Noncoding RNAs and epigenetic mechanisms during X-chromosome inactivation.

In mammals, the process of X-chromosome inactivation ensures equivalent levels of X-linked gene expression between males and females through the silencing of one of the two X chromosomes in female cells. The process is established early in development and is initiated by a unique locus, which produces a long noncoding RNA, Xist. The Xist transcript triggers gene silencing in cis by coating the future inactive X chromosome. It also induces a cascade of chromatin changes, including posttranslational histone modifications and DNA methylation, and leads to the stable repression of all X-linked genes throughout development and adult life. We review here recent progress in our understanding of the molecular mechanisms involved in the initiation of Xist expression, the propagation of the Xist RNA along the chromosome, and the cis-elements and trans-acting factors involved in the maintenance of the repressed state. We also describe the diverse strategies used by nonplacental mammals for X-chromosome dosage compensation and highlight the common features and differences between eutherians and metatherians, in particular regarding the involvement of long noncoding RNAs.

More sex chromosomes than autosomes in the Amazonian frog Leptodactylus pentadactylus.

Heteromorphic sex chromosomes are common in eukaryotes and largely ubiquitous in birds and mammals. The largest number of multiple sex chromosomes in vertebrates known today is found in the monotreme platypus (Ornithorhynchus anatinus, 2n = 52) which exhibits precisely 10 sex chromosomes. Interestingly, fish, amphibians, and reptiles have sex determination mechanisms that do or do not involve morphologically differentiated sex chromosomes. Relatively few amphibian species carry heteromorphic sex chromosomes, and when present, they are frequently represented by only one pair, either XX:XY or ZZ:ZW types. Here, in contrast, with several evidences, from classical and molecular cytogenetic analyses, we found 12 sex chromosomes in a Brazilian population of the smoky jungle frog, designated as Leptodactylus pentadactylus Laurenti, 1768 (Leptodactylinae), which has a karyotype with 2n = 22 chromosomes. Males exhibited an astonishing stable ring-shaped meiotic chain composed of six X and six Y chromosomes. The number of sex chromosomes is larger than the number of autosomes found, and these data represent the largest number of multiple sex chromosomes ever found among vertebrate species. Additionally, sequence and karyotype variation data suggest that this species may represent a complex of species, in which the chromosomal rearrangements may possibly have played an important role in the evolution process.

X inactivation in a mammal species with three sex chromosomes.

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

The XY Body of the Cat (Felis catus): Structural Differentiations and Protein Immunolocalization.

The heteromorphic X and Y chromosomes behave in a special way in mammalian spermatocytes; they form the XY body and synapse only partially. The aim of this article was to study the origin and the role of the special differentiations in the XY pair of the domestic cat during pachytene by analyzing its fine structural characteristics and the immunolocalization of the main meiotic proteins SYCP3, SYCP1, SYCE3, SMC3, γ-H2AX, BRCA1, H3K27me3, and MLH1. The cat XY body shows particularly striking structures: an extreme degree of axial fibrillation in late pachynema and a special location of SYCP3-containing fibrils, bridging different regions of the main X axis, as well as one bridge at the inner end of the pairing region that colocalizes with the single mandatory MLH1 focus. There are sequential changes, first bullous expansions, then subdivision into fibrils, all involving axial thickening. The chromatin of the XY body presents the usual features of meiotic sex chromosome inactivation. An analysis of the XY body of many eutherians and metatherians suggests that axial thickenings are primitive features. The sequential changes in the mass and location of SYCP3-containing fibers vary among the clades because of specific processes of axial assembly/disassembly occurring in different species.

Dissociation of the X chromosome from the synaptonemal complex in the XY body of the rodent Galea musteloides.

