMCID: PMC1420679  PMID: 16596168
10.  B Chromosomes Have a Functional Effect on Female Sex Determination in Lake Victoria Cichlid Fishes 
PLoS Genetics  2011;7(8):e1002203.
The endemic cichlid fishes in Lake Victoria are a model system for speciation through adaptive radiation. Although the evolution of the sex-determination system may also play a role in speciation, little is known about the sex-determination system of Lake Victoria cichlids. To understand the evolution of the sex-determination system in these fish, we performed cytogenetic analysis in 11 cichlid species from Lake Victoria. B chromosomes, which are present in addition to standard chromosomes, were found at a high prevalence rate (85%) in these cichlids. In one species, B chromosomes were female-specific. Cross-breeding using females with and without the B chromosomes demonstrated that the presence of the B chromosomes leads to a female-biased sex ratio in this species. Although B chromosomes were believed to be selfish genetic elements with little effect on phenotype and to lack protein-coding genes, the present study provides evidence that B chromosomes have a functional effect on female sex determination. FISH analysis using a BAC clone containing B chromosome DNA suggested that the B chromosomes are derived from sex chromosomes. Determination of the nucleotide sequences of this clone (104.5 kb) revealed the presence of several protein-coding genes in the B chromosome, suggesting that B chromosomes have the potential to contain functional genes. Because some sex chromosomes in amphibians and arthropods are thought to be derived from B chromosomes, the B chromosomes in Lake Victoria cichlids may represent an evolutionary transition toward the generation of sex chromosomes.
Author Summary
The diversity of sex chromosomes among animal species is well known, but how these sex chromosomes emerged during evolutionary history remains to be solved. One hypothesis for the origin of sex chromosomes is that a portion of the sex chromosome was derived from B chromosomes. In about 10% of eukaryotes, B chromosomes are found in addition to standard chromosomes (sex chromosomes and autosomes). B chromosomes have been thought to be selfish genetic elements with no functional effect on the phenotype of individuals and have been thought to lack protein-coding genes. Although B chromosomes share unique features with sex chromosomes, concrete evidence describing which B chromosomes have evolved to gain a function in sex determination has not been reported. In this study, we found that B chromosomes in one cichlid species from Lake Victoria have a functional effect on sex determination. Moreover, we found that they contained multiple protein-coding genes including morphogenetic related genes. These findings support the hypothesis that a portion of the sex chromosomes has been derived from B chromosomes and shed light on the study of the evolution of sex chromosomes.
doi:10.1371/journal.pgen.1002203
PMCID: PMC3158035  PMID: 21876673
11.  Convergent Evolution of Chicken Z and Human X Chromosomes by Expansion and Gene Acquisition 
Nature  2010;466(7306):612-616.
In birds, as in mammals, one pair of chromosomes differs between the sexes. In birds, males are ZZ and females ZW. In mammals, males are XY and females XX. Like the mammalian XY pair, the avian ZW pair is believed to have evolved from autosomes, with most change occurring in the chromosomes found in only one sex – the W and Y chromosomes1–5. By contrast, the sex chromosomes found in both sexes – the Z and X chromosomes – are assumed to have diverged little from their autosomal progenitors2. Here we report findings that overturn this assumption for both the chicken Z and human X chromosomes. The chicken Z chromosome, which we sequenced essentially to completion, is less gene-dense than chicken autosomes but contains a massive tandem array containing hundreds of duplicated genes expressed in testes. A comprehensive comparison of the chicken Z chromosome to the finished sequence of the human X chromosome demonstrates that each evolved independently from different portions of the ancestral genome. Despite this independence, the chicken Z and human X chromosomes share features that distinguish them from autosomes: the acquisition and amplification of testis-expressed genes, as well as a low gene density resulting from an expansion of intergenic regions. These features were not present on the autosomes from which the Z and X chromosomes originated but were instead acquired during the evolution of the Z and X as sex chromosomes. We conclude that the avian Z and mammalian X chromosomes followed convergent evolutionary trajectories, despite their evolving with opposite (female vs. male) systems of heterogamety. More broadly, in birds and mammals, sex chromosome evolution involved not only gene loss in sex-specific chromosomes, but also marked expansion and gene acquisition in sex chromosomes common to males and females.
