The Interactive Fly

Zygotically transcribed genes

Sex Determination and Dosage Compensation Genes

A simplified model for sex determination in the somatic gonad and germline


Sex Determination
Sex determination across evolution -- connecting the dots: Evolution of sex determination mechanisms
Nonautonomous sex determination controls sexually dimorphic development of the Drosophila gonad
Specialized cells tag sexual and species identity in Drosophila melanogaster
Somatic control of germline sexual development is mediated by the JAK/STAT pathway
The establishment of sexual identity in the Drosophila germline
Drosophila Sex lethal gene initiates female development in germline progenitors
m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination
m6A modulates neuronal functions and sex determination in Drosophila

Dosage Compensation
Dosage compensation and demasculinization of X chromosomes in Drosophila
X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila
'Jump Start and Gain' model for dosage compensation in Drosophila based on direct sequencing of nascent transcripts
POF regulates the expression of genes on the fourth chromosome in Drosophila melanogaster by binding to nascent RNA
Dosage compensation via transposable element mediated rewiring of a regulatory network
The epigenome of evolving Drosophila neo-sex chromosomes: dosage compensation and heterochromatin formation
siRNAs from an X-linked satellite repeat promote X-chromosome recognition in Drosophila melanogaster
High-affinity sites form an interaction network to facilitate spreading of the MSL complex across the X chromosome in Drosophila
Modulation of heterochromatin by male specific lethal proteins and roX RNA in Drosophila melanogaster males
Male-killing spiroplasma alters behavior of the dosage compensation complex during Drosophila melanogaster embryogenesis
Sex chromosome-wide transcriptional suppression and compensatory cis-regulatory evolution mediate gene expression in the Drosophila male germline
The essential Drosophila CLAMP protein differentially regulates non-coding roX RNAs in male and females
Expansion of GA dinucleotide repeats increases the density of CLAMP binding sites on the X-Chromosome to promote Drosophila dosage compensation
Effects of gene dose, chromatin, and network topology on expression in Drosophila melanogaster
PionX sites mark the X chromosome for dosage compensation
Satellite repeats identify X chromatin for dosage compensation in Drosophila melanogaster males
Genes involved in sex determination

  • Establishing the X:A ratio (the X chomosome:autosome ratio)

  • Somatic sex determination

  • Germ-line sex determination and differentiation

  • Dosage compensation

  • Other

  • Genes affecting courtship behavior including courship conditioning


  • Sex determination across evolution -- connecting the dots: Evolution of sex determination mechanisms

    The variety of primary sex determination cues was appreciated long before the advent of molecular genetics. The two broadest categories are genetic sex determination (GSD), in which the sex of offspring is set by a sex chromosome or an autosomal gene, and environmental sex determination (ESD), in which sex is determined by temperature (as with turtles), local sex ratio (as with some tropical fish), or population density (as with mermithid nematodes). Though little is known about the molecular mechanisms of ESD, within the GSD systems many different mechanisms have been uncovered. Dual sex chromosome systems, in which either the female (ZW/ZZ) or the male (XX/XY) is heterogametic, are common, as are systems set by the ratio of the number of X chromosomes to sets of autosomes (X:A). There are also systems in which heterozygosity at a single locus is required for female development (known as complementary sex determination), as well as systems involving sex determination via multiple genes with additive effects (Haag, 2005; see full text of article).

    Molecular genetic investigations of GSD in model systems such as Drosophila, Caenorhabditis, and mice have revealed a clear lack of conservation, underscoring the diversity. For example, although the primary sex determination signal in both D. melanogaster and C. elegans is the X:A ratio, the fruit fly pathway consists of a cell-autonomous cascade of regulated mRNA splicing, while that of the nematode follows a Hedgehog-like intercellular signaling pathway. GSD in mammals depends (with some interesting exceptions upon a Y-specific dominant gene (Sry) encoding a transcription factor. In the face of such impressive differences, perhaps the assumption of homology should be questioned: could it be that sex determination in different taxa has arisen independently over and over again in evolution? Until 1998, this seemed like a good bet (Haag, 2005).

    The discovery of the homology of the key sex-determining genes doublesex in Drosophila and mab-3 in C. elegans provided the first evidence for a common evolutionary basis of sex determination in animals. Soon, related doublesex-mab-3 (DM)-family genes with roles in male sexual development were discovered in vertebrates and even cnidarians. Here at last was a smoking gun that could link the diverse metazoan sex determination systems. But as satisfying as the result was, it immediately gave birth to another mystery: if the enormous diversity of sex determination systems are all derived from a common ancestor, how could they possibly have been modified so radically? After all, sexual differentiation and reproduction are hardly unimportant developmental processes (Haag, 2005).

    To understand how such diversity came to be, differences between closely related species must be examined. This approach allows the discovery and interpretation of small-scale sex determination changes before they are obscured by subsequent changes. The processes discovered in this way might then be reasonably extrapolated to explain the seemingly unrelated systems of more deeply diverged taxa. Work in dipterans has revealed three evolutionary phenomena that characterize shorter-term sex determination evolution (Haag, 2005).

    The first of these is the often astounding rate of molecular evolution at the level of nucleotide and aminoacid sequences. Although some sex-determining genes are well conserved, many show unprecedented substitution rates. An extreme example is the central integrator of the X:A ratio in Caenorhabditis, xol-1. The xol-1 orthologues of the closely related nematodes C. elegans and C. briggsae are a mere 22% identical, even though genes surrounding xol-1 are much better conserved. Remarkably, the 3′ neighbor of xol-1, the immunoglobulin dim-1, is only 5 kb away and is essentially identical between species (Haag, 2005).

    A second phenomenon, best exemplified by dipteran insects, is the modification of genetic control pathways through the gain or loss of key pathway components. In Drosophila, the first gene to respond to the X:A ratio is Sxl, whose transcription is regulated by both autosomal and X-linked factors very early in development. When X: A = 1 (i.e., in female embryos), Sxl transcription occurs and produces Sxl protein. Later in development, transcription from a second promoter occurs in both sexes, but these transcripts cannot be productively spliced without the earlier burst of Sxl expression. As a result, only females sustain Sxl expression, and in turn only females can productively splice the mRNA of tra, its downstream target. Productive splicing of tra is required to produce the female-specific form of dsx, a founding member of the DM family mentioned above (Haag, 2005).

    In a series of groundbreaking papers, Saccone and colleagues investigated the pathway in the more distantly related heterogametic Mediterranean fruit fly Ceratitis capitata. The first surprise was that although a highly conserved Sxl homologue exists in Ceratitis, it does not undergo sex-specific regulation similar to that of Drosophila, which suggests that it does not play a key switch role (Saccone, 1998). Similar results have also been found for the housefly, Musca domestica, indicating that the role of Sxl in sex determination may be restricted to Drosophila and its closest relatives. In contrast, tra and dsx are key sex regulators in all dipterans examined thus far (Haag, 2005).

    A further surprise came when the Ceratitis tra homologue was characterized. In the case of this gene, clear evidence for sex-specific regulation was found, and as with Drosophila, only females productively splice tra mRNA. However, this splicing difference can be explained nicely by a positive feedback, similar to that seen in Drosophila Sxl, in which Tra protein regulates its own splicing. It has been proposed that the dominant, male-specifying M factor on the Y chromosome inhibits this autoregulation. As a result, males cannot make functional Tra protein, and the male form of Dsx is produced. These experiments show not only how a pathway can evolve, but also, importantly, how X:A and heterogametic GSD systems can be interconverted by modifying the cue that regulates a conserved molecular switch gene (the splicing of tra mRNA) (Haag, 2005).

    Finally, recent studies of Caenorhabditis nematodes have shed light on the genetic basis of the convergent evolution of sex determination related to mating system adaptations. An important factor in this area are new phylogenies of the genus, which consistently suggest the surprising possibility that the closely related hermaphroditic species C. elegans and C. briggsae acquired self-fertilization independently, from distinct gonochoristic (male/female) ancestors. Although this scenario is somewhat uncertain purely on parsimony grounds, recent work on the genetic control of the germline bisexuality that defines hermaphroditism has tipped the balance toward parallel evolution (Haag, 2005).

    C. elegans fog-2, a gene required for spermatogenesis in hermaphrodites but not in males, has been cloned. It became clear that fog-2 is part of a large family of F-box genes and was produced by several recent rounds of gene duplication. The C. briggsae genome sequence suggested that while C. briggsae possesses a similarly large family of F-box proteins, the duplication event giving rise to fog-2 was specific to the C. elegans lineage. This work has been extended by the rigorous demonstration that fog-2 is indeed absent in C. briggsae. A short, C-terminal domain has been identified that makes FOG-2 uniquely able to perform its germline sex-determining function. This domain is probably derived from a frame-shifting mutation in an ancestral gene. Working with C. briggsae, evidence has been found of important species-specific regulation of germline sex determination. RNA interference and gene knockout approaches have shown that while C. elegans requires the male-promoting genes fem-2 and fem-3 to produce sperm in hermaphrodites, C. briggsae requires neither. Given that both genes have conserved roles in male somatic sex determination, this suggests that C. briggsae evolved hermaphroditism in a way that bypasses these genes (Haag, 2005).

    The long-standing mystery of sex determination and its diversity began by comparisons between distantly related species. Recent work on closer relatives has uncovered processes that through a reasonable extrapolation enable the connection of these disparate dots into a fascinating picture of developmental evolution. Though the divergence is extreme, it is likely that a better understanding of the evolution of sex determination genes and pathways holds lessons about the evolution of development in general. The next major challenge will be to integrate the comparative developmental data with the ecological and population processes that are driving the evolution of sex determination. Only then will it be possible to say that the picture is complete (Haag, 2005).

    Dosage compensation and demasculinization of X chromosomes in Drosophila

    The X chromosome of Drosophila shows a deficiency of genes with male-biased expression (see Sturgill, 2007), whereas mammalian X chromosomes are enriched for spermatogenesis genes expressed premeiosis and multicopy testis genes. Meiotic X-inactivation and sexual antagonism can only partly account for these patterns. This study shows that dosage compensation (DC) in Drosophila may contribute substantially to the depletion of male genes on the X. To equalize expression between X-linked and autosomal genes in the two sexes, male Drosophila hypertranscribe their single X, whereas female mammals silence one of their two X chromosomes. Fine-scale mapping data of dosage compensated regions was combined with genome-wide expression profiles to show that most male-biased genes on the D. melanogaster X are located outside dosage compensated regions. Additionally, X-linked genes that have newly acquired male-biased expression in D. melanogaster are less likely to be dosage compensated, and parental X-linked genes that gave rise to an autosomal male-biased retrocopy are more likely located within compensated regions. This suggests that DC contributes to the observed demasculinization of X chromosomes in Drosophila, both by limiting the emergence of male-biased expression patterns of existing X genes, and by contributing to gene trafficking of male genes off the X (Bachtrog, 2010).

    This study found compelling evidence that dosage compensation influences patterns of sex-biased expression in Drosophila, and contributes to movement of male-biased genes off the X. This analysis suggests that the deficiency of male-biased genes on the Drosophila X does not simply reflect a lack of dosage compensation at some genes but instead can partly be accounted for by dosage compensation directly interfering with further upregulation of MSL-bound, already hypertranscribed X-linked genes in males. The X chromosome in male Drosophila is encumbered by the MSL complex and its chromatin structure is modified globally, which may limit subsequent transcription factor binding or chromatin remodeling, and thus inhibit further transcriptional activation. Indeed, direct interference between chromatin remodeling complexes and the dosage compensation machinery has been reported in Drosophila. Additionally, male-biased gene expression originates mainly by increasing transcription of nonbiased genes in males (rather than downregulation in females, and higher expression levels may be harder to achieve on an already hypertranscribed chromosome. High-expression male-biased genes are located less often on the X than low-expression male-biased genes. This is expected if limits in rates of transcription prevent the accumulation of male-biased genes on the X, given that such limitations are less likely to affect genes that are transcribed only at low levels (Bachtrog, 2010).

    Not all organisms show a deficiency of male-biased genes on the X. In particular, mammalian X chromosomes are enriched for single-copy genesis genes that are expressed premeiosis, and multicopy testis genes showing postmeiotic expression. This difference in X-chromosomal gene content between taxa could result from fundamental differences in the mechanisms of dosage compensation. Dosage compensation in mammals is achieved by first doubling global expression levels of the X in both sexes, followed by inactivation of one X in females. The chromatin structure of the active X in mammals and baseline transcription rates of X-linked genes thus appear the same between the sexes (even though they might differ from average autosomal rates of transcription), therefore imposing no male-specific restrictions on the evolution of sex-biased expression patterns. Thus, the difference in X-chromosomal gene content between Drosophila and mammals -- with a deficiency versus an accumulation of male-biased genes -- may be understood in light of their vastly different dosage compensation mechanisms (Bachtrog, 2010).