The XY body from spermatocytes of the rodent Galea musteloides shows progressive changes of the synaptonemal complex (SC) axes and the X-chromatin during pachynema. There is a gross thickening of the X-axis and the formation of a large X chromosome loop at mid and late pachytene stages. The SC proteins synaptonemal complex protein 3 (SYCP3), synaptonemal complex protein 1, and synaptonemal complex central element protein 3 and the proteins breast cancer 1, MutL homolog 1 (MLH1), and radiation-repair 51 (related to meiotic processes), the cohesin structural maintenance of chromosome 3, the centromeric protein (with CREST antibody), and the silenced chromatin (with phosphorylated (139ph) H2A histone family, member X (γ-H2AX) antibody) were analyzed in this XY body. The thick X-axis, including the interstitial loop, becomes formed by four to six laminae showing a cross-striation with a periodicity of about 20 nm. The whole length of the gross X-axis shows no significant changes during pachynema, but the interstitial chromatin of the X chromosome and the X centromere are included in the large loop, and it becomes separated from the SC. A conventional SC formed by the Y-axis, a central region and a thin lateral element originally corresponding to the X-axis, remains undisturbed up to the end of pachynema. A single MLH1 focus develops either at the distal or the proximal region of the loop end attached to the conventional SC. The chromatin surrounding the thickened axis is labeled with γ-H2AX. It is shown that most of the SYCP3 protein associated with the X chromosome loop is not involved in the SC maintenance, but it is located with the cohesin axis separated from the SC proper.

Synapsis, recombination, and chromatin remodeling in the XY body of armadillos.

Three xenarthrans species Chaetophractus villosus, Chaetophractus vellerosus, and Zaedyus pichiy have been used for the analysis of the structure, behavior, and immunochemical features of the XY body during pachytene. In all these species, the sex chromosomes form an XY body easily identifiable in thin sections by the special and regular packing of the chromatin fibers of the internal region of the XY body ("differential" regions) and those of the peripheral region (synaptic region). Spermatocyte spreads show a complete synapsis between the X- and the Y-axis, which lasts up to the end of pachytene. From the early pachytene substages to the late ones, the X-axis develops prominent branches, which in late pachytene span the synaptic region. Synapsis is regular as shown by SYCP1 labeling. Axial development is followed by SYCP3 labeling and in the asynaptic region of the X-axis by BRCA1. Gamma-H2AX labels exclusively the differential (asynaptic) region of the X chromosome. A single focus is labeled by MLH1 in the synaptic region. The location of this MLH1 focus spans from 0.3 to 1.6 µm from the telomere in the analyzed xenarthrans, covering approximately half of the Y-axis length. It is concluded that xenarthrans, as basal placental mammals, harbor the largest pseudoautosomal regions of presently analyzed mammals, and shows the typical features of meiotic sex chromosome inactivation (MSCI).

Multiple independent evolutionary losses of XY pairing at meiosis in the grey voles.

In many eutherian mammals, X-Y chromosome pairing and recombination is required for meiotic progression and correct sex chromosome disjunction. Arvicoline rodents present a notable exception to this meiotic rule, with multiple species possessing asynaptic sex chromosomes. Most asynaptic vole species belong to the genus Microtus sensu lato. However, many of the species both inside and outside the genus Microtus display normal X-Y synapsis at meiosis. These observations suggest that the synaptic condition was present in the common ancestor of all voles, but gaps in current taxonomic sampling across the arvicoline phylogeny prevent identification of the lineage(s) along which the asynaptic state arose. In this study, we use electron and immunofluorescent microscopy to assess heterogametic sex chromosome pairing in 12 additional arvicoline species. Our sample includes ten species of the tribe Microtini and two species of the tribe Lagurini. This increased breadth of sampling allowed us to identify asynaptic species in each major Microtine lineage. Evidently, the ability of the sex chromosomes to pair and recombine in male meiosis has been independently lost at least three times during the evolution of Microtine rodents. These results suggest a lack of evolutionary constraint on X-Y synapsis in Microtini, hinting at the presence of alternative molecular mechanisms for sex chromosome segregation in this large mammalian tribe.

A 2D crossover-based map of the human X chromosome as a model for map integration.

We have constructed a two-dimensional map of 243 markers on the X chromosome. The average distance between markers ordered by two recombinants is 5.4 centiMorgans (cM), which is reduced to 3.2 cM using a less stringent criterion of one recombinant. Map resolution is enhanced by replacing the usual reference marker format with a 2D format, and the two-recombinant rule is more conservative than the lod 3.0 criterion for order. Taken together, crossover mapping and the 2D format produces maps with greater reliability and higher resolution than maps constructed using currently accepted standards. This first high-density crossover-based map of an entire human chromosome provides a model for integrating physical and genetic maps.

Telomere-associated chromosome fragmentation: applications in genome manipulation and analysis.