doi:10.1038/nature09172
PMCID: PMC2943333  PMID: 20622855
12.  A Complex Suite of Forces Drives Gene Traffic from Drosophila X Chromosomes 
Theoretical studies predict X chromosomes and autosomes should be under different selection pressures, and there should therefore be differences in sex-specific and sexually antagonistic gene content between the X and the autosomes. Previous analyses have identified an excess of genes duplicated by retrotransposition from the X chromosome in Drosophila melanogaster. A number of hypotheses may explain this pattern, including mutational bias, escape from X-inactivation during spermatogenesis, and the movement of male-favored (sexually antagonistic) genes from a chromosome that is predominantly carried by females. To distinguish among these processes and to examine the generality of these patterns, we identified duplicated genes in nine sequenced Drosophila genomes. We find that, as in D. melanogaster, there is an excess of genes duplicated from the X chromosome across the genus Drosophila. This excess duplication is due almost completely to genes duplicated by retrotransposition, with little to no excess from the X among genes duplicated via DNA intermediates. The only exception to this pattern appears within the burst of duplication that followed the creation of the Drosophila pseudoobscura neo-X chromosome. Additionally, we examined genes relocated among chromosomal arms (i.e., genes duplicated to new locations coupled with the loss of the copy in the ancestral locus) and found an excess of genes relocated off the ancestral X and neo-X chromosomes. Interestingly, many of the same genes were duplicated or relocated from the independently derived neo-X chromosomes of D. pseudoobscura and Drosophila willistoni, suggesting that natural selection favors the traffic of genes from X chromosomes. Overall, we find that the forces driving gene duplication from X chromosomes are dependent on the lineage in question, the molecular mechanism of duplication considered, the preservation of the ancestral copy, and the age of the X chromosome.
doi:10.1093/gbe/evp018
PMCID: PMC2817413  PMID: 20333188
gene duplication; retrotransposition; sex chromosomes; neo-X chromosomes; X-inactivation
13.  Targeting Determinants of Dosage Compensation in Drosophila 
PLoS Genetics  2006;2(2):e5.
The dosage compensation complex (DCC) in Drosophila melanogaster is responsible for up-regulating transcription from the single male X chromosome to equal the transcription from the two X chromosomes in females. Visualization of the DCC, a large ribonucleoprotein complex, on male larval polytene chromosomes reveals that the complex binds selectively to many interbands on the X chromosome. The targeting of the DCC is thought to be in part determined by DNA sequences that are enriched on the X. So far, lack of knowledge about DCC binding sites has prevented the identification of sequence determinants. Only three binding sites have been identified to date, but analysis of their DNA sequence did not allow the prediction of further binding sites. We have used chromatin immunoprecipitation to identify a number of new DCC binding fragments and characterized them in vivo by visualizing DCC binding to autosomal insertions of these fragments, and we have demonstrated that they possess a wide range of potential to recruit the DCC. By varying the in vivo concentration of the DCC, we provide evidence that this range of recruitment potential is due to differences in affinity of the complex to these sites. We were also able to establish that DCC binding to ectopic high-affinity sites can allow nearby low-affinity sites to recruit the complex. Using the sequences of the newly identified and previously characterized binding fragments, we have uncovered a number of short sequence motifs, which in combination may contribute to DCC recruitment. Our findings suggest that the DCC is recruited to the X via a number of binding sites of decreasing affinities, and that the presence of high- and moderate-affinity sites on the X may ensure that lower-affinity sites are occupied in a context-dependent manner. Our bioinformatics analysis suggests that DCC binding sites may be composed of variable combinations of degenerate motifs.
Synopsis
In fruit flies, just like in humans, the two sexes are distinguished by different sex chromosomes. Females have two X chromosomes and hence a double dose of all X-linked genes when compared to males, which only have a single X chromosome. This different gene dosage needs to be compensated for by adjusting transcription levels such that male and female cells synthesize equal amounts of gene products. In Drosophila melanogaster, dosage compensation occurs by doubling the transcription of many genes on the single male X. This chromosome-wide control is achieved by a male-specific dosage compensation complex (DCC), which contains enzymes, structural proteins, and non-coding RNA. How is the DCC able to distinguish the X chromosome from the autosomes for selective interaction? In the following article, the authors identify and characterize several novel DNA sequences on the X chromosome that can recruit the DCC. Their results suggest that the X chromosome contains a large number of binding sites for the DCC, which are made up of combinations of degenerate sequence elements. These elements constitute binding sites with varying affinities for the complex. Collectively, their abundance on the X chromosome restricts the action of DCC to the X chromosomal territory.
doi:10.1371/journal.pgen.0020005
PMCID: PMC1359073  PMID: 16462942
14.  The Chromosomal High-Affinity Binding Sites for the Drosophila Dosage Compensation Complex 
PLoS Genetics  2008;4(12):e1000302.
Dosage compensation in male Drosophila relies on the X chromosome–specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC). The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called “high-affinity sites” (HAS). Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing high-resolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on non-coding parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features.