    Somatic control of germline sexual development is mediated by the JAK/STAT pathway

    Germ cells must develop along distinct male or female paths to produce the sperm or eggs required for sexual reproduction. In both mouse and Drosophila, sexual identity of germ cells is influenced by the sex of the surrounding somatic tissue, but little is known about how the soma controls germline sex determination. This study shows that the JAK/STAT pathway provides a sex-specific signal from the soma to the germline in the Drosophila embryonic gonad. The somatic gonad expresses a JAK/STAT ligand, unpaired (upd), in a male-specific manner, and activates the JAK/STAT pathway in male germ cells at the time of gonad formation. Furthermore, the JAK/STAT pathway is necessary for male-specific germ cell behavior during early gonad development, and is sufficient to activate aspects of male germ cell behavior in female germ cells. This work provides direct evidence that the JAK/STAT pathway mediates a key signal from the somatic gonad that regulates male germline sexual development (Wawersik, 2005).

    While investigating communication between the somatic gonad and germline, the JAK/STAT pathway was found to be specifically activated in male, but not female, germ cells. In Drosophila, JAK/STAT signaling is initiated when an UPD or UPD-like ligand binds a transmembrane receptor (Domeless), activating the JAK Hopscotch (HOP), which phosphorylates the STAT92E transcription factor. STAT activation has been shown to regulate stat gene expression and can induce upregulation of the STAT92E protein, which can be used as an assay for JAK/STAT pathway activation. STAT92E is upregulated specifically in male, but not female germ cells at the time of gonad formation. This reflects male-specific activation of the JAK/STAT pathway since (1) the activated form of STAT92E (phospho-STAT92E) is also detected in only male germ cells, and (2) JAK activity is necessary and sufficient for STAT92E expression. Expression of a JAK inhibitor, Socs36E, results in loss of STAT92E expression in male germ cells and expression of constitutively active JAK (hopTumL) induces STAT92E in female germ cells. The male-specific activation of STAT92E at this time is distinct from STAT92E activation in germ cells in the early embryo, which is not sex-specific and is regulated by the MAP kinase pathway (Wawersik, 2005).

    It was also found that STAT92E expression in male germ cells is dependent on their association with the somatic gonad. STAT92E is not detected in germ cells that are migrating to the gonad, but is detected in male germ cells after they contact the somatic gonad. STAT92E expression is greatly reduced or absent in eya mutants, where somatic gonad identity is initiated, but not maintained. Furthermore, STAT92E is not detected in germ cells found outside the gonad in wild type embryos or in mis-localized germ cells in wunen and HMG-CoA reductase mutants which lack guidance cues that target germ cells to the somatic gonad. However, in these same mutants, STAT92E is detected in the few germ cells that contact the somatic gonad in male embryos (Wawersik, 2005).

    STAT92E expression in the germline is dependent on the sex of the surrounding soma. When XX (normally female) germ cells were present in a soma that was masculinized by expression of the male form of the somatic sex determination gene doublesex (dsx), germ cells now expressed STAT92E. dsx does not play an autonomous role in germ cells themselves, indicating that STAT92E induction in these embryos is caused by masculinization of the soma. Conversely, when the somatic gonad of an XY (normally male) embryo is feminized by expression of the sex determination gene transformer (tra) in the mesoderm, but not germ cells, STAT92E expression is no longer observed in XY germ cells. Taken together, these data indicate that the male somatic gonad is necessary and sufficient to activate the JAK/STAT pathway in either XX or XY germ cells (Wawersik, 2005).

    Consistent with this, it was found that the JAK/STAT ligand, upd, is expressed specifically in the male, but not female, somatic gonad. Expression of STAT92E in male germ cells was no longer detected in embryos in which upd and two homologs, upd2 and upd3, are deleted [Df(os1a]. Since male germ cells from embryos mutant for upd alone still express STAT92E, JAK/STAT activation in the germline may be regulated redundantly by upd and one or more of its homologs. In addition, expression of upd in either the mesoderm or germ cells is sufficient to induce STAT92E expression in XX germ cells. Expression of upd2 or upd3 is also capable of inducing STAT92E in germ cells (Wawersik, 2005).

    upd is also important for embryonic patterning and somatic sex determination. Interestingly, upd promotes female identity in the soma, but promotes male development in the germline. To verify that the effects of upd on the germline are not indirectly caused by other effects of upd, indicators of embryonic segmentation (Engrailed), somatic sex determination (Sex lethal), somatic gonad identity (Eyes absent), and somatic gonad sexual identity (Sox100B) were examined. Df(os1a) hemizygous male embryos exhibit segmentation defects as expected, but form gonads that express normal somatic and sex-specific markers. Embryos ectopically expressing upd are normal in all respects examined (Wawersik, 2005).

    Whether activation of the JAK/STAT pathway by the male somatic gonad regulates male-specific development of germ cells was examined. In adult testes, the JAK/STAT pathway is required for maintenance of germline stem cells, making it difficult to assess the role of this pathway on male germ cell identity at this stage. Instead, germ cells were examined during embryogenesis and early larval stages, when germ cell development first becomes sexually dimorphic. In the mouse, the earliest manifestation of sex determination in the germline is differential regulation of the germline cell cycle by the soma. In Drosophila, germ cells undergo 1-2 divisions after their formation, but are arrested in the cell cycle during germ cell migration and only resume division shortly after the gonad has formed. Since larval testes contain more germ cells than larval ovaries, whether proliferation is regulated differently in male and female germ cells was examined. Indeed, sex-specific analysis of a mitotic marker (phosphohistone-H3) in the germline indicates that germ cell proliferation is entirely male-specific during early stages of gonad development. Furthermore, male-specific germ cell division is dependent on the male somatic gonad. Male germ cells do not proliferate in eya mutants that lack the somatic gonad, or in lost germ cells within wunen mutant embryos. XX germ cells in a masculinized soma (dsxD/ dsx1) proliferate, while XY germ cells in a feminized soma (UAS-traF; twist-Gal4) do not. Thus, the pattern of germ cell proliferation correlates exactly with activity of the JAK/STAT pathway in germ cells (Wawersik, 2005).

    To assess whether JAK/STAT signaling regulates male-specific germ cell division, embryos lacking zygotic Stat92E activity were examined and a dramatic decrease was observed in male germ cell proliferation. Similar reductions in germ cell proliferation are observed in the upd/upd-like mutant (Df(os1a)) and in embryos where the JAK inhibitor Socs36E is expressed in germ cells. Thus, JAK/STAT activity is required within germ cells for proper male-specific germ cell division in the gonad. Expression of upd in the germline is sufficient to induce proliferation in female germ cells. Thus, the JAK/STAT pathway can induce XX germ cells to exhibit this male-specific germ cell behavior (Wawersik, 2005).

    Whether the JAK/STAT pathway regulates other aspects of male germ cell development was examined. male germline marker-1 (mgm-1) is a lacZ enhancer trap line that is expressed in male germ cells, but not female germ cells, and therefore is a marker for male germ cell identity. Inhibiting the JAK/STAT pathway by removing zygotic Stat92E activity does not affect mgm-1 expression in the embryo, which is as expected since initial mgm-1 expression is dependent on germ cell autonomous cues. However, removal of zygotic Stat92E activity completely abolished mgm-1 expression in first instar larvae. In wild-type first instar male larvae, mgm-1 expression is observed in most germ cells, which are likely to be developing male germline stem cells and spermatogonia. No mgm-1 expression is observed in Stat92E-mutant larvae, and β-galactosidase expression is only observed in the soma, not the germline, in the pattern expected from the Stat92E P element allele. In an experiment where 25% of larvae were expected to be both male and contain the mgm-1 enhancer trap, 23.2% (n=262) of wild type larvae exhibited mgm-1 expression in the germ cells, while no Stat92E mutant larvae exhibited germ cell mgm-1 expression; this is significantly different from wild type siblings. Thus, Stat92E mutants exhibit a strong effect on male germline development, and some male germline cell types are either missing, or have an altered identity (Wawersik, 2005).

    Finally, the extent to which activation of the JAK/STAT pathway can masculinize female germ cells was assessed. Female germ cells expressing upd are not expected to be fully masculinized because, although a male-specific signal is being activated, these germ cells are otherwise still in a female somatic environment and retain female germ cell autonomous cues. Indeed, such embryos give rise to fertile adult females, indicating that at least some germ cells retain, or revert back to, a female identity. This may be due, in part, to the failure of the upd construct to be expressed in the adult female germline. However, upd is sufficient to induce male-specific gene expression in embryonic XX germ cells. While mgm-1 is normally expressed only in germ cells in males, mgm-1 was expressed in all embryos when upd was ectopically expressed. In addition, two new male germline markers, disc proliferation abnormal (dpa) and minichromosome maintenance 5 (mcm5), were identified, that can also be induced by upd. Whereas these genes are normally expressed in germ cells only in males, female embryos exhibit germ cell expression of these genes when upd is ectopically expressed. In an experiment where only 50% of embryos are expected to express ectopic upd in the germline, 32.5% of female embryos expressed dpa and 21.3% expressed mcm5. Therefore, upd expression is sufficient to activate male-specific gene expression in female germ cells (Wawersik, 2005).

    These data indicate that the JAK/STAT pathway mediates a critical signal from the male somatic gonad that is required for male germ cell development. This signal likely acts together with male germ cell autonomous cues to promote male germline identity and spermatogenesis. This signal is also sufficient to activate the male pattern of proliferation and gene expression in female germ cells, even when these germ cells retain female germ cell autonomous cues and are present in an otherwise female soma. It will be very interesting in the future to identify additional (e.g. female) somatic signals, along with germ cell autonomous cues, and to assess the relative contribution of these factors to proper germline sexual development. Since one of the earliest aspects of sex-specific germ cell behavior in both Drosophila and mouse is the regulation of the germline cell cycle by the somatic gonad, it will be of further interest to determine if the somatic signals operating in Drosophila play a similar role in germline sex determination in mammals (Wawersik, 2005).

    Nonautonomous sex determination controls sexually dimorphic development of the Drosophila gonad

    Sex determination in Drosophila is commonly thought to be a cell-autonomous process, where each cell decides its own sexual fate based on its sex chromosome constitution (XX versus XY). This is in contrast to sex determination in mammals, which largely acts nonautonomously through cell-cell signaling. This study examined how sexual dimorphism is created in the Drosophila gonad by investigating the formation of the pigment cell precursors, a male-specific cell type in the embryonic gonad surrounding the testis. Surprisingly, sex determination in the pigment cell precursors, as well as the male-specific somatic gonadal precursors, was found to be non-cell autonomous. Male-specific expression of Wnt2 within the somatic gonad triggers pigment cell precursor formation from surrounding cells. These results indicate that nonautonomous sex determination is important for creating sexual dimorphism in the Drosophila gonad, similar to the manner in which sex-specific gonad formation is controlled in mammals (DeFalco, 2008).

    This study has shown that two distinct male-specific cell types in the Drosophila gonad exhibit nonautonomous sex determination. For both the male specific somatic gonadal precursors (msSGPs) and the pigment cell (PC) precursors, the sex determination pathway does not act in these cells themselves, and both are dependent on sex-specific signaling from the SGPs in order to develop properly as male or female. These findings are in contrast to the commonly held view that sex determination in Drosophila is a cell-autonomous process, and demonstrate the similarity in sex-specific gonad development between flies and mammals (DeFalco, 2008).

    This study has identified a novel, sex-specific cell type in the Drosophila embryonic gonad, the PC precursors, and studied the mechanism by which the sex determination switch controls the sex-specific development of these cells. The data indicate that male-specific expression of Wnt2 in the SGPs of the gonad signals nonautonomously to the fat body to form PC precursors. dsx ensures that PC formation is male-specific by repressing Wnt2 expression in female gonads in late-stage embryos (stage 17). The sex of the fat body itself does not affect PC precursor formation, since cells with a female identity can form PC precursors when associated with a male gonad or with a female gonad that expresses Wnt2. Furthermore, Wnt2 acts directly on the fat body, since blocking Wnt signaling in male fat body cells prevents them from forming PC precursors. PC precursor identity in the fat body is regulated by ems acting upstream of Sox100B in response to the Wnt2 signal. An interesting question is whether Wnt2 is a direct downstream target of DSX in controlling sexual dimorphism. The DNA binding specificity for DSX has been determined, and there are a number of sites upstream of the Wnt2 start of transcription that either exactly match or closely match the DSX binding consensus sequence. Several of these sites are conserved between different Drosophila species. However, a fragment of the Wnt2 promoter has not yet been identified that allows testing of whether Wnt2 expression in the somatic gonad is directly regulated by DSX, since the upstream region that includes the putative DSX binding sites does not promote expression in the gonad (DeFalco, 2008).