Telomere-associated chromosome fragmentation (TACF) is a new approach for chromosome mapping based on the non-targeted introduction of cloned telomeres into mammalian cells. TACF has been used to generate a panel of somatic cell hybrids with nested terminal deletions of the long arm of the human X chromosome, extending from Xq26 to the centromere. This panel has been characterized using a series of X chromosome loci. Recovery of the end clones by plasmid rescue produces a telomeric marker for each cell line and partial sequencing will allow the generation of sequence tagged sites (STSs). TACF provides a powerful and widely applicable method for genome analysis, a general way of manipulating mammalian chromosomes and a first step towards constructing artificial mammalian chromosomes.

Isolation of a GCC repeat showing expansion in FRAXF, a fragile site distal to FRAXA and FRAXE.

Three folate-sensitive fragile sites, termed FRAXA, FRAXE and FRAXF, have been identified on the distal end of chromosome Xq. The first two contain expanded, hypermethylated and unstable CGG (or GCC) repeats within CpG islands. We now report the isolation of similar sequences responsible for the third fragile site, FRAXF. A 5-kilobase EcoRI fragment derived from a cosmid coincident with the cytogenetic anomaly detects expanded, methylated and unstable sequences in five individuals who exhibit fragile sites in distal Xq; these individuals have normal repeat lengths at both FRAXA and FRAXE. By sequence analysis, the expanded region contains a GCC repeat. PCR and sequence analysis of chromosomes from the general population indicates that the repeat is polymorphic (6 to 29 triplets), and is stable upon transmission.

Highly homologous loci on the X and Y chromosomes are hot-spots for ectopic recombinations leading to XX maleness.

In 80% of XX males, maleness is due to the presence of Y-specific DNA including the SRY gene and results from an abnormal terminal X-Y interchange during paternal meiosis. Here we address the molecular basis of this ectopic recombination through the analysis of the X-Y junction in two class 3 XX males. We show that each of the rearrangements has involved X-Y highly homologous loci on the sex-specific part of these chromosomes (98.7% and 96% sequence identity over 1.2 and 1.1 kb respectively). Moreover in five out of six other XX males, the X-Y junctions are located in the same rearranged restriction fragment as in either of these patients. These fragments thus define two hot-spots of ectopic recombination which together could account for about one third of XX males. Evolution of these loci in primates is discussed.

Chromosomal evolution in Raphicerus antelope suggests divergent X chromosomes may drive speciation through females, rather than males, contrary to Haldane's rule.

Chromosome structural change has long been considered important in the evolution of post-zygotic reproductive isolation. The premise that karyotypic variation can serve as a possible barrier to gene flow is founded on the expectation that heterozygotes for structurally distinct chromosomal forms would be partially sterile (negatively heterotic) or show reduced recombination. We report the outcome of a detailed comparative molecular cytogenetic study of three antelope species, genus Raphicerus, that have undergone a rapid radiation. The species are largely conserved with respect to their euchromatic regions but the X chromosomes, in marked contrast, show distinct patterns of heterochromatic amplification and localization of repeats that have occurred independently in each lineage. We argue a novel hypothesis that postulates that the expansion of heterochromatic blocks in the homogametic sex can, with certain conditions, contribute to post-zygotic isolation. i.e., female hybrid incompatibility, the converse of Haldane's rule. This is based on the expectation that hybrids incur a selective disadvantage due to impaired meiosis resulting from the meiotic checkpoint network's surveillance of the asymmetric expansions of heterochromatic blocks in the homogametic sex. Asynapsis of these heterochromatic regions would result in meiotic silencing of unsynapsed chromatin and, if this persists, germline apoptosis and female infertility.

The X chromosome dosage compensation program during the development of cynomolgus monkeys.

X chromosome dosage compensation ensures balanced gene dosage between the X chromosome and autosomes and between the sexes, involving divergent mechanisms among mammals. We elucidated a distinct mechanism for X chromosome inactivation (XCI) in cynomolgus monkeys, a model for human development. The trophectoderm and cytotrophoblast acquire XCI around implantation through an active intermediate bearing repressive modifications and compacted structure, whereas the amnion, epiblast, and hypoblast maintain such an intermediate protractedly, attaining XCI by a week after implantation. Males achieve X chromosome up-regulation (XCU) progressively, whereas females show XCU coincidentally with XCI, both establishing the X:autosome dosage compensation by 1 week after implantation. Conversely, primordial germ cells undergo X chromosome reactivation by reversing the XCI pathway early during their development. Our findings establish a foundation for clarifying the dosage compensation mechanisms in primates, including humans.