Author Summary
In sexually dimorphic species, unequal distribution of sex chromosomes requires adjustment of gene expression levels between the sexes. Male flies enhance transcription from the single X chromosome to meet the levels in females (XX). The specific recognition of sex chromosomes is a crucial step in this dosage compensation process. Intuitively, one might assume that sex chromosomes harbor distinct DNA sequence motifs for recruitment of the modulating machinery; however, no clearly defined motifs capable of fulfilling this role have yet been found. One explanation for this shortcoming could be our failure to date to identify a sufficiently large set of sites that serve as specific docking stations. In the following study, we have systematically mapped the strongest recruitment sites of the Drosophila dosage compensation complex (DCC) and identified shared sequence elements. The closer a gene resides to one of these sites the more robust is regulation by the DCC, which documents the function of our inventory of high-affinity binding sites.
doi:10.1371/journal.pgen.1000302
PMCID: PMC2586088  PMID: 19079572
15.  Adaptive Evolution of Genes Duplicated from the Drosophila pseudoobscura neo-X Chromosome 
Molecular Biology and Evolution  2010;27(8):1963-1978.
Drosophila X chromosomes are disproportionate sources of duplicated genes, and these duplications are usually the result of retrotransposition of X-linked genes to the autosomes. The excess duplication is thought to be driven by natural selection for two reasons: X chromosomes are inactivated during spermatogenesis, and the derived copies of retroposed duplications tend to be testis expressed. Therefore, autosomal derived copies of retroposed genes provide a mechanism for their X-linked paralogs to “escape” X inactivation. Once these duplications have fixed, they may then be selected for male-specific functions. Throughout the evolution of the Drosophila genus, autosomes have fused with X chromosomes along multiple lineages giving rise to neo-X chromosomes. There has also been excess duplication from the two independent neo-X chromosomes that have been examined—one that occurred prior to the common ancestor of the willistoni species group and another that occurred along the lineage leading to Drosophila pseudoobscura. To determine what role natural selection plays in the evolution of genes duplicated from the D. pseudoobscura neo-X chromosome, we analyzed DNA sequence divergence between paralogs, polymorphism within each copy, and the expression profiles of these duplicated genes. We found that the derived copies of all duplicated genes have elevated nonsynonymous polymorphism, suggesting that they are under relaxed selective constraints. The derived copies also tend to have testis- or male-biased expression profiles regardless of their chromosome of origin. Genes duplicated from the neo-X chromosome appear to be under less constraints than those duplicated from other chromosome arms. We also find more evidence for historical adaptive evolution in genes duplicated from the neo-X chromosome, suggesting that they are under a unique selection regime in which elevated nonsynonymous polymorphism provides a large reservoir of functional variants, some of which are fixed by natural selection.
doi:10.1093/molbev/msq085
PMCID: PMC2915643  PMID: 20351054
X chromosome; gene duplication; Drosophila; natural selection
16.  Chromosomal divergence and evolutionary inferences in Rhodniini based on the chromosomal location of ribosomal genes 
Memórias do Instituto Oswaldo Cruz  2013;108(3):376-382.
In this study, we used fluorescence in situ hybridisation to determine the chromosomal location of 45S rDNA clusters in 10 species of the tribe Rhodniini (Hemiptera: Reduviidae: Triatominae). The results showed striking inter and intraspecific variability, with the location of the rDNA clusters restricted to sex chromosomes with two patterns: either on one (X chromosome) or both sex chromosomes (X and Y chromosomes). This variation occurs within a genus that has an unchanging diploid chromosome number (2n = 22, including 20 autosomes and 2 sex chromosomes) and a similar chromosome size and genomic DNA content, reflecting a genome dynamic not revealed by these chromosome traits. The rDNA variation in closely related species and the intraspecific polymorphism in Rhodnius ecuadoriensis suggested that the chromosomal position of rDNA clusters might be a useful marker to identify recently diverged species or populations. We discuss the ancestral position of ribosomal genes in the tribe Rhodniini and the possible mechanisms involved in the variation of the rDNA clusters, including the loss of rDNA loci on the Y chromosome, transposition and ectopic pairing. The last two processes involve chromosomal exchanges between both sex chromosomes, in contrast to the widely accepted idea that the achiasmatic sex chromosomes of Heteroptera do not interchange sequences.
doi:10.1590/S0074-02762013000300017
PMCID: PMC4005576  PMID: 23778665
chromosomal evolution; Chagas disease vectors; Triatominae; holocentric chromosomes; rDNA variability
17.  Comparative Genomic Hybridization (CGH) Reveals a Neo-X Chromosome and Biased Gene Movement in Stalk-Eyed Flies (Genus Teleopsis) 
PLoS Genetics  2010;6(9):e1001121.