    The creation of sexual dimorphism in the PC precursors differs from that of the msSGPs. While the PC precursors are apparently only specified in males and recruited to form part of the testis, msSGPs are initially specified in both sexes, and are only present in the male gonad because they undergo programmed cell death specifically in females. Furthermore, the germline stem cell niche in the testis (the hub) is formed from a population of anterior SGPs that are present in the gonads of both sexes, but only form the hub in males and presumably form part of the ovary in females. These events are all regulated by dsx, and demonstrate the diverse cellular mechanisms that a sex determination gene can utilize to control sexual dimorphism. Interestingly, in dsx null mutant embryos each of these cell types develops as if it were male. Thus, the male mode of development can at least be initiated in these cell types in the absence of dsx function, and dsx primarily acts in females to repress male development. dsx is clearly required in males at some point for proper testis formation, therefore some cell types in the gonad may not be entirely masculinized in dsx mutants (DeFalco, 2008).

    The nonautonomous nature of PC precursor specification contrasts with the commonly held view that sex determination in Drosophila is a cell-autonomous process, where 'every cell decides for itself' whether it should develop as male or female based on its own intrinsic sex chromosome constitution. This study has shown that the msSGPs undergo nonautonomous sex determination. The data indicate that a male-specific survival signal coming from the SGPs allows the msSGPs to survive and join the male gonad, while they undergo apoptosis in females. Finally, it has been shown that nonautonomous sex determination in the germ cells requires a male-specific signal from the SGPs that acts through the JAK/STAT pathway. Thus, not only does non-cell autonomous sex determination occur in the Drosophila gonad, it appears to be the predominant mechanism of sex determination. Of the cell types tested so far, only the hub cells, which form from a subset of SGPs, appear to decide their sexual fate in an autonomous manner. The current model is that the SGPs determine their sex in a cell-autonomous manner, and then signal to other cell types in the gonad (PC precursors, msSGPs, and germ cells) to control the sex-specific development of these cells via nonautonomous sex determination (DeFalco, 2008).

    Nonautonomous sex determination is not limited to the gonad. Other tissues have been shown to decide their sex through cell-cell signaling. In the genital imaginal disc, the sexual identity of a signaling center, the A/P organizer, largely determines whether the disc will develop in the male or female mode. This is controlled non-cell autonomously through Wingless and Decapentaplegic signaling. In addition, sex-specific migration of mesodermal cells into the male genital disc is regulated by male-specific expression of the Fibroblast Growth Factor Branchless in the genital disc. Finally, in the nervous system, male neurons can non-cell autonomously induce the formation of the male-specific muscle of Lawrence from female muscle precursors. Given the large number of tissues and cell types that undergo nonautonomous sex determination, it seems that the conventional view can be abandoned that sex determination in Drosophila is an obligatorily cell-autonomous process; while some cell types utilize a cell-autonomous mechanism, many cell types clearly do not (DeFalco, 2008).

    One reason why sex determination has been traditionally thought of as a cell-autonomous process in Drosophila is due to its relationship with X chromosome dosage compensation. This is the process by which gene expression from the single X chromosome in males is regulated to match that from the two X chromosomes in females. Both sex determination and X chromosome dosage compensation are regulated by the number of X chromosomes, acting through the master control gene Sex lethal (Sxl). It is likely that most or all cells count their X chromosomes and use this information to control X chromosome dosage in a cell-autonomous manner. However, as discussed above, it is now clear that cells do not necessarily use this information to decide their sex. Consistent with this idea, the expression of dsx, a key regulator of sex determination downstream of Sxl, is surprisingly tissue-specific. Within the embryo, dsx is only expressed in the SGPs and msSGPs of the gonad. Thus, not all cells even express the machinery to translate their sex chromosome constitution into sexual identity, and it is therefore necessary that sex-specific development of many cell types be controlled nonautonomously (DeFalco, 2008).

    The nonautonomous cell-cell interactions that control gonad sexual dimorphism in Drosophila show great similarity to sex-specific gonad development in other species. In mammals, somatic sex determination is based on the presence or absence of the Y chromosome. The critical Y chromosome gene Sry is mainly expressed in a subset of cells in the somatic gonad in the mouse embryo, similar to dsx expression in the Drosophila embryonic gonad. Sry is only thought to be important for formation of Sertoli cells in males, and the sexually dimorphic development of all other cell types is thought to be regulated by local cell-cell interaction or hormonal cues. An excellent example of nonautonomous sex determination in the mouse is the recruitment of cells from the neighboring mesoderm (mesonephros) to form specific cell types in the mouse testis. Recruitment of these cells is dependent on the sex of the gonad, not the sex of the mesonephros. In addition, sex-specific development of other somatic cell types in the mouse gonad is regulated nonautonomously by cell-cell interaction, as is sexual identity in the germline. Thus, the use of non-cell autonomous sex determination and sex-specific cell recruitment are common mechanisms for creating gonad sexual dimorphism in flies and mice (DeFalco, 2008).

    Nonautonomous sex determination in the mouse also utilizes signaling through the Wnt pathway. Wnt4 acts as a 'pro-female' gene that opposes Fibroblast growth factor 9 to regulate sex determination in the gonad. In early stages of gonad development, Wnt4 knockout females form a male-specific coelomic blood vessel and exhibit ectopic migratory steroidogenic cells, suggesting that Wnt4 acts to inhibit endothelial cell and steroid cell migration from the mesonephros into the female gonad. Interestingly, Wnt4 also has been shown to have a role in the male gonad, as male knockout mice show defects in Sertoli cell differentiation, downstream of Sry but upstream of Sox9. Wnt7a also has been implicated in sexual dimorphism in the reproductive tract, as Wnt7a knockout mice fail to express Mullerian-inhibiting substance (MIS) type II receptor in the Mullerian duct mesenchyme, which is required for regression of the duct in male embryos. In addition, a number of Wnt genes have been found to be expressed sex-specifically in the gonad through sex-specific gene profiling, indicating that other Wnt family members play a role in creating sexual dimorphism in the mammalian gonad (DeFalco, 2008).

    It is also interesting that several conserved transcription factors act during gonad development in diverse species. Sox100B is the fly homolog of SOX9/Sox9, a critical regulator of sex determination and male gonad development in humans and mice. Similarly, a mouse homolog of ems, Emx2, is expressed in the developing gonad and is required for development of the urogenital system. Lastly, dsx homologs of the DMRT family have been implicated in sex-specific gonad development in diverse species. Thus, not only are the cellular mechanisms, such as non-cell autonomous sex determination and cell-cell recruitment, common between flies and mice, but the specific genes that regulate sexually dimorphic gonad development may also be conserved. Since the formation of testes versus ovaries, and sperm versus egg, are critical features of sexual reproduction, they may represent processes that are highly conserved across the animal kingdom (DeFalco, 2008).

    Specialized cells tag sexual and species identity in Drosophila melanogaster

    Social interactions depend on individuals recognizing each other, and in this context many organisms use chemical signals to indicate species and sex. Cuticular hydrocarbon signals are used by insects, including Drosophila, to distinguish conspecific individuals from others. These chemicals also contribute to intraspecific courtship and mating interactions. However, the possibility that sex and species identification are linked by common chemical signalling mechanisms has not been formally tested. This study provides direct evidence that a single compound is used to communicate female identity among flies, and to define a reproductive isolation barrier between Drosophila melanogaster and sibling species. A transgenic manipulation eliminated cuticular hydrocarbons by ablating the oenocytes (see Insect oenocytes: a model system for studying cell-fate specification by Hox genes), specialized cells required for the expression of these chemical signals. The resulting oenocyte-less (oe-) females elicited the normal repertoire of courtship behaviours from males, but were actually preferred over wild-type females by courting males. In addition, wild-type males attempted to copulate with oe- males. Thus, flies lacking hydrocarbons are a sexual hyperstimulus. Treatment of virgin females with the aversive male pheromone cis-vaccenyl acetate (cVA) significantly delayed mating of oe- females compared to wild-type females. This difference was eliminated when oe- females were treated with a blend of cVA and the female aphrodisiac (7Z,11Z)-heptacosadiene (7,11-HD), showing that female aphrodisiac compounds can attenuate the effects of male aversive pheromones. 7,11-HD also was shown to have a crucial role in heterospecific encounters. Specifically, the species barrier was lost because males of other Drosophila species courted oe- D. melanogaster females, and D. simulans males consistently mated with them. Treatment of oe(-) females with 7,11-HD restored the species barrier, showing that a single compound can confer species identity. These results identify a common mechanism for sexual and species recognition regulated by cuticular hydrocarbons (Billeter, 2009).

    D. melanogaster produces hydrocarbons of various chain lengths, including unbranched alkanes, methyl-branched alkanes, alkenes and derivatives thereof. The alkenes are expressed sex-specifically, and have been associated with both sex and species discrimination. Compared to females, males express high levels of the monoalkene (Z)-7-tricosene (7-T), which has been reported to increase females' receptivity to mating attempts. Moreover, 7-T is repulsive to other males and may prevent male-male interactions. In contrast, females produce sex-specific dienes such as (7Z,11Z)-heptacosadiene (7,11-HD) and (7Z,11Z)-nonacosadiene (7,11-ND), which act as aphrodisiac pheromones for D. melanogaster males. Hydrocarbons are strongly associated with sexual recognition, because wild-type males court males that have been genetically modified to express female hydrocarbons, indicating that the mutants are perceived as females hydrocarbons (Billeter, 2009).

    There are still large gaps in knowledge of the functions of individual hydrocarbons and the tissues where these compounds are synthesized. As in other insects, specialized cells called oenocytes, located on the inner surface of the abdominal cuticle, are thought to be the site of hydrocarbon biosynthesis in D. melanogaster. Consistent with this hypothesis, desaturase 1 (desat1), which encodes an enzyme involved in hydrocarbon synthesis, is expressed in Drosophila oenocytes (Marcillac, 2005). Previous studies have demonstrated that genetic feminization of these cells results in production of female hydrocarbons by male flies; however, these and other manipulations have been confounded by the concurrent feminization of cells in many other sexually dimorphic tissues, including the central nervous system. To test the hypothesis that these cells are required for production of chemical signals used in sexual and species recognition, the Gal4-UAS system was used to target transgene expression specifically to the adult oenocytes. An oenocyte Gal4 driver was generated, derived from the regulatory sequence of one of the desat1 promoters (Marcillac, 2005) that is expressed specifically in oenocytes of adult females. The driver is also expressed in the larval oenocytes and in the reproductive organs of adult males. This driver was used to ablate adult oenocytes by inducing expression of the pro-apoptotic gene head involution defective (hid). This approach initially caused lethality in larvae, probably due to the destruction of the larval oenocytes. To circumvent this problem blocked the driver's action was blocked during development using the Tubulin-Gal80ts transgene. Using this method, flies were generated without oenocytes (oe-). Analysis of whole-body hydrocarbon extracts confirmed that both oe- males and females were essentially devoid of these compounds, showing that the oenocytes are necessary for hydrocarbon display in D. melanogaster. The male pheromone cis-vaccenyl acetate (cVA) was unaffected in oe- males because this compound is synthesized in the ejaculatory bulb. The oe- transgenic strain therefore provided a 'blank slate' for evaluating the role of hydrocarbons in intra- and interspecific communication hydrocarbons (Billeter, 2009).

    Sexual behaviour of oe- flies was assayed to test hydrocarbon function during reproduction. Despite the association of hydrocarbon signals and Drosophila courtship, absence of these signals did not alter courtship behaviours per se. The oe- males displayed normal courtship behaviour towards wild-type females, but slightly less intense than control males. However, wild-type females were less receptive to oe- males than control males, with oe- males taking almost four times as long to achieve mating. Thus, hydrocarbons of males do not seem to affect their own courtship behaviour, but rather, influence the receptivity of females to their mating attempts. However, the influence of non-oenocyte cells within the male reproductive organs that may have been affected by the ablation cannot be excluded. Notably, oe- males elicited courtship and copulation attempts from both wild-type males and other oe- males, indicating that oe- males were perceived as females, even though all other male characteristics were present. The vigorous courtship of oe- males by each other resulted in unnatural behaviours such as engaging one another by rotating in a head-to-head orientation, and males attempting copulation with each other's heads. These behaviours were suppressed by treatment of oe- males with synthetic 7-T, confirming the function of 7-T in inhibiting male-male interactions hydrocarbons (Billeter, 2009).

    Wild-type males exhibited normal courtship behaviour towards oe- females, apparently undeterred by the lack of female . However, mating latency was significantly shorter, and when given a choice between an oe- and a control female, wild-type males preferred oe- females. Together, these data indicate that females lacking hydrocarbons are more attractive than those with a normal hydrocarbon profile. This suggests that female hydrocarbons normally act to slow down male mating attempts, facilitating assessment of a potential partner's species and fitness. Thus, any oe- fly, irrespective of its development as female or male, seems to sexually hyperstimulate males. It is hypothesized that hydrocarbons normally act to superimpose sexual identity on an otherwise attractive fly substrate hydrocarbons (Billeter, 2009).