Single-cell nuclear architecture across cell types in the mouse brain.

Diverse cell types in tissues have distinct gene expression programs, chromatin states, and nuclear architectures. To correlate such multimodal information across thousands of single cells in mouse brain tissue sections, we use integrated spatial genomics, imaging thousands of genomic loci along with RNAs and epigenetic markers simultaneously in individual cells. We reveal that cell type­specific association and scaffolding of DNA loci around nuclear bodies organize the nuclear architecture and correlate with differential expression levels in different cell types. At the submegabase level, active and inactive X chromosomes access similar domain structures in single cells despite distinct epigenetic and expression states. This work represents a major step forward in linking single-cell three-dimensional nuclear architecture, gene expression, and epigenetic modifications in a native tissue context.

Microdissection and chromosome painting of X and B chromosomes in Locusta migratoria.

Acquisition of knowledge of the nature and DNA content of B chromosomes has been triggered by a collection of molecular techniques, one of which, microdissection, has provided interesting results in a number of B chromosome systems. Here we provide the first data on the molecular composition of B chromosomes in Locusta migratoria, after microdissection of the B and X chromosomes, DNA amplification by one (B) or two (X) different methods, and chromosome painting. The results showed that B chromosomes share at least two types of repetitive DNA sequences with the A chromosomes, suggesting that Bs in this species most likely arose intraspecifically. One of these repetitive DNAs is located on the heterochromatic distal half of the B chromosome and in the pericentromeric regions of about half of the A chromosomes, including the X. The other type of repetitive DNA is located interspersedly over the non-centromeric euchromatic regions of all A chromosomes and in an interstitial part of the proximal euchromatic half of the B chromosome. Chromosome painting, however, did not provide results sufficiently reliable to determine, in this species, which A chromosome gave rise to the B; this might be done by detailed analysis of the microdissected DNA sequences.

Racial divergence of a rare laboratory-evolved centromeric fission Cytorace of nasuta-albomicans complex of Drosophila.

Fissioncytorace-1, a member of the nasuta-albomicans complex of Drosophila is an evolutionary product of centric fission, which had occurred in the chromosome X3 of Cytorace 1, a hydridization product of Drosophila nasuta nasuta male (2n=8) and Drosophila nasuta albomicans female (2n=6). Cytorace 1 (males 2n=7; females 2n=6) has inherited this chromosome from its D. n. albomicans parent. The chromosome X3 of D. n. albomicans is a derivative of a centric fusion between the acrocentric chromosome 3 and the chromosome X of D. n. nasuta. The Fissioncytorace-1 has crossed 200 generations from the time of its evolution in the laboratory environment. When this centromeric fission race was subjected to some of the morphophenotypic and fitness assessment to find its overall population fitness showed, increased body size, sternopleural bristle, ovarioles, lifetime fecundity and fertility with reduced interspecific competitive ability and hatching success when compared with its parent (Cytorace 1). These results suggest that the hybrid races must have encountered an early event of recombinational raciation during their evolution in the laboratory environment, which is a unique observation in animal system illustrating the increase in the tempo of evolution after the event of hybridization.

Interaction study of the male specific lethal (MSL) complex and trans-acting dosage effects in metafemales of Drosophila melanogaster.

The effect of ectopic expression of male specific lethal 2 (msl2) on chromatin modification and gene expression was studied in Drosophila diploid females and metafemales (3X;2A). Results show that ectopic expression of MSL2 in transgenic msl2 females and metafemales sequesters the MOF histone acetylase to the X, which occurs concordantly with an increase of histone acetylation. Gene expression studies indicate that the X-linked genes are not affected by direct targeting of the MSL complex and the resulting increased H4Lys16 acetylation on the X chromosomes, suggesting one function of the MSL complex is to nullify the effect of a high level of histone acetylation. These results are not consistent with the hypothesis that the presence of the MSL complex conditions a two-fold upregulation. Autosomal gene expression is generally decreased in ectopically expressed MSL2 females, which correlates with the reduced autosomal histone acetylation. Metafemales show dosage compensation of X-linked genes with some autosomal reductions in expression. Interestingly, in metafemales with ectopically expressed MSL2, the autosomal expression is returned to a more normal level. There is a lower autosomal level of histone acetylation compared to the normal metafemales, suggesting a nullifying effect on the negative dosage effect of the X chromosome as previously hypothesized to occur in normal males.