Chromosomal location has a significant effect on the evolutionary dynamics of genes involved in sexual dimorphism, impacting both the pattern of sex-specific gene expression and the rate of duplication and protein evolution for these genes. For nearly all non-model organisms, however, knowledge of chromosomal gene content is minimal and difficult to obtain on a genomic scale. In this study, we utilized Comparative Genomic Hybridization (CGH), using probes designed from EST sequence, to identify genes located on the X chromosome of four species in the stalk-eyed fly genus Teleopsis. Analysis of log2 ratio values of female-to-male hybridization intensities from the CGH microarrays for over 3,400 genes reveals a strongly bimodal distribution that clearly differentiates autosomal from X-linked genes for all four species. Genotyping of 33 and linkage mapping of 28 of these genes in Teleopsis dalmanni indicate the CGH results correctly identified chromosomal location in all cases. Syntenic comparison with Drosophila indicates that 90% of the X-linked genes in Teleopsis are homologous to genes located on chromosome 2L in Drosophila melanogaster, suggesting the formation of a nearly complete neo-X chromosome from Muller element B in the dipteran lineage leading to Teleopsis. Analysis of gene movement both relative to Drosophila and within Teleopsis indicates that gene movement is significantly associated with 1) rates of protein evolution, 2) the pattern of gene duplication, and 3) the evolution of eyespan sexual dimorphism. Overall, this study reveals that diopsids are a critical group for understanding the evolution of sex chromosomes within Diptera. In addition, we demonstrate that CGH is a useful technique for identifying chromosomal sex-linkage and should be applicable to other organisms with EST or partial genomic information.
Author Summary
The distribution and organization of genes on chromosomes vary widely among animals. Chromosomes can change in number and size, as well as gene composition, over short evolutionary time scales. Furthermore, chromosome location can influence how genes are expressed in various tissues and how they evolve. The sex chromosomes, in particular, have a dynamic impact on gene movement, expression, and evolution. Uncovering the chromosomal location of genes has traditionally been difficult for non-model organism species. In this study, we assess sex chromosome linkage using a new method that hybridizes DNA from males and females to probes representing over 3,400 genes in stalk-eyed flies. This technique identifies 533 genes (15%) that are located on the X chromosome with the remaining genes located on two autosomes. Comparison of these genes with their location in Drosophila indicates that the X chromosome in stalk-eyed flies is nearly completely homologous to the autosome 2L in D. melanogaster. This result reveals the formation of a neo-X chromosome in the lineage leading to stalk-eyed flies and indicates that stalk-eyed flies provide a valuable new system to study the origin and evolution of sex chromosomes.
doi:10.1371/journal.pgen.1001121
PMCID: PMC2940734  PMID: 20862308
18.  The role of LINEs and CpG islands in dosage compensation on the chicken Z chromosome 
Chromosome Research  2009;17(6):727-736.
Most avian Z genes are expressed more highly in ZZ males than ZW females, suggesting that chromosome-wide mechanisms of dosage compensation have not evolved. Nevertheless, a small percentage of Z genes are expressed at similar levels in males and females, an indication that a yet unidentified mechanism compensates for the sex difference in copy number. Primary DNA sequences are thought to have a role in determining chromosome gene inactivation status on the mammalian X chromosome. However, it is currently unknown whether primary DNA sequences also mediate chicken Z gene compensation status. Using a combination of chicken DNA sequences and Z gene compensation profiles of 310 genes, we explored the relationship between Z gene compensation status and primary DNA sequence features. Statistical analysis of different Z chromosomal features revealed that long interspersed nuclear elements (LINEs) and CpG islands are enriched on the Z chromosome compared with 329 other DNA features. Linear support vector machine (SVM) classifiers, using primary DNA sequences, correctly predict the Z compensation status for >60% of all Z-linked genes. CpG islands appear to be the most accurate classifier and alone can correctly predict compensation of 63% of Z genes. We also show that LINE CR1 elements are enriched 2.7-fold on the chicken Z chromosome compared with autosomes and that chicken chromosomal length is highly correlated with percentage LINE content. However, the position of LINE elements is not significantly associated with dosage compensation status of Z genes. We also find a trend for a higher proportion of CpG islands in the region of the Z chromosome with the fewest dosage-compensated genes compared with the region containing the greatest concentration of compensated genes. Comparison between chicken and platypus genomes shows that LINE elements are not enriched on sex chromosomes in platypus, indicating that LINE accumulation is not a feature of all sex chromosomes. Our results suggest that CpG islands are not randomly distributed on the Z chromosome and may influence Z gene dosage compensation status.