    The results described above suggested that female attractiveness depends on a balance between attraction/stimulation and repulsion/deterrence. This was investigated by treating females with the aphrodisiac compound 7,11-HD, and with cVA, which males transfer to females via the ejaculate to deter further mating attempts by other males. Whereas cVA decreases the probability that females will re-mate, wild-caught females produce offspring from multiple sires, indicating that polyandry is common and that the effect of cVA is not absolute. O- flies were treated with doses of these compounds approximating wild-type levels. The mating latency of wild-type males with oe- females treated with 7,11-HD was not different from that with untreated oe- females, indicating that 7,11-HD alone does not affect attractiveness of oe- females. As expected, treating wild-type females with increasing doses of cVA delayed mating accordingly, and the effect was even more pronounced with oe- females treated with the same doses of cVA. This effect was not due to differences in the rates of release of cVA from the control and oe- flies, as shown by the profiles of cumulative loss of cVA over time for the two genotypes. Instead, the exaggerated effect of cVA on oe- females is consistent with the hypothesis that the aversive effects of this compound are normally moderated by the presence of other hydrocarbons. Indeed, when cVA and 7,11-HD were applied together, the mating latencies of oe- and wild-type females were indistinguishable. Apparently, 7,11-HD mitigated the deterrent effects of cVA. The data suggest that a male's perception of a female's availability is normally regulated by a mixture of attractive and aversive signals. From an evolutionary perspective, the combined effect of a female attractant with a male deterrent may illustrate an instance of post-copulatory sexual conflict in which the attractant solicits additional mates despite the first male's effort to render a female unattractive by marking her with cVA hydrocarbons (Billeter, 2009).

    In addition to mediating conspecific reproductive interactions, the hydrocarbons of female D. melanogaster have an important role in reproductive isolation between species. For example, within the nine species of the melanogaster subgroup, only D. melanogaster, D. sechellia and D. erecta produce female-specific dienes. Females in the rest of the subgroup express the same hydrocarbons as males. Males of species with non-sexually dimorphic hydrocarbons generally do not court females from dimorphic species, indicating that the dienes might act as reproductive isolation barriers between these species groups. Furthermore, males from dimorphic species do not vigorously court females from non-dimorphic species. In contrast, males of all species in the melanogaster subgroup have similar hydrocarbons, including abundant 7-T. Finally, D. melanogaster females lacking hydrocarbons are courted by at least two sibling species, D. simulans and D. mauritiana. The behaviour of males from other species in the melanogaster subgroup towards oe- females was tested, to assess the contribution of oenocytes and hydrocarbons to reproductive isolating mechanisms. D. simulans and D. yakuba were tested as test species because they represent species in which the females lack dienes. D. erecta was included because it differs from D. melanogaster in the pattern of dienes expressed hydrocarbons (Billeter, 2009).

    Males of all three species courted oe- D. melanogaster females, but exhibited limited or no courtship towards control D. melanogaster females. This indicates that oenocytes and their hydrocarbon products are major components of the reproductive isolation barrier, ensuring that courtship and mating attempts are only initiated between conspecifics. It has been proposed that 7,11-HD functions to create this barrier in D. melanogaster. To test this directly, oe- D. melanogaster females and wild-type females from the different species were treated with synthetic 7,11-HD. Treatment suppressed courtship by males of all three species, demonstrating that 7,11-HD alone is sufficient to create a species barrier. Interestingly, D. erecta males were blocked by 7,11-HD, despite the fact that hydrocarbons of D. erecta females include other dienes in common with those of D. melanogaster. Furthermore, D. melanogaster males actively courted D. erecta females, possibly because the diene 7,11-ND is also expressed by D. melanogaster females. D. simulans and D. yakuba females treated with 7,11-HD elicited strong courtship from D. melanogaster males. These results demonstrate the multifunctional role of 7,11-HD as an attractant and/or stimulant for some species and as a deterrent for others hydrocarbons (Billeter, 2009).

    Despite attempting copulation, D. erecta males never mated with oe- females, suggesting that signals other than hydrocarbons are required to induce receptivity in these females. However, within a 24-h period, nearly all oe- D. melanogaster females mated with D. simulans males, whereas no control D. melanogaster females mated with these males. Treatment of oe- females with 7,11-HD completely blocked interspecific mating with D. simulans males, even at a dose five times lower than the amount found in females of wild-type D. melanogaster strain. Similar treatment of D. simulans females with 7,11-HD only delayed mating by D. simulans males. It is hypothesized that 7-T counters the effect of 7,11-HD in D. simulans females. This is because 7-T functions as an aphrodisiac for D. simulans males and is expressed in higher quantities in D. simulans females than in D. melanogaster females. D. simulans males were assayed with oe- females treated with either 7-T alone, or in combination with 7,11-HD. Synthetic 7-T alone induced a slight decrease in mating latency, indicating that 7-T is an attractant for D. simulans males. However, the striking effect of 7-T was to reduce the effect of 7,11-HD in a dose-dependent manner. These data parallel the balancing effect of 7,11-HD on cVA for D. melanogaster males. Thus, this study has demonstrated that female hydrocarbons orchestrate mating both within and between the species. Whereas a single compound such as 7,11-HD may be enough to establish a species barrier, the effect of this compound is moderated by the relative quantity of other signals. Indeed, the effects of 7,11-HD are particularly noteworthy because it functions as an attractant in an intraspecific context, whereas in an interspecific context, it aids in species recognition, thereby placing social communication and speciation on the same continuum hydrocarbons (Billeter, 2009).

    The logic of pheromonal communication in Drosophila seems to be based on a foundation that imparts general attractiveness to a fly. In this study female oenocytes are the primary organ for communicating species and sex identity to males. Others have shown that males use species-specific acoustic tags within their love song for females during courtship. Thus, both acoustic and pheromonal tags establish a context for social interactions by regulating sex and species recognition. Given that individual flies regulate their own hydrocarbon display in accord with their social surroundings, it is plausible that these compounds also function in individual recognition hydrocarbons (Billeter, 2009).

    The establishment of sexual identity in the Drosophila germline

    The establishment of sexual identity is a crucial step of germ cell development in sexually reproducing organisms. Sex determination in the germline is controlled differently than in the soma, and often depends on communication from the soma. To investigate how sexual identity is established in the Drosophila germline, a molecular screen was conducted for genes expressed in a sex-specific manner in embryonic germ cells. Sex-specific expression of these genes is initiated at the time of gonad formation (stage 15), indicating that sexual identity in the germline is established by this time. Experiments where the sex of the soma was altered relative to that of the germline (by manipulating transformer) reveal a dominant role for the soma in regulating initial germline sexual identity. Germ cells largely take on the sex of the surrounding soma, although the sex chromosome constitution of the germ cells still plays some role at this time. The male soma signals to the germline through the JAK/STAT pathway, while the nature of the signal from the female soma remains unknown. The genes ovo and ovarian tumor (otu) are expressed in a female-specific manner in embryonic germ cells, consistent with their role in promoting female germline identity. However, removing the function of ovo and otu, or reducing germline function of Sex lethal, had little effect on establishment of germline sexual identity. This is consistent with findings that signals from the soma are dominant over germline autonomous cues at the initial stage of germline sex determination (Casper, 2009).

    This analysis demonstrates that a sex-specific program of gene expression is present in the germline soon after the time of gonad formation [stage 15, ~12 hours after egg laying (AEL)]. Examples were identified of both male-specific germline genes and female-specific germline genes, with other genes being expressed equally in the two sexes. Therefore, it is likely that the sex-specific pattern of gene expression in the germline represents the establishment of true sexual identity in the germline, as opposed to other differences that might be observed in the germline between the sexes, such as proliferation status or transcriptional competence. It is concluded that sexual identity in the germline is established at least as early as stage 15. The observation that many genes examined initiate sex-specific germline expression at this time might indicate that this is when germline sexual identity is first established. However, it was also found that the soma signals to the germline in a sex-specific manner just prior to gonad formation (stage 13, ~10 hours AEL), indicating that germ cells could determine their sex even earlier. Because the zygotic genome is only robustly activated in the germline during gastrulation (stage 9, ~4 hours AEL), this sets a narrow time window during which sexual identity is established in the germline (Casper, 2009).

    It is significant that germline sexual identity appears to be first manifested as the germ cells contact the somatic gonad. This is also the time that sexual dimorphism is first observed in the somatic gonad and that it exhibits sex-specific patterns of gene expression, Jheh2 and CG5149). The finding that germ cells might establish their sexual identity only as they contact the somatic gonad is consistent with the strong role of the soma in determining germline sexual identity. In addition, signaling from the germline back to the soma also occurs at this time (Kitadate, 2007; Casper, 2009).

    The establishment of a sex-specific pattern of gene expression in the germline requires these cells to acquire germ cell identity, in addition to sexual identity. Previous work has established that germ cell-specific transcription is independent of the somatic environment, and is autonomously regulated in the germ cells. Indeed, it was seen that germ cell expression of the genes reported in this study can be independent of the somatic gonad. Germ cell-specific gene expression is likely to be regulated by the germ plasm, and several maternally expressed germline transcription factors have recently been identified (Yatsu, 2008; Casper, 2009).

    Previously, it was known that sexual identity in the germline requires autonomous cues, along with non-autonomous cues from the surrounding soma, but little was known about how the signals work together to establish sexual identity. XX germ cells cannot develop normally in a male soma, and XY germ cells cannot develop normally in a female soma. Although evidence was found for both autonomous and non-autonomous regulation of germline sexual identity in the embryo, it appears that non-autonomous signals from the soma are dominant over germline autonomous cues. When XX germ cells are present in a male soma (tra mutants), they exhibit a clear increase in expression of male germ cell genes, and decreased expression of the female-specific gene otu. Similarly, XY germ cells present in a female soma (UAS-tra) exhibit a strong repression of male germ cell genes, while otu expression is increased. Thus, in each case the germ cells largely take on the sexual identity of the surrounding soma, independent of their own sex chromosome constitution. However, subtle differences in gene expression between XX and XY germ cells remain in these situations, indicating some germ cell autonomous control of sexual identity. In addition, when germ cells are present outside of the gonad (srp mutants), some differences are also observed between XX and XY germ cells. When outside of the gonad, XY germ cells are more likely than XX germ cells to express male-specific genes, and XX germ cells maintain some otu expression, while otu is largely off in XY germ cells. Thus, there is at least some autonomous contribution of the germ cell sex chromosome genotype to germline sexual identity at this early stage (Casper, 2009).

    The genes of the ovarian tumor loci, ovo, otu, stil and Sxl, are thought to contribute to autonomous germline sexual identity by promoting female identity in XX germ cells. At this early stage, no change was observed in sex-specific germ cell gene expression in mutants for ovo, otu or stil, or in embryos with mutations that reduce the function of Sxl in the germline. Although this could indicate that these genes do not play a role in the initial establishment of germline sexual identity, these observations could also be due to the dominant effects of the soma on sex-specific germ cell gene expression at this time. These mutations would be expected to masculinize XX germ cells, causing them to exhibit a more male-like pattern of gene expression. However, even fully male (XY) germ cells exhibit a female pattern of gene expression when in a female soma. Thus, the dominant effect of the female soma might mask any masculinizing effects from the removal of ovarian tumor locus genes at this time (Casper, 2009).

    Evidence is seen for at least two types of non-autonomous regulation of germline sexual identity by the soma, one coming from the male soma and the other from the female soma. Both XY and XX germ cells can activate male-specific gene expression when not in contact with the somatic gonad, but expression of these genes does not appear to be as robust as when germ cells are in contact with a male soma. Furthermore, when XX germ cells are in contact with a male soma, expression of the female germ cell gene otu is partially repressed. Thus, the male soma is required for full levels of male gene expression in the germ cells, and for the repression of female genes (Casper, 2009).

    The signal from the male soma to the germline appears to be primarily, and perhaps exclusively, acting through the JAK/STAT pathway. Previously, it was shown that the male soma activates the JAK/STAT pathway in the germ cells, and that this is required for proper male germ cell behavior (Wawersik, 2005). This pathway is necessary for the proper male-specific proliferation of embryonic germ cells, and for the maintenance, but not the initiation, of male-specific mgm-1 expression (male germline marker-1, which appears to be an enhancer trap in the escargot locus). The JAK/STAT ligand UPD is also sufficient for the induction of germ cell proliferation and mgm-1 expression, and for partial induction of mcm5 and dpa expression. This study found that, when the JAK/STAT pathway is inactivated, sex-specific gene expression in the germline resembles that of germ cells that are not in contact with the somatic gonad. Ectopic expression of upd in females is able to induce some aspects of male-specific gene expression in XX germ cells, but not as robustly as when XX germ cells are present in a male soma. This is likely to be due to the fact that, when upd is expressed in an otherwise female soma, it is in competition with female signals that repress male gene expression. It is concluded that the JAK/STAT pathway is important for the regulation of germline sex determination by the male soma, and may be the primary or only signal from the male soma to the embryonic germ cells. It is essential for the male-specific pattern of germ cell proliferation, and for the robust initiation and maintenance of male-specific gene expression (Casper, 2009).