Three-dimensional organization of Drosophila melanogaster interphase nuclei. II. Chromosome spatial organization and gene regulation.

In the preceding article we compared the general organization of polytene chromosomes in four different Drosophila melanogaster cell types. Here we describe experiments aimed at testing for a potential role of three-dimensional chromosome folding and positioning in modulating gene expression and examining specific chromosome interactions with different nuclear structures. By charting the configurations of salivary gland chromosomes as the cells undergo functional changes, it is shown that loci are not repositioned within the nucleus when the pattern of transcription changes. Heterologous loci show no evidence of specific physical interactions with one another in any of the cell types. However, a specific subset of chromosomal loci is attached to the nuclear envelope, and this subset is extremely similar in at least two tissues. In contrast, no specific interactions between any locus and the nucleolus are found, but the base of the X chromosome, containing the nucleolar organizer, is closely linked to this organelle. These results are used to evaluate models of gene regulation that involve the specific intranuclear positioning of gene sequences. Finally, data are presented on an unusual class of nuclear envelope structures, filled with large, electron-dense particles, that are usually associated with chromosomes.

Three-dimensional reconstruction of painted human interphase chromosomes: active and inactive X chromosome territories have similar volumes but differ in shape and surface structure.

This study provides a three-dimensional (3D) analysis of differences between the 3D morphology of active and inactive human X interphase chromosomes (Xa and Xi territories). Chromosome territories were painted in formaldehyde-fixed, three-dimensionally intact human diploid female amniotic fluid cell nuclei (46, XX) with X-specific whole chromosome compositive probes. The colocalization of a 4,6-diamidino-2-phenylindole dihydrochloride-stained Barr body with one of the two painted X territories allowed the unequivocal discrimination of the inactive X from its active counterpart. Light optical serial sections were obtained with a confocal laser scanning microscope. 3D-reconstructed Xa territories revealed a flatter shape and exhibited a larger and more irregular surface when compared to the apparently smoother surface and rounder shape of Xi territories. The relationship between territory surface and volume was quantified by the determination of a dimensionless roundness factor (RF). RF and surface area measurements showed a highly significant difference between Xa and Xi territories (P < 0.001) in contrast to volume differences (P > 0.1). For comparison with an autosome of similar DNA content, chromosome 7 territories were additionally painted. The 3D morphology of the chromosome 7 territories was similar to the Xa territory but differed strongly from the Xi territory with respect to RF and surface area (P < 0.001).

The total length of spindle microtubules depends on the number of chromosomes present.

We extracted chromosomes by micromanipulation from Melanoplus differentialis spermatocytes, producing metaphase spindles with only one or a few chromosomes instead of the usual complement of 23. Cells with various numbers of chromosomes were prepared for electron microscopy, and spindle microtubule length was measured. A constant increment of microtubule length was lost upon the removal of each chromosome; we estimate that only approximately 40% of the original length would remain in the total absence of chromosomes. Unexpectedly, kinetochore microtubules were not the only ones affected when chromosomes were removed: nonkinetochore microtubules accounted for a substantial fraction of the total length lost. No compensatory increase in microtubule length outside the spindle was found. Studies by others show that the kinetochore microtubules of extracted chromosomes are left behind in the cell and dissassemble. The resulting increase in subunit concentration would be expected from in vitro studies to drive microtubule assembly until the original total microtubule length was restored, but that did not happen in these living cells. We conclude that the assembly of a certain, large fraction of microtubule subunits into stable microtubules is dependent on the presence of chromosomes. Possible explanations include (a) limits on microtubule length that prevent any net assembly of the subunits released after chromosomes are removed or (b) a promotion of microtubule assembly by chromosomes, which therefore is reduced in their absence. Chromosome-dependent regulation of microtubule length may account for some features of normal mitosis.