Electronic supplementary material
The online version of this article (doi:10.1007/s10577-009-9068-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s10577-009-9068-4
PMCID: PMC2759020  PMID: 19672682
dosage compensation; Z chromosome; DNA sequence; LINEs; CpG; chicken; sex chromosome; X chromosome
19.  An apparent excess of sex- and reproduction-related genes on the human X chromosome. 
We describe here the results of a search of Mendelian inheritance in man, GENDIAG and other sources which suggest that, in comparison with autosomes 1, 2, 3, 4 and 11, the X chromosome may contain a significantly higher number of sex- and reproduction-related (SRR) genes. A similar comparison between X-linked entries and a subset of randomly chosen entries from the remaining autosomes also indicates an excess of genes on the X chromosome with one or more mutations affecting sex determination (e.g. DAX1), sexual differentiation (e.g. androgen receptor) or reproduction (e.g. POF1). A possible reason for disproportionate occurrence of such genes on the X chromosome could be that, during evolution, the 'choice' of a particular pair of homomorphic chromosomes for specialization as sex chromosomes may be related to the number of such genes initially present in it or, since sex determination and sexual dimorphism are often gene dose-dependent processes, the number of such genes necessary to be regulated in a dose-dependent manner. Further analysis of these data shows that XAR, the region which has been added on to the short arm of the X chromosome subsequent to eutherian-marsupial divergence, has nearly as high a proportion of SRR genes as XCR, the conserved region of the X chromosome. These observations are consistent with current hypotheses on the evolution of sexually antagonistic traits on sex chromosomes and suggest that both XCR and XAR may have accumulated SRR traits relatively rapidly because of X linkage.
PMCID: PMC1689664  PMID: 10097393
20.  Sex-Specific Embryonic Gene Expression in Species with Newly Evolved Sex Chromosomes 
PLoS Genetics  2014;10(2):e1004159.
Sex chromosome dosage differences between females and males are a significant form of natural genetic variation in many species. Like many species with chromosomal sex determination, Drosophila females have two X chromosomes, while males have one X and one Y. Fusions of sex chromosomes with autosomes have occurred along the lineage leading to D. pseudoobscura and D. miranda. The resulting neo-sex chromosomes are gradually evolving the properties of sex chromosomes, and neo-X chromosomes are becoming targets for the molecular mechanisms that compensate for differences in X chromosome dose between sexes. We have previously shown that D. melanogaster possess at least two dosage compensation mechanisms: the well- characterized MSL-mediated dosage compensation active in most somatic tissues, and another system active during early embryogenesis prior to the onset of MSL-mediated dosage compensation. To better understand the developmental constraints on sex chromosome gene expression and evolution, we sequenced mRNA from individual male and female embryos of D. pseudoobscura and D. miranda, from ∼0.5 to 8 hours of development. Autosomal expression levels are highly conserved between these species. But, unlike D. melanogaster, we observe a general lack of dosage compensation in D. pseudoobscura and D. miranda prior to the onset of MSL-mediated dosage compensation. Thus, either there has been a lineage-specific gain or loss in early dosage compensation mechanism(s) or increasing X chromosome dose may strain dosage compensation systems and make them less effective. The extent of female bias on the X chromosomes decreases through developmental time with the establishment of MSL-mediated dosage compensation, but may do so more slowly in D. miranda than D. pseudoobscura. These results also prompt a number of questions about whether species with more sex-linked genes have more sex-specific phenotypes, and how much transcript level variance is tolerable during critical stages of development.
Author Summary
Many animals have sex-specific combinations of chromosomes. In humans, for example, females have two X chromosomes while males have one X and one Y. In most species with XX:XY systems, the Y chromosome is degenerate and gene-poor while the X encodes a large number of functional genes. A variety of systems have evolved to ensure that males with one X chromosome and females with two X chromosomes have the same gene expression level for X-linked genes. The vinegar fly D. melanogaster has at least two dosage compensation systems: one that acts early in development, and another active in later stages. In this paper, we determine expression levels for thousands of genes in male and female embryos at different developmental stages in two species, D. pseudoobscura and D. miranda, that have unusually large fractions of their genomes in X or X-like chromosomes. We show that dosage compensation is established slowly during embryogenesis, and that in these species, dosage compensation appears to be absent in early development. This may be due to a lineage-specific loss or gain of compensation mechanism, or possibly because the machinery of dosage compensation cannot effectively handle the increased demand in these species.
doi:10.1371/journal.pgen.1004159
PMCID: PMC3923672  PMID: 24550743
21.  Female Meiotic Sex Chromosome Inactivation in Chicken 
PLoS Genetics  2009;5(5):e1000466.