    It is clear that the female soma also plays a key role in regulating the sexual identity of the germline. Both XX and XY germ cells exhibit some level of male-specific gene expression when outside of the gonad, but this expression is dramatically repressed when in contact with a female soma. This repression has also been observed with mgm-1. In addition, XY germ cells exhibit some female-specific otu expression when in contact with a female soma. Therefore, the female soma is essential for the proper sex-specific regulation of germline gene expression, although the nature of the signal from the female soma to the germ cells remains unknown (Casper, 2009). The source of both the male and female somatic signals to the germline is likely to be the somatic gonad. The germ cells show signs of receiving the JAK/STAT signal only when in contact with male somatic gonad, and germ cells outside of the gonad do not appear to receive the proper sex-specific signals (e.g. srp mutants). Furthermore, it is known that somatic regulation of germline sex is controlled by the sex determination cascade, acting through tra and dsx. However, the only cells to express DSX within the embryo are part of the somatic gonad. Thus, the somatic control over germline sex determination is likely to represent local signaling within the gonad environment, rather than long-range signaling from other somatic cell types (Casper, 2009).

    Somatic control over germline sex determination is a common feature of germ cell sexual development. In the mouse, germ cells first manifest sex-specific behaviors as they contact the somatic gonad in the genital ridge. The initial behavior of the germ cells is dependent on the sex of the soma, as male germ cells exhibit female behavior (early meiosis) when contacting a female soma, and female germ cells exhibit male behavior when in contact with a male soma. At least some aspects of this sex-specific behavior are regulated by retinoic acid (RA) levels controlled by the soma; female germ cells receive a high level of RA signal, but the male soma degrades RA so that male germ cells receive less of this signal. Interestingly, this study found that the somatic gonad in Drosophila expresses Jheh2 in a male-specific manner. JHEH2 hydrolyzes Juvenile Hormone (JH), which is structurally related to RA. JH analogs have also been observed to influence sex determination in crustaceans, which further supports this interesting parallel between vertebrates and invertebrates (Casper, 2009).

    In mammals, the germline sex chromosome constitution is also important for germ cell development. As in Drosophila, mouse and human Y chromosome genes are crucial for spermatogenesis. However, the number of X chromosomes also appears to play an autonomous role in germline sexual development in mammals. XX germ cells in a male soma [e.g. in Sex reversed (Sxr) mice appear initially male and do not enter meiosis, but eventually die. XO germ cells in a male soma survive and progress further in spermatogenesis. Similarly, having two X chromosomes promotes female germ cell identity. XX germ cells are predisposed to enter meiosis on the female timetable compared with XO germ cells under the same conditions, and are biased towards a female pattern of imprinting. Thus, as in Drosophila, the germ cell genotype contributes to germ cell sex determination in the mouse and depends on the number of X chromosomes. Similarly, humans with altered sex chromosome constitutions, such as those with Turner's Syndrome (XO females) or Klinefelter's Syndrome (XXY males) have relatively normal somatic development but exhibit severe germline defects, indicating that the proper number of X chromosomes in the germline is essential for germline development (Casper, 2009).

    However, in other species, the sex chromosome constitution of germ cells does not play an important role in germline sex determination. In the housefly, Musca domestica, transplanted germ cells develop normally according to the somatic sex of their host and produce fertile gametes. Some species also exhibit dramatic sexual plasticity in the germline, with the same individual being able to produce both sperm and egg, such as in hermaphrodites (e.g., C. elegans or in species that exhibit natural sex reversal. i.e., wrasses and gobies). Given the diversity of mechanisms animals use for establishing sex determination in the soma, it is not surprising that there might be considerable diversity in the germline as well. However, it appears that an important role for the soma in controlling sexual identity in the germline is a common theme in germ cell development. Whether or not the sex chromosome constitution of the germline is also important for fertility in an organism places additional constraints on the evolution of sex determination mechanisms, sexual plasticity and the sex chromosomes (Casper, 2009).

    Drosophila Sex lethal gene initiates female development in germline progenitors

    Sex determination in the Drosophila germ line is regulated by both the sex of the surrounding soma and cell-autonomous cues. How primordial germ cells (PGCs) initiate sexual development via cell-autonomous mechanisms is unclear. This study demonstrates that, in Drosophila, the Sex lethal (Sxl) gene acts autonomously in PGCs to induce female development. Sxl is transiently expressed in PGCs during their migration to the gonads; this expression, which was detected only in XX PGCs, is necessary for PGCs to assume a female fate. Ectopic expression of Sxl in XY PGCs was sufficient to induce them to enter oogenesis and produce functional eggs when transplanted into an XX host. These data provide powerful evidence that Sxl initiates female germline fate during sexual development (Hashiyama, 2011).

    Primordial germ cells (PGCs) are able to differentiate into eggs or sperm. It is thought that PGCs do not assume a sexual fate until they reach the gonads, where sexual dimorphism is imposed by both the sex of the surrounding soma and cell-autonomous cues. In Drosophila, pole cells or PGCs differentiate to a male fate in response to JAK/STAT signaling from the gonadal soma. The method by which female sexual development is initiated in pole cells, however, has not been elucidated. To clarify the mechanism that initiates a female fate in pole cells, a female-specific marker for this cell type was identified. Although several sex-specific markers, including mgm-1, disc proliferation abnormal, and minichromosome maintenance 5, have been reported, they are all expressed only in male pole cells after gonad formation (stage 15), based on signals from the male gonadal soma. lesswright (lwr), a gene that regulates posttranslational modification of proteins by small ubiquitin-related modifiers, is expressed in pole cells during embryogenesis. lwr is not characterized by sex-specific expression. When a dominant-negative form of lwr (lwrDN) was expressed in the pole cells of either sex, however, apoptosis was induced only in female (XX) pole cells during migration to the gonads. This effect caused a significant reduction in the number of XX pole cells in the gonads. Introduction of female-specific germline apoptosis induced by a dominant-negative form of lwr (f-gal) provides a previously uncharacterized marker of female sexual identity in migrating pole cells (Hashiyama, 2011).

    Sex determination is controlled by the Sex lethal (Sxl) gene, which is first expressed at the blastodermal stage in the embryonic soma. Sxl encodes an RNA binding protein involved in alternative splicing and translation. In the soma of XX embryos, it functions through transformer (tra) and transformer-2 (tra-2), which in turn regulate alternative splicing of the doublesex (dsx) gene to produce a female-specific form of Dsx. In male (XY) embryos, this pathway is turned off, and a male-specific form of Dsx is produced by default. These Dsx proteins determine the sexual identity of somatic tissues. Previous reports, however, suggested that Sxl does not induce female sexual development in the germ line, as it does in the soma. Although Sxl is autonomously required for female sexual development, constitutive mutations in Sxl (SxlM) that cause XY animals to undergo sexual transformation from male to female do not necessarily interfere with male germline development. Moreover, tra, tra-2, and dsx are not required for female germline development. Finally, female-specific Sxl expression has been detected later in gametogenesis, but not in early germline development (Hashiyama, 2011).

    Contrary to previous observations, this study found that Sxl was expressed in XX but not XY pole cells during their migration to the gonads. In the soma, Sxl transcripts are first expressed from the establishment promoter (Sxl-Pe) in a female-specific manner. Using a probe specific to the early transcript derived from Sxl-Pe, in situ hybridization signals were detected in migrating XX pole cells at around stage 9/10. Transgenic embryos, which expressed enhanced green fluorescent protein (EGFP) under the control of the Sxl-Pe promoter, were used to further confirm this female-specific Sxl-Pe activation. RTPE and sequencing analyses in pole cells were used to detect early Sxl transcripts that had the same sequence as the transcripts expressed in the soma (Hashiyama, 2011).

    Next, it was determined whether Sxl feminize early pole cells using f-gal as a marker for female identity. It was found that the loss-of-function mutation SxlfP7B0 represses f-gal in XX pole cells. This repression is unlikely to result from sexual transformation of the soma, because an amorphic tra-2 mutation, which alters somatic sex, did not affect f-gal. Conversely, when the expression of Sxl together with lwrDN is forced in pole cells from stage 9 onward by using nanos-Gal4 and UAS-Sxl, f-gal is ectopically observed in XY pole cells. Sxl alone does not induce apoptosis or developmental defects in pole cells. These observations suggest that female sexual identity of migrating pole cells is regulated cell-autonomously by Sxl (Hashiyama, 2011).

    It was then determined whether Sxl induces female development in XY pole cells. Because XY soma produces signals that direct XX germline cells to a male fate, XY pole cells expressing Sxl were transplanted into XX females, and their developmental fate was examined. Even in the presence of a gain-of-function Sxl mutation (SxlM1) that causes XY soma to transform from male to female, XY (or XO) pole cells enter the spermatogenic pathway when transplanted into XX females. These results suggest that Sxl is not sufficient to activate female germline development. SxlM1 mutations, however, do not affect transcription from the Sxl-Pe promoter, but instead structurally alter the late transcript from the Sxl maintenance promoter (Sxl-Pm), which allows Sxl protein production in both males and females. Consistent with this observation, Sxl transcripts derived from Sxl-Pe were detected in the pole cells of only female SxlM1 embryos. Thus, the SxlM1 mutation does not result in Sxl expression in XY pole cells as early as in XX pole cells (Hashiyama, 2011).

    Instead, nanos-Gal4 and UAS-Sxl were used to induce Sxl expression in XY pole cells. Three types of XY pole cells were transplanted, each characterized by a different duration of Sxl expression: (1) XY pole cells in which Sxl was expressed from stage 9 until stage 16/17 using maternal nanos-Gal4 (XY-mSxl), (2) XY pole cells in which Sxl was expressed from stage 15/16 onward using zygotic nanos-Gal4 (XY-zSxl), and (3) XY pole cells in which Sxl was expressed from stage 9 onward using both maternal and zygotic nanos-Gal4 (XY-mzSxl). XY-mzSxl and XY-mSxl pole cells entered the oogenic pathway and produced mature oocytes in XX females. These oocytes contributed to progeny production. Thus, the XY pole cells produced functional eggs, even though oogenesis and egg production were reduced compared with XX pole cells. In contrast, XY-zSxl pole cells did not enter the oogenic pathway in almost all (92.3%) of the XX female hosts and instead were characterized by a tumorous phenotype, an indication of XY germline cells that have maintained male characteristics. Control XY pole cells from the embryos expressing Sxl only in the soma (XY-nullo-Sxl) showed a similar phenotype to that of XY-zSxl pole cells. These observations demonstrate that Sxl expression in XY pole cells during embryogenesis induces functional egg differentiation in the female soma (Hashiyama, 2011).

    Sxl-specific double-stranded RNA (UAS-SxlRNAi) under the control of maternal nanos-Gal4 was used to reduce Sxl activity in XX pole cells during embryogenesis. Introducing UAS-SxlRNAi resulted in tumorous and agametic phenotypes in female adults, indicating that the XX germ line lost female characteristics. Taken together, these results show that Sxl acts as a master gene necessary and sufficient to induce female development in pole cells (Hashiyama, 2011).

    XY-mzSxl pole cells adopted a male fate and executed spermatogenesis when they developed in an XY male soma. This observation suggests that the male soma plays a dominant role in determining the male germline fate, overriding the feminizing effect of Sxl. Another possibility is that the XX female soma plays a critical role in maintaining the Sxl-initiated female germline fate. Indeed, an XX germ line in the male soma shows a male gene-expression profile, whereas an XY germ line in the female soma exhibits a female expression profile, although these germ lines does not execute gametogenesis. Thus, female germline development requires interactions between the germline and somatic cells, in addition to germline-autonomous mechanisms involving Sxl (Hashiyama, 2011).