During meiotic prophase in male mammals, the heterologous X and Y chromosomes remain largely unsynapsed, and meiotic sex chromosome inactivation (MSCI) leads to formation of the transcriptionally silenced XY body. In birds, the heterogametic sex is female, carrying Z and W chromosomes (ZW), whereas males have the homogametic ZZ constitution. During chicken oogenesis, the heterologous ZW pair reaches a state of complete heterologous synapsis, and this might enable maintenance of transcription of Z- and W chromosomal genes during meiotic prophase. Herein, we show that the ZW pair is transiently silenced, from early pachytene to early diplotene using immunocytochemistry and gene expression analyses. We propose that ZW inactivation is most likely achieved via spreading of heterochromatin from the W on the Z chromosome. Also, persistent meiotic DNA double-strand breaks (DSBs) may contribute to silencing of Z. Surprisingly, γH2AX, a marker of DSBs, and also the earliest histone modification that is associated with XY body formation in mammalian and marsupial spermatocytes, does not cover the ZW during the synapsed stage. However, when the ZW pair starts to desynapse, a second wave of γH2AX accumulates on the unsynapsed regions of Z, which also show a reappearance of the DSB repair protein RAD51. This indicates that repair of meiotic DSBs on the heterologous part of Z is postponed until late pachytene/diplotene, possibly to avoid recombination with regions on the heterologously synapsed W chromosome. Two days after entering diplotene, the Z looses γH2AX and shows reactivation. This is the first report of meiotic sex chromosome inactivation in a species with female heterogamety, providing evidence that this mechanism is not specific to spermatogenesis. It also indicates the presence of an evolutionary force that drives meiotic sex chromosome inactivation independent of the final achievement of synapsis.
Author Summary
Meiosis is a sequence of two specialized cell divisions during which haploid gametes are generated. During meiotic prophase, homologous chromosomes pair and recombine to allow proper separation of chromosomes during the first meiotic division. The pairing mechanism is challenged by the presence of the largely nonhomologous sex chromosomes in spermatocytes of male mammals, since X and Y pair only in the short regions of homology. The unpaired nonhomologous regions are recognized and transcriptionally silenced, which leads to the formation of the so-called XY body. In mammalian females, which carry two homologous X chromosomes, no such structure is formed and the sex chromosomes are both active in oocytes. We asked whether meiotic silencing of sex chromosomes also occurs during gametogenesis in chickens. In this species, males carry two Z chromosomes, and females are ZW. We show that Z and W fully pair in oocytes, despite the overall lack of sequence homology. Surprisingly, the ZW pair is transcriptionally silenced during meiotic prophase and remains inactive until the two chromosomes have largely separated. Reactivation of Z at this stage may be necessary to allow expression of genes that are required for further oocyte development. These data show that meiotic sex chromosome silencing occurs also in species with female heterogamety.
doi:10.1371/journal.pgen.1000466
PMCID: PMC2678266  PMID: 19461881
22.  Breakage-fusion-bridge Cycles and Large Insertions Contribute to the Rapid Evolution of Accessory Chromosomes in a Fungal Pathogen 
PLoS Genetics  2013;9(6):e1003567.
Chromosomal rearrangements are a major driver of eukaryotic genome evolution, affecting speciation, pathogenicity and cancer progression. Changes in chromosome structure are often initiated by mis-repair of double-strand breaks in the DNA. Mis-repair is particularly likely when telomeres are lost or when dispersed repeats misalign during crossing-over. Fungi carry highly polymorphic chromosomal complements showing substantial variation in chromosome length and number. The mechanisms driving chromosome polymorphism in fungi are poorly understood. We aimed to identify mechanisms of chromosomal rearrangements in the fungal wheat pathogen Zymoseptoria tritici. We combined population genomic resequencing and chromosomal segment PCR assays with electrophoretic karyotyping and resequencing of parents and offspring from experimental crosses to show that this pathogen harbors a highly diverse complement of accessory chromosomes that exhibits strong global geographic differentiation in numbers and lengths of chromosomes. Homologous chromosomes carried highly differentiated gene contents due to numerous insertions and deletions. The largest accessory chromosome recently doubled in length through insertions totaling 380 kb. Based on comparative genomics, we identified the precise breakpoint locations of these insertions. Nondisjunction during meiosis led to chromosome losses in progeny of three different crosses. We showed that a new accessory chromosome emerged in two viable offspring through a fusion between sister chromatids. Such chromosome fusion is likely to initiate a breakage-fusion-bridge (BFB) cycle that can rapidly degenerate chromosomal structure. We suggest that the accessory chromosomes of Z. tritici originated mainly from ancient core chromosomes through a degeneration process that included BFB cycles, nondisjunction and mutational decay of duplicated sequences. The rapidly evolving accessory chromosome complement may serve as a cradle for adaptive evolution in this and other fungal pathogens.
Author Summary
Chromosomal rearrangements are a hallmark of genetic differences between species. But changes in chromosome structure can also occur spontaneously within species, within populations, or even within individuals. The causes and consequences of chromosomal rearrangements affecting natural populations are poorly understood. We investigated a class of fungal chromosomes called accessory chromosomes that are not shared among all individuals within a species. Using a fungal pathogen possessing numerous accessory chromosomes as a model, we assessed chromosome diversity based on whole-genome sequencing and a PCR assay of chromosomal segments that included a global collection of isolates. We show that the accessory chromosomes are highly variable in their gene content and that geographic differences correlate with the number and the structure of the chromosomes. We applied the same approach to document chromosomal rearrangements occurring during sexual reproduction. We identified viable offspring carrying a novel chromosome that originated from a large duplication affecting the majority of the chromosome. Our study showed that chromosomal structure can evolve rapidly within a species to generate a highly diverse set of accessory chromosomes. This chromosomal diversity may contribute significantly to the adaptive potential of fungal pathogens.
doi:10.1371/journal.pgen.1003567
PMCID: PMC3681731  PMID: 23785303
23.  ATM Promotes the Obligate XY Crossover and both Crossover Control and Chromosome Axis Integrity on Autosomes 
PLoS Genetics  2008;4(5):e1000076.
During meiosis in most sexually reproducing organisms, recombination forms crossovers between homologous maternal and paternal chromosomes and thereby promotes proper chromosome segregation at the first meiotic division. The number and distribution of crossovers are tightly controlled, but the factors that contribute to this control are poorly understood in most organisms, including mammals. Here we provide evidence that the ATM kinase or protein is essential for proper crossover formation in mouse spermatocytes. ATM deficiency causes multiple phenotypes in humans and mice, including gonadal atrophy. Mouse Atm−/− spermatocytes undergo apoptosis at mid-prophase of meiosis I, but Atm−/− meiotic phenotypes are partially rescued by Spo11 heterozygosity, such that ATM-deficient spermatocytes progress to meiotic metaphase I. Strikingly, Spo11+/−Atm−/− spermatocytes are defective in forming the obligate crossover on the sex chromosomes, even though the XY pair is usually incorporated in a sex body and is transcriptionally inactivated as in normal spermatocytes. The XY crossover defect correlates with the appearance of lagging chromosomes at metaphase I, which may trigger the extensive metaphase apoptosis that is observed in these cells. In addition, control of the number and distribution of crossovers on autosomes appears to be defective in the absence of ATM because there is an increase in the total number of MLH1 foci, which mark the sites of eventual crossover formation, and because interference between MLH1 foci is perturbed. The axes of autosomes exhibit structural defects that correlate with the positions of ongoing recombination. Together, these findings indicate that ATM plays a role in both crossover control and chromosome axis integrity and further suggests that ATM is important for coordinating these features of meiotic chromosome dynamics.
Author Summary
Meiosis is the specialized cell division that gives rise to reproductive cells such as sperm and eggs. During meiosis in most organisms, genetic information is exchanged between homologous maternal and paternal chromosomes through the process of homologous recombination. This recombination forms connections between homologous chromosomes that allow them to segregate accurately when the meiotic cell divides. Recombination defects can result in reproductive cells with abnormal chromosome numbers, which are a major cause of developmental disorders and spontaneous abortions in humans. Meiotic recombination is tightly controlled such that each pair of chromosomes undergoes at least one crossover recombination event despite a low average number of crossovers per chromosome. Moreover, multiple crossovers on the same chromosome tend to be evenly and widely spaced. Mechanisms of this control are not well understood, but here we provide evidence that ATM protein is required for normal operation of this process(es) in male mice. ATM has long been known to be involved in cellular responses to DNA damage. Our studies reveal a new function for this protein and also provide new insight into the mechanisms by which meiotic cells ensure accurate transmission of genetic material from one generation to the next.
doi:10.1371/journal.pgen.1000076
PMCID: PMC2374915  PMID: 18497861
24.  The genomic landscape of meiotic crossovers and gene conversions in Arabidopsis thaliana 
eLife  2013;2:e01426.
Knowledge of the exact distribution of meiotic crossovers (COs) and gene conversions (GCs) is essential for understanding many aspects of population genetics and evolution, from haplotype structure and long-distance genetic linkage to the generation of new allelic variants of genes. To this end, we resequenced the four products of 13 meiotic tetrads along with 10 doubled haploids derived from Arabidopsis thaliana hybrids. GC detection through short reads has previously been confounded by genomic rearrangements. Rigid filtering for misaligned reads allowed GC identification at high accuracy and revealed an ∼80-kb transposition, which undergoes copy-number changes mediated by meiotic recombination. Non-crossover associated GCs were extremely rare most likely due to their short average length of ∼25–50 bp, which is significantly shorter than the length of CO-associated GCs. Overall, recombination preferentially targeted non-methylated nucleosome-free regions at gene promoters, which showed significant enrichment of two sequence motifs.