    In mice, germline sexual identity is also regulated by both germline-autonomous and somatic signals. In the coelenterate Hydra, the germline sex is not influenced by the surrounding soma, and the germ line determines the phenotypic sex of the polyp. Thus, germline-autonomous regulation of sex has probably been present throughout the evolution of animals, and somatic control may have evolved with the emergence of mesodermal tissues, including gonadal soma. Sxl does not appear to play a key role in sex determination in non-drosophilid animals. Nevertheless, future studies should determine whether Sxl homologs are expressed in the germ line of non-drosophilids. Moreover, it would be of particular interest to identify downstream targets of Sxl in the Drosophila germ line and to test whether these genes have a widespread role in germline sex determination (Hashiyama, 2011).

    m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination

    N6-methyladenosine (m6A) is the most common internal modification of eukaryotic messenger RNA (mRNA) and is decoded by YTH domain proteins. Drosophila mRNA m6A methylosome consists of Ime4 and KAR4 (Inducer of meiosis 4 and Karyogamy protein 4), and Female-lethal (2)d (Fl(2)d) and Virilizer (Vir). In Drosophila, fl(2)d and vir are required for sex-dependent regulation of alternative splicing of the sex determination factor Sex lethal (Sxl). However, the functions of m6A in introns in the regulation of alternative splicing remain uncertain. This study shows that m6A is absent in the mRNA of Drosophila lacking Ime4. In contrast to mouse and plant knockout models, Drosophila Ime4-null mutants remain viable, though flightless, and show a sex bias towards maleness. This is because m6A is required for female-specific alternative splicing of Sxl, which determines female physiognomy, but also translationally represses male-specific lethal 2 (msl-2) to prevent dosage compensation in females. The m6A reader protein YT521-B decodes m6A in the sex-specifically spliced intron of Sxl, as its absence phenocopies Ime4 mutants. Loss of m6A also affects alternative splicing of additional genes, predominantly in the 5' untranslated region, and has global effects on the expression of metabolic genes. The requirement of m6A and its reader YT521-B for female-specific Sxl alternative splicing reveals that this hitherto enigmatic mRNA modification constitutes an ancient and specific mechanism to adjust levels of gene expression (Haussmann, 2016).

    m6A modulates neuronal functions and sex determination in Drosophila

    N6-methyladenosine RNA (m6A) is a prevalent messenger RNA modification in vertebrates. Although its functions in the regulation of post-transcriptional gene expression are beginning to be unveiled, the precise roles of m6A during development of complex organisms remain unclear. This study carried out a comprehensive molecular and physiological characterization of the individual components of the methyltransferase complex, as well as of the YTH domain-containing nuclear reader protein in Drosophila melanogaster. The member of the split ends protein family, Spenito, was identified as a novel bona fide subunit of the methyltransferase complex. Important roles of this complex were demonstrated in neuronal functions and sex determination, and the nuclear YT521-B protein was implicated as a main m6A effector in these processes. Altogether, this work substantially extends knowledge of m6A biology, demonstrating the crucial functions of this modification in fundamental processes within the context of the whole animal (Lence, 2016).

    X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila

    The evolution of sex chromosomes has resulted in numerous species in which females inherit two X chromosomes but males have a single X, thus requiring dosage compensation. MSL (Male-specific lethal) complex increases transcription on the single X chromosome of Drosophila males to equalize expression of X-linked genes between the sexes. The biochemical mechanisms used for dosage compensation must function over a wide dynamic range of transcription levels and differential expression patterns. It has been proposed that the MSL complex regulates transcriptional elongation to control dosage compensation, a model subsequently supported by mapping of the MSL complex and MSL-dependent histone 4 lysine 16 acetylation to the bodies of X-linked genes in males, with a bias towards 3' ends. However, experimental analysis of MSL function at the mechanistic level has been challenging owing to the small magnitude of the chromosome-wide effect and the lack of an in vitro system for biochemical analysis. This study used global run-on sequencing (GRO-seq) to examine the specific effect of the MSL complex on RNA Polymerase II (RNAP II) on a genome-wide level. Results indicate that the MSL complex enhances transcription by facilitating the progression of RNAP II across the bodies of active X-linked genes. Improving transcriptional output downstream of typical gene-specific controls may explain how dosage compensation can be imposed on the diverse set of genes along an entire chromosome (Larschan, 2011).

    In addition to increasing the transcription of X-linked genes for dosage compensation, MSL complex also positively regulates the roX noncoding RNA components of the complex, to promote their male-specificity. roX1 expression is low in the SL2 cell line, but GRO-seq data indicate that active transcription of roX2 is highly dependent on MSL2 as predicted. Interestingly, there is a strong GRO-seq peak at the 3′ roX2 DHS which contains sequences important for targeting MSL complex to the X chromosome. Sites of roX gene transcription are thought to be critical for MSL complex assembly. Therefore, it is possible that paused RNAP II at the roX2 DHS could promote an open chromatin structure that facilitates MSL complex targeting or incorporation of noncoding roX2 RNA into the complex (Larschan, 2011).

    In summary, it is hypothesized that MSL complex functions on the male X chromosome to promote progression and processivity of RNAP II through the nucleosomal template. Improving transcriptional output downstream of typical gene-specific regulation makes biological sense when compensating the diverse set of genes found along an entire chromosome (Larschan, 2011).

    'Jump Start and Gain' model for dosage compensation in Drosophila based on direct sequencing of nascent transcripts

    Dosage compensation in Drosophila is mediated by the MSL complex, which increases male X-linked gene expression approximately 2-fold. The MSL complex preferentially binds the bodies of active genes on the male X, depositing H4K16ac with a 3' bias. Two models have been proposed for the influence of the MSL complex on transcription: one based on promoter recruitment of RNA polymerase II (Pol II), and a second featuring enhanced transcriptional elongation. This study utilize nascent RNA sequencing to document dosage compensation during transcriptional elongation. Also, comparison was made of X and autosomes from published data on paused and elongating polymerase in order to assess the role of Pol II recruitment. The results support a model for differentially regulated elongation, starting with release from 5' pausing and increasing through X-linked gene bodies. The results highlight facilitated transcriptional elongation as a key mechanism for the coordinated regulation of a diverse set of genes (Ferrari, 2013).

    This study has systematically dissected the mechanism of DC during distinct steps of transcription. Multiple high-resolution, genome-wide approaches converge on the following model: paused Pol II is not augmented in general on the male X, but Pol II release from pausing ('jump-start' in the model) appears to be a key rate-limiting step that is facilitated by X-specific enrichment of H4K16ac in gene bodies. The increasing MSL and H4K16ac levels over the bodies of genes further reduce steric hindrance, leading to a 'gain' of Pol II density. Currently, it cannot be determined whether this gain is the result of (1) increased processivity (reduced termination) or (2) positive feedback to 5' Pol II, to further increase pausing release. In either case, it is believed that facilitated elongation through an acetylated chromatin template enables coordinate control of X-linked genes with widely differing mechanisms of individual, gene-specific regulation (Ferrari, 2013).

    POF regulates the expression of genes on the fourth chromosome in Drosophila melanogaster by binding to nascent RNA

    In Drosophila, two chromosome-wide compensatory systems have been characterized: the dosage compensation system that acts on the male X chromosome and the chromosome-specific regulation of genes located on the heterochromatic fourth chromosome. Dosage compensation in Drosophila is accomplished by hypertranscription of the single male X chromosome mediated by the male-specific lethal (MSL) complex. The mechanism of this compensation is suggested to involve enhanced transcriptional elongation mediated by the MSL complex, while the mechanism of compensation mediated by the Painting of fourth (POF) protein on the fourth chromosome has remained elusive. This study shows that POF binds to nascent RNA, and this binding is associated with increased transcription output from chromosome 4. Genes located in heterochromatic regions spend less time in transition from the site of transcription to the nuclear envelope. These results provide useful insights into the means by which genes in heterochromatic regions can overcome the repressive influence of their hostile environment (Johansson, 2012).

    Aneuploidy of entire chromosomes and chromosome segments is an important evolutionary driving force that increases variation but is accompanied by problems associated with changes in gene dosage and genomic instability. The evolution of buffering systems that compensate for dosage differences must therefore allow for a balance between allowing genomic variability and avoiding genomic instability. Buffering systems of this kind have been described in Drosophila; in haploid conditions, they cause the transcription output to increase by a factor of approximately 1.4. However, the evolution of heteromorphic sex chromosomes, such as the X and Y chromosome pair in flies and mammals, is accompanied by an expression problem that requires more extensive compensation. Since most genes on the X chromosome should be expressed at the same levels in males and females, dosage compensation mechanisms coevolve as the X-Y chromosome pair is formed. Notably, while the ancient homology between the mammalian X and Y is clear, the evolutionary origin of the Drosophila Y is more complicated. The evolution of dosage compensation mechanisms is attributable to evolutionary pressures that act at all levels of expression to compensate for the losses of functional gene copies. Two dosage compensation systems have been studied in Drosophila: the male-specific lethal (MSL) complex, which targets and upregulates the male X chromosome, and the painting of fourth (POF) protein, which stimulates the expression of the fourth chromosome in Drosophila melanogaster but is believed to have originated from a dosage-compensating mechanism. The MSL complex and POF coexist; they are on different chromosomes in, e.g., D. melanogaster, but are colocalized on the same chromosome in, e.g., Drosophila ananassae. Their coexistence suggests that they probably act on different levels of gene regulation (Johansson, 2012).

    The MSL complex is a ribonucleoprotein complex consisting of five male-specific lethal proteins (MSL1, MSL2, MSL3, MLE, and MOF) and two noncoding RNAs, roX1 and roX2. Because expression of MSL2 is sex restricted, the complex is formed only in males and specifically targets the male X chromosome. A model of its action has been proposed: MOF (a histone acetyltransferase) acetylates H4K16, and this modification leads to decompaction of the chromatin and hypertranscription of the male X chromosome genes. The prevailing idea is that the MSL complex stimulates transcriptional elongation. This idea is supported by genome-wide mappings showing that expression of the MSL complex and the associated H4K16 acetylation are both enhanced within gene bodies with a bias to their 3' end. A recent study confirmed that the MSL complex enhances transcription by facilitating the progression of RNP2 across the active X chromosomal genes (Johansson, 2012).

    Less is known about the regulatory level at which POF acts. POF is a 55-kDa protein containing an RNA recognition motif (RRM). Like the MSL complex, POF binds within the bodies of expressed genes. However, in D. melanogaster, POF specifically targets the fourth chromosome in both males and females. The targeting of POF to the fourth chromosome is associated with a chromosome-specific increase in transcript levels that primarily affects differentially expressed genes. Flies can survive without POF or missing one copy of the fourth. However, haplo-fourth animals die if they also lack POF. Importantly, the expression of nonubiquitously expressed genes on the fourth chromosome has been shown to be compensated by POF in haplo-4 flies; suppressing or eliminating this compensation causes haplo-fourth lethality (Johansson, 2012).

    The fourth chromosome of D. melanogaster has several unique characteristics. It is the smallest chromosome in the Drosophila genome, with an approximate size of 5 Mb. Of these 5 Mb, 3 to 4 Mb consists exclusively of simple AT-rich satellite repeats and does not contain any known genes. The sequenced part of chromosome 4 is only 1.3 Mb and represents the banded, polytenized, and gene-rich portion corresponding to cytological sections 101E to 102F. In principle, the entire fourth chromosome can be considered heterochromatic; more specifically, it consists of the HP1 enriched 'green chromatin' as defined by van Steensel and coworkers. This means that the fourth chromosome is enriched in the heterochromatin protein HP1 and in specific histone modification markers of heterochromatin, e.g., methylated H3K9. In keeping with its heterochromatic nature, the polytenized part of chromosome 4 contains large blocks of repeated sequences and transposable elements that are interspersed with the genes. Transgenes inserted on the fourth chromosome often show partially silenced, variegated expression. In fact, the structure and sequence composition of the fourth chromosome, with its scattered repetitive elements, is more reminiscent of the organization of mammalian chromosomes than that of the other D. melanogaster autosomes. It appears as though the genes located on the fourth chromosome have adapted to function in this repressive milieu (Johansson, 2012).

    To study the fundamental compensatory processes acting on the fourth chromosome, RNA immunoprecipitation (RIP) experiments followed by tiling array analysis (RIP-chip) and transcript profiling experiments were performed. This study shows that POF associates with the newly transcribed RNA produced from the fourth chromosome. The data indicate that POF binds to the spliced form of the transcript and that this binding is associated with an increase in the amount of chromosome 4 transcripts. It was also shown that transcripts encoded by the fourth chromosome or pericentromeric heterochromatin have a shorter transition time from the site of transcription to the nuclear envelope (Johansson, 2012).

    In Drosophila, two chromosome-wide compensatory systems have been characterized: the MSL complex, which targets and stimulates the expression of the X chromosome in males, and POF, which targets and stimulates the expression of the fourth chromosome. It has been hypothesized that the MSL complex stimulates expression by facilitating transcriptional elongation, and this hypothesis was recently confirmed experimentally. This study has used RNA immunoprecipitation and transcriptome profiling techniques to further clarify the mechanism by which POF stimulates gene expression (Johansson, 2012).

    Previous studies have shown that POF binds to active genes on the fourth chromosome. Since previously reported ChIP-chip results were obtained using cross-linked extracts, they could not be used to determine whether POF associates directly with the chromatin or binds via interactions with other components and was frozen in place by the cross-linking. In the work reported in this study, a relationship was observed between the binding of the POF protein to the chromosome and that of the RNA polymerase. This connection with transcription, together with the fact that POF possesses an RNA binding domain, suggested that POF binds to RNA rather than directly to chromatin. To test this hypothesis, a genome-wide POF RIP-chip experiment was performed. The POF-RIPs verified the association of POF with chromosome 4 transcripts (Johansson, 2012).

    Two components of the MSL complex (MSL2 and MOF) were also investigated, both as controls and to determine whether the MSL complex also binds to transcripts from the chromosome it regulates, i.e., the male X chromosome. In contrast to the observed association of POF with transcripts from the fourth chromosome, no unambiguous evidence was found to support any binding of MSL2 or MOF to X-linked transcripts. However, at this point, it cannot be excluded that other components of the MSL complex, especially the more loosely bound MLE, may have a general affinity for transcripts of the X chromosome. A slight reduction was also noticed in the number of transcripts from the fourth chromosome in both the MSL2-RIPs and the MOF-RIPs, suggesting that the general RNA affinity of the MSL complex is less pronounced for the fourth chromosome than for the other chromosomes. This relative reduction in the number of chromosome 4 transcripts associated with the MSL complex might reflect the binding of POF to these transcripts, which could block their association with other RNA binding proteins (Johansson, 2012).

    The POF-RIP results, together with the previously reported strong link between POF and the fourth chromosome, suggested that POF binds to nascent RNAs, while these RNAs are still bound to the RNP2. The most parsimonious explanation of the high similarity between the ChIP-chip and RIP-chip profiles for POF is that POF associates with nascent RNA and the ChIP profile is indicative of chromatin linked via RNP2. However, at this point it cannot be excluded that POF in addition to binding nascent RNAs also associates with fully processed mature mRNAs en route to the cytoplasm. It also remains possible that POF, in addition to binding nascent RNA, also associates with chromatin via interactions with other proteins (for instance, HP1). No evidence of physical association between POF and HP1 has so far been reported, but POF and Setdb1 (the HKMT responsible for H3K9me on the fourth chromosome) have been shown to interact in vitro. Thus, POF may be an element of an adaptor system linking histone marks to nascent RNA via HP1 and Setdb1, in a fashion similar to MRG15 and PTB. The fact that POF binding to chromatin is RNase resistant may be explained by a stabilization of POF via interaction with a chromatin-associated factor like HP1 or Setdb1. The RNase resistance may also be caused by inaccessibility, i.e., the nascent RNA associated with POF is not accessible to the RNase (Johansson, 2012).

    POF was observed to bind to RNA from other chromosomes, indicating that it possesses a general affinity for RNA. This association with transcripts other than those from the fourth chromosome is more pronounced in the native samples, suggesting that it occurs during sample preparation as an equilibrium reaction rather than accurately reflecting in vivo associations. It is speculated that during the preparation of the native samples, some POF molecules are released from their normal target sites and become free to associate with any transcript in the nucleoplasm. In contrast, in the cross-linked samples, the in vivo POF binding is 'frozen' before any sample treatment steps are performed. Consequently, POF will only be observed to bind to chromosome 4 transcripts, and the apparent enrichment of RNAs encoded from other chromosomes is lost (Johansson, 2012).

    Splicing is commonly regarded as a process that takes place after transcription. However, cotranscriptional splicing was visualized in Drosophila more than 25 years ago. More recent studies have revealed that cotranscriptional splicing is more common than was previously believed and also that splicing can begin as a cotranscriptional event and continue posttranscriptionally . Therefore, the strong exon bias reported in this study is compatible with POF binding to nascent RNAs. Cotranscriptional splicing is believed to depend on cooperation between exon recognition and the speed of transcription by RNP2. Further, it has been shown that the density of nucleosomes is higher within exons; they may thus function as 'speed bumps' to slow down the RNP2 elongation rate. Since POF is connected to nascent RNAs, this reduction in the speed of transcription over exons would explain the exon bias observed at the chromatin level in ChIP-chip experiments (Johansson, 2012).

    Given that POF presumably binds to nascent RNA via its RRM1 domain, it is interesting to consider hypothetical mechanisms by which POF might regulate the expression of genes on chromosome 4. The binding of POF to nascent RNA may directly or indirectly (i.e., via chromatin structure modifications) stimulate transcription. There are several examples of interactions between chromatin-associated proteins recognizing histone modification marks and proteins that bind nascent RNA (Johansson, 2012).

    To explore the potential difference in engaged RNP2 distribution on chromosome 4 compared to other autosomes, the previously published GRO-seq data (that maps the position, amount, and orientation of transcriptionally engaged polymerases genome wide) for Drosophila S2 cells was used. That study shows that the male X chromosome has a higher elongation density index (EdI) than the autosomes, which is interpreted as an enhanced transcription elongation. In contrast, comparing the fourth chromosome to all other autosomes, a significantly decreased EdI was found, consistent with a less efficient transcription elongation. The fourth chromosome also shows a decreased pausing index (PI). This is, on the other hand, in line with an increased transcription output from chromosome 4 genes. It is tempting to speculate that the heterochromatic nature of the fourth chromosome, with HP1 enriched over gene bodies of active genes, causes the decreased EdI. Considering that the fourth chromosome is expressed at levels equal to (or slightly higher than) those of the other chromosomes, this elongation disadvantage may be counteracted by a decreased RNP2 pausing. Since POF is bound to in principle all active chromosome 4 genes, it remains elusive whether POF is connected to the observed decrease in chromosome 4 PI (Johansson, 2012).

    It is also possible that the binding of POF to nascent RNA has posttranscriptional effects. There are at least three possible posttranscriptional scenarios we must consider: splicing, protection, and transport. If splicing were the main function of POF targeting, we would expect a difference in the transcriptome profiles between Pof mutants and the wild type. However, the transcriptome profiles of Pof mutants are very similar to those of the wild type, and there is no evidence of an increased rate of incorrect splicing or more frequent use of introns in the Pof mutant. The only striking difference between the transcriptome profile of the Pof mutant and that of the wild type is the reduction in the amount of processed transcripts from chromosome 4. The same reduction is observed whether we look at the 5' end, 3' end, or middle part of the genes and is thus less consistent with the hypothesis that chromosome 4 transcripts are more prone to degradation in Pof mutants. This demonstrates that POF has a positive effect on the amount of chromosome 4 transcripts which is not caused by improved splicing efficiency (Johansson, 2012).

    Analysis of the ratio of input RNA levels from WCFA (whole cells) to input RNA levels from FA (nuclei) revealed that the relative amounts of chromosome 4 transcripts and transcripts from genes in the pericentromeric region are higher in the cytoplasm than in the nucleoplasm, in relation to transcripts from the other chromosomes. Notably, although export rates per se have not been measured, the results are consistent with pericentromeric and chromosome 4 transcripts being more efficiently exported. Chromatin is highly organized within the nucleus: euchromatic blocks are preferentially located in the center, while heterochromatic regions, such as the fourth chromosome and pericentromeric heterochromatin, tend to localize closer to the nuclear rim. Whether the nuclear periphery is a repressive or permissive environment for gene expression has been debated. The transition time for the export of transcribed mRNAs from the site of synthesis to the nuclear pore would be minimized for chromosomal regions close to the nuclear pore. The gene-gating hypothesis postulates that nucleoporins associate with active genes and facilitate the export of the corresponding mRNAs. Components of the nuclear pore complex (nucleoporins) have been reported to interact with transcriptionally active genes. It has been shown in a mammalian system that transport of an mRNA from the site of transcription to the nuclear pore occurs within a time frame of 5 to 40 min. In the same study, no pileup of mRNAs at the nuclear pore was found, and export through the pore was rapid (0.5 s). Thus, a closer proximity to the nuclear pore may increase transcription output, especially in rapidly dividing cells, since reducing the transition time would allow a more rapid initiation of protein synthesis after cell division. It was observed that the whole cell/nuclei ratio for transcripts produced from the fourth chromosome and the pericentromeric heterochromatin was increased compared to the transcript ratio of the entire genome. It should be stressed that although these genes are located in seemingly repressive environments, both the number of expressed genes and gene expression are comparable to euchromatic chromosome regions. It may be that genes located in these heterochromatic regions benefit from their relative proximity to the nuclear pore, which would facilitate the export of transcribed mRNA. This may in fact be one reason why genes located in pericentric heterochromatin, such as light and rolled, are repressed by rearrangements that move them into euchromatic surroundings (Johansson, 2012).

    It is tempting to speculate that the evolution of more efficient logistics for the export of transcripts from the fourth chromosome was driven by the need to facilitate the expression of its genes. This study has also shown that the quantity of chromosome 4 transcripts is reduced in Pof mutants, and the data indicate that this decrease is not caused by splicing defects or increased degradation. It is not yet known whether the binding of POF to nascent RNAs increases the efficiency of transcription or whether it facilitates their efficient export. However, it should be stressed that transcription levels are probably influenced by a number of stimulatory and repressive influences and that during the course of evolution these factors become increasingly interdependent (Johansson, 2012).

    Dosage compensation via transposable element mediated rewiring of a regulatory network

    Transposable elements (TEs) may contribute to evolutionary innovations through the rewiring of networks by supplying ready-to-use cis regulatory elements. Genes on the Drosophila X chromosome are coordinately regulated by the male specific lethal (MSL) complex to achieve dosage compensation in males. This study shows that the acquisition of dozens of MSL binding sites on evolutionarily new X chromosomes, in Drosophila miranda, was facilitated by the independent co-option of a mutant helitron TE that attracts the MSL complex (TE domestication). The recently formed neo-X recruits helitrons that provide dozens of functional, but suboptimal, MSL binding sites, whereas the older XR chromosome has ceased acquisition and appears to have fine-tuned the binding affinities of more ancient elements for the MSL complex. Thus, TE-mediated rewiring of regulatory networks through domestication and amplification may be followed by fine-tuning of the cis-regulatory element supplied by the TE and erosion of nonfunctional regions (Ellison, 2013).

    Active transposable elements (TEs) impose a substantial mutational burden on the host genome. However, there is growing evidence implicating TEs as drivers of key evolutionary innovations by creating or rewiring regulatory networks. Many TEs harbor a variety of regulatory motifs, and TE amplification may allow for the rapid accumulation of a specific motif throughout the genome, thus recruiting multiple genes into a single regulatory network (Ellison, 2013).

    In Drosophila miranda, multiple sex chromosome/autosome fusions have created a series of X chromosomes of differing ages. The ancestral X chromosome, XL, is homologous to the D. melanogaster X and is at least 60 million years old. Chromosome XR became a sex chromosome ~15 million years ago and is shared among members of the affinis and pseudoobscura subgroups, whereas the neo-X chromosome is specific to D. miranda and originated only 1 million years ago. The male specific lethal (MSL) complex coordinates gene expression on the Drosophila male X to achieve dosage compensation. This complex is recruited to the X chromosome in males to high-affinity chromatin entry sites (CES) containing a conserved GA-rich sequence motif, roughly 21-base pair (bp) in length, termed the MSL recognition element (MRE). Once bound, the MSL-complex spreads from the CES in cis to actively transcribed genes, where it catalyzes the deposition of the activating histone modification H4K16ac, which ultimately results in a chromosome-wide twofold increase in gene expression levels. D. miranda males show MSL binding specific to the X chromosomes, associated with full dosage compensation of chromosomes XL and XR. In contrast, the neo-X shows incomplete dosage compensation (Ellison, 2013).

    The evolution of dosage compensation on XR and the neo-X involved co-option of the MSL machinery (Marin, 1996) and the creation of CES capable of recruiting this machinery, via MRE sequence motifs at a few hundred locations along the two X chromosomes. This study used chromatin immunoprecipitation sequencing (ChIP-seq) profiling of MSL binding to conservatively define 132 CES on chromosome XL, 215 on XR, and 68 on the neo-X, and a more realistic estimate identifies 219 CES on XL, 383 on XR, and 175 on the neo-X; these two groups are referred to as 'strict' versus 'broad' set of CES. The CES on XR and the neo-X likely arose within the past 15 and 1 million years, respectively, after these chromosomes became X-linked in an ancestor of D. miranda (Ellison, 2013).

    Comparison of the genomic regions at strict neo-X CES sequences to their homologous regions in D. pseudoobscura, which are not X-linked and do not recruit the MSL complex, identified the mutational paths responsible for the formation of a MRE at 41 CES on the neo-X. In half of these sites, point mutations and short indels at prebinding sites created a stronger MRE. For the remaining half, however, the new MREs appeared to have been gained via a relatively large (~1 kb), D. miranda-specific insertion. Sanger resequencing and manual curation of the genome assembly at these sites allowed it to be determined that these insertions are derived from a transposable element (homologous to the ISY element) that is highly abundant in the genome of D. miranda and its relatives (>1000 copies in D. miranda and D. pseudoobscura). The ISY element (~1150 bp) is a nonautonomous helitron, a class of DNA-transposable elements that replicate through a rolling-circle mechanism. All 21 elements found at strict CES on the neo-X share a 10-bp deletion relative to the consensus ISY element, and the ISY sequence containing this deletion is referred to as ISX. ISX is also found at 24 of broad CES and is present at 43% of strict CES and 30% of broad CES on the neo-X. This 10-bp deletion creates a sequence motif more similar to the consensus MRE motif inferred from XL relative to the consensus ISY sequence and thus might create a strong recruitment signal for the MSL-complex. The ISX element - but not ISY - is specific to D. miranda and highly enriched on the neo-X relative to other chromosomes and strongly bound by the MSL complex in vivo. Additionally, the sequence similarity among ISX elements found at CES on the neo-X is consistent with their recent acquisition on the neo-X, after the formation of the neo-sex chromosomes. Together, these results suggest that within the past 1 million years, the D. miranda lineage was invaded by a domesticated helitron that recruits hundreds of genes into the MSL regulatory network on the neo-X. This process involved the formation of a high-affinity MRE sequence motif via a 10-bp deletion, followed by amplification and fixation of this element at dozens of sites along the neo-X chromosome(Ellison, 2013).

    A transgenic assay was used in D. melanogaster to functionally verify that the ISX element attracts the MSL complex and functions as a CES. The construct was targeted to the previously characterized autosomal landing site 37B7 in D. melanogaster. Immunostaining of male polytene chromosomes shows that the ISX element can recruit the MSL complex of D. melanogaster, but no staining was detected with the ISY elemen. A higher affinity of the MSL complex to ISX versus ISY was also confirmed by means of ChIP-quantitative polymerase chain reaction. Mutagenesis assays were used to convert this ISX element into ISY by inserting the 10-bp sequence (ISX ->: ISY), and the 10-bp fragment was deleted from the ISY element to create ISX (ISY ->: ISX). Immunostaining confirmed that the ISX ->: ISY construct could no longer recruit the MSL-complex to an autosomal location, whereas the ISY ->: ISX transgene was now able to attract MSL to an autosomal landing site in D. melanogaster. Thus, the ISX element alone is able and sufficient to attract the MSL complex, and the 10-bp deletion creates a functional MSL recruitment site. This experimentally confirms that the amplification of this TE along the neo-X chromosome may have resulted in the rapid wiring of neo-X-linked genes into the dosage compensation network. Dosage compensation of neo-X genes is advantageous because ~40% of homologous neo-Y genes are pseudogenized; however, because of its ability to recruit the MSL complex and induce dosage compensation, the ISX element should be selected against from autosomal locations. Indeed, out of a total of 82 copies of the ISX element, only two exist on an autosome, within repeat-rich and supposedly silenced regions on the dot chromosome (Ellison, 2013).

    In the ancestor of the affinis and pseudoobscura subgroups (~15 million years ago), Muller element D became incorporated into the dosage compensation network after it fused to the ancestral X to form chromosome XR. All CES sequences on XR were compared to determine whether they were enriched for sequence elements besides the MRE motif that would be indicative of a TE burst. Three repeat elements were present in ~22% of strict (and in 14.4% of broad) XR CES sequences, but not in the homologous regions from D. subobscura, where this chromosome is an autosome. Furthermore, these elements were all determined to be conserved fragments from a single TE (hereafter referred to as ISXR), which is derived from the same helitron family as the ISY/ISX elements. Individual ISXR copies are less similar to each other than the ISX elements, and sequence divergence among the different copies of this TE is consistent with a burst of transposition activity coinciding with the formation of chromosome XR. Additionally, ISXR is enriched on chromosome XR, and similar to ISX/ISY, its autosomal homologs show less sequence similarity to the MRE consensus motif and cannot recruit the MSL-complex in vivo. ISXR contains a ~350-bp region that is not present in any of the ISY or ISX elements, and this region specific to ISXR contains an additional MRE motif in close proximity to the MRE whose location is conserved between the ISX and ISXR elements. In addition, although the location of the 3' ISXR MRE is conserved with ISX, there is no evidence of the 10-bp deletion seen in ISX. The presence of this particular sequence region suggests that although ISX and ISXR evolved from a similar helitron progenitor TE, they represent independent TE domestications and chromosomal expansions at different time points. Consistent with the more ancient expansion of ISXR, nonfunctional parts of the TE are severely eroded (Ellison, 2013).

    Similarity-based clustering of the MRE consensus motifs from each helitron subtype reveal that both ISXR MRE motifs are more similar to the canonical XL MRE motif, compared with the ISX MRE motif. This suggests that MSL binding motifs supplied by ISX may be suboptimal, whereas ISXR binding affinity is optimized. A large number of substitutions observed at MRE motifs among ISXR copies across the genome and elevated rate of evolution at homologous ISXR MRE sites relative to XL MREs across species suggest that the ISXR element initially may have also harbored a suboptimal MRE motif. Over time, mutation and selection may have fine-tuned the nucleotide composition at ISXR independently across elements and species, to maximize MSL recruitment by increasing their similarity to the canonical XL MRE motif. In agreement with this observation, the TE-derived XR CES show a higher affinity for MSL complex in vivo as compared with that of those on the neo-X (Ellison, 2013).

    The recently formed sex chromosomes of D. miranda provide insights into the role of TEs in rewiring regulatory networks. The evolutionary pressure driving the acquisition of dosage compensation as well as the molecular mechanism of MSL function and targeting provide clear expectations of which genes should be recruited into the dosage compensation network, as well as when and how. Additionally, the comparison of XR and the neo-X allows a study of the dynamic process of TE-mediated wiring of chromosomal segments into the dosage-compensation network at two different evolutionary stages: both the initial incorporation of the neo-X chromosome by amplification of a domesticated TE and possible subsequent fine-tuning of the regulatory element supplied by the TE on XR, together with the erosion of TE sequence not required for MSL-binding. The data support a three-step model for TE-mediated rewiring of regulatory networks (domestication, amplification, and potential fine-tuning) followed by erosion of nonfunctional parts of the transposon. Eventually, the footprints left behind by TE-mediated rewiring will completely vanish, and many ancient bursts of domesticated TEs that rewired regulatory networks are likely to go undetected. Indeed, no TE relics within the CES of chromosome XL were detected that acquired MSL-mediated dosage compensation over 60 million years ago, either because they evolved via a different mechanism or deletions and substitutions have degraded the signal of TE involvement to the point at which they are no longer recognizable (Ellison, 2013).

    The epigenome of evolving Drosophila neo-sex chromosomes: dosage compensation and heterochromatin formation

    Sex chromosomes originated from autosomes but have evolved a highly specialized chromatin structure. Drosophila Y chromosomes are composed entirely of silent heterochromatin, while male X chromosomes have highly accessible chromatin and are hypertranscribed as a result of dosage compensation. This study dissected the molecular mechanisms and functional pressures driving heterochromatin formation and dosage compensation of the recently formed neo-sex chromosomes of Drosophila miranda. The onset of heterochromatin formation on the neo-Y is triggered by an accumulation of repetitive DNA. The neo-X has evolved partial dosage compensation and it was found that diverse mutational paths have been utilized to establish several dozen novel binding consensus motifs for the dosage compensation complex on the neo-X, including simple point mutations at pre-binding sites, insertion and deletion mutations, microsatellite expansions, or tandem amplification of weak binding sites. Spreading of these silencing or activating chromatin modifications to adjacent regions results in massive mis-expression of neo-sex linked genes, and little correspondence between functionality of genes and their silencing on the neo-Y or dosage compensation on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those becoming heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities along the ancestral chromosome that formed the sex chromosome to adopt active or repressive chromatin configurations. These findings have broad implications for current models of sex chromosome evolution, and demonstrate how mechanistic constraints can limit evolutionary adaptations. The study also highlights how evolution can follow predictable genetic trajectories, by repeatedly acquiring the same 21-bp consensus motif for recruitment of the dosage compensation complex, yet utilizing a diverse array of random mutational changes to attain the same phenotypic outcome (Zhou, 2013).

    D. miranda has a unique karyotype, harboring three sex chromosomes of different ages: XL is the ancestral X chromosome in the genus Drosophila and >60 MY old, XR became X-linked about 15 MY ago, is entirely dosage compensated and its former homolog is completely degenerated (i.e., all genes on XR are hemizygous in males). This implies that a former autosome can become completely transformed into a heteromorphic sex chromosome within only 15 MY in Drosophila. The much younger neo-sex chromosomes are at an earlier stage of this evolutionary transition, and the neo-Y is only partially degenerated and the neo-X has evolved incomplete dosage compensation. This provides a unique opportunity to study the evolutionary processes driving the differentiation of sex chromosomes, and this study investigated how changes to the DNA sequence result in novel epigenetic modifications of the diverging neo-sex chromosomes that affect levels of transcription of neo-sex linked genes. Recruitment of the dosage compensation complex to the neo-X requires the acquisition of a 21-bp consensus motif, and this study uncovered diverse mutational paths that have led to the evolution of novel CES on the neo-X. This highlights how evolution can follow predictable genetic trajectories by repeatedly acquiring the same 21-bp consensus motif for recruitment of the dosage compensation complex, yet utilizing a diverse array of random mutational changes to attain the same phenotypic outcome. It was further shown that heterochromatin formation is triggered by an accumulation of repetitive DNA on the neo-Y, and silences adjacent genes (Zhou, 2013).

    siRNAs from an X-linked satellite repeat promote X-chromosome recognition in Drosophila melanogaster

    Highly differentiated sex chromosomes create a lethal imbalance in gene expression in one sex. To accommodate hemizygosity of the X chromosome in male fruit flies, expression of X-linked genes increases twofold. This is achieved by the male- specific lethal (MSL) complex, which modifies chromatin to increase expression. Mutations that disrupt the X localization of this complex decrease the expression of X-linked genes and reduce male survival. The mechanism that restricts the MSL complex to X chromatin is not understood. The siRNA pathway has been shown to contribute to localization of the MSL complex, raising questions about the source of the siRNAs involved. The X-linked 1.688 g/cm3 satellite related repeats (1.688X repeats; 359-bp repeat unit) are restricted to the X chromosome and produce small RNA, making them an attractive candidate. RNA from these repeats was tested for a role in dosage compensation, and ectopic expression of single-stranded RNAs from 1.688X repeats was found to enhance the male lethality of mutants with defective X recognition. In contrast, expression of double-stranded hairpin RNA from a 1.688X repeat generated abundant siRNA and dramatically increased male survival. Consistent with improved survival, X localization of the MSL complex was largely restored in these males. The striking distribution of 1.688X repeats, which are nearly exclusive to the X chromosome, suggests that these are cis-acting elements contributing to identification of X chromatin (Menon, 2014: PubMed).

    High-affinity sites form an interaction network to facilitate spreading of the MSL complex across the X chromosome in Drosophila

    Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation toward targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses, this study shows that high-affinity sites (HAS), landing platforms of the male-specific lethal (MSL) complex, are enriched around topologically associating domain (TAD) boundaries on the X chromosome and harbor more long-range contacts in a sex-independent manner. Ectopically expressed roX1 and roX2 RNAs target HAS on the X chromosome in trans and, via spatial proximity, induce spreading of the MSL complex in cis, leading to increased expression of neighboring autosomal genes. It was shown that the MSL complex regulates nucleosome positioning at HAS, therefore acting locally rather than influencing the overall chromosomal architecture. The study proposes that the sex-independent, three-dimensional conformation of the X chromosome poises it for exploitation by the MSL complex, thereby facilitating spreading in males (Ramírez, 2015).

    This study provides a first step toward understanding the role of chromosome conformation in dosage compensation in D. melanogaster. HAS, the landing regions of the MSL complex on the X chromosome, frequently reside in proximity to TAD boundaries. HAS are enriched in Hi-C contacts to each other and to other X chromosomal regions and that this organization remains comparable between male and female cells (Ramírez, 2015).

    This analysis revealed that HAS are characterized by a combination of DNA sequence (MREs), chromatin state (active), and gene architecture, which drives the specificity of the MSL complex toward the X chromosome. The data suggest that when the MSL complex binds to HAS, it then spreads (either via an active mechanism or via diffusion) to spatially close regions to place the histone H4 lysine 16 acetylation (H4K16ac) mark on active genes. A 'conformation-based affinity' model is proposed based on the strategic location of HAS at highly interconnected regions of the D. melanogaster X chromosome that efficiently distribute the MSL complex over the X chromosome by attracting the MSL complex to cis-interacting HAS on the X chromosome. This system ensures that only this chromosome is specifically and globally targeted. By spreading from those HAS over short (3D) distances, all active genes on the X chromosome are then reached and acetylated without influencing the autosomes. It is suggested that this system is resilient to major perturbations, exemplified by the large autosomal insertion from chromosome 3L and the ectopic expression of the roX genes that produce viable cells and flies, respectively (Ramírez, 2015).

    MNase-seq analysis shows a direct effect of the MSL complex on nucleosome organization specifically on HAS and not on the TSS, despite prominent binding of MSL1/2 to promoter regions. The MSL complex may act similar to a pioneer DNA binding protein to establish nucleosome patterns at HAS and may act on neighboring active regions rather than modifying TAD boundaries. This system may be unique to flies because the Drosophila dosage compensation evolved a fine-tuning transcription activation mechanism rather than a complete shutdown of gene transcription as seen in mammalian X chromosome inactivation. It would be very interesting to see how nucleosome positioning is affected upon Xist binding in mammals (Ramírez, 2015).

    Although many factors, including the CCCTC-binding factor (CTCF) as well as tRNA and housekeeping genes, have been shown to be enriched at boundaries, by dissecting the targeting and