DOI: http://dx.doi.org/10.7554/eLife.01426.001
eLife digest
Most living organisms package their DNA into bundles called chromosomes. These chromosomes generally form pairs, with each chromosome in the pair containing the same number of genes. The genes also come in the same order, but the exact sequence of DNA bases within the genes can be different.
When sex cells—such as egg, sperm or pollen cells—are made, each pair of chromosomes is separated so that the each sex cell contains only half the normal number of chromosomes. However, before they are separated, the pairs swap lengths of DNA via recombination events. These can involve exchanging large chunks of the chromosomes: this is called a ‘crossover’. Alternatively, short stretches of one chromosome can be replaced by the corresponding region from the other in the pair. When these ‘non-crossovers’ cause a change in the DNA sequence they are known as gene conversions.
Long-standing questions in the field of plant biology include: how common are gene conversions? How much DNA is typically exchanged? And where in the chromosomes do these events happen most? Now, Wijnker et al. have addressed these questions by focusing on the accurate detection of recombination events, with a special emphasis on gene conversions, in the plant biologist’s favourite species: Arabidopsis.
Searching for recombination events is a challenge because, when piecing together an entire genome from lots of shorter stretches of DNA—typically called ‘reads’, it is easy to misplace some of the pieces. However, meticulous examination of these short DNA reads allowed Wijnker et al. to reliably identify gene conversions on a genome-wide scale. In Arabidopsis, gene conversion appears to be unexpectedly rare—with approximately one gene conversion detected per 140–240 non-crossovers. Recombination tends to occur in regions of the chromosomes where the DNA is only loosely packaged, is not heavily modified by the process of ‘DNA methylation’, and also near the start of genes. Furthermore, two specific sequences of DNA bases were identified that marked ‘hot spots’ in the chromosomes, where recombination happens more frequently.
Wijnker et al. suggest that the low number of gene conversions detected indicates that non-crossovers tend to exchange very short stretches of DNA. However, future research may point to additional mechanisms that explain the low incidence of gene conversion in Arabidopsis.
DOI: http://dx.doi.org/10.7554/eLife.01426.002
doi:10.7554/eLife.01426
PMCID: PMC3865688  PMID: 24347547
meiotic recombination; gene conversion; next generation sequencing; double strand break repair; meiotic tetrads; Arabidopsis
25.  Sex-Biased Evolutionary Forces Shape Genomic Patterns of Human Diversity 
PLoS Genetics  2008;4(9):e1000202.
Comparisons of levels of variability on the autosomes and X chromosome can be used to test hypotheses about factors influencing patterns of genomic variation. While a tremendous amount of nucleotide sequence data from across the genome is now available for multiple human populations, there has been no systematic effort to examine relative levels of neutral polymorphism on the X chromosome versus autosomes. We analyzed ∼210 kb of DNA sequencing data representing 40 independent noncoding regions on the autosomes and X chromosome from each of 90 humans from six geographically diverse populations. We correct for differences in mutation rates between males and females by considering the ratio of within-human diversity to human-orangutan divergence. We find that relative levels of genetic variation are higher than expected on the X chromosome in all six human populations. We test a number of alternative hypotheses to explain the excess polymorphism on the X chromosome, including models of background selection, changes in population size, and sex-specific migration in a structured population. While each of these processes may have a small effect on the relative ratio of X-linked to autosomal diversity, our results point to a systematic difference between the sexes in the variance in reproductive success; namely, the widespread effects of polygyny in human populations. We conclude that factors leading to a lower male versus female effective population size must be considered as important demographic variables in efforts to construct models of human demographic history and for understanding the forces shaping patterns of human genomic variability.
Author Summary
Like many primate species, the mating system of humans is considered to be moderately polygynous (i.e., males exhibit a higher variance in reproductive success than females). As a consequence, males are expected to have a lower effective population size (Ne) than females, and the proportion of neutral genetic variation on the X chromosome (relative to the autosomes) should be higher than expected under the assumption of strict neutrality and an equal breeding sex ratio. We test for the effects of polygyny by measuring levels of neutral polymorphism at 40 independent loci on the X chromosome and autosomes in six human populations. To correct for mutation rate heterogeneity among loci, we divide our diversity estimates within human populations by divergence with orangutan at each locus. Consistent with expectations under a model of polygyny, we find elevated levels of X-linked versus autosomal diversity. While it is possible that multiple demographic processes may contribute to the observed patterns of genomic diversity (i.e., background selection, changes in population size, and sex-specific migration), we conclude that an historical excess of breeding females over the number of breeding males can by itself explain most of the observed increase in effective population size of the X chromosome.
doi:10.1371/journal.pgen.1000202
PMCID: PMC2538571  PMID: 18818765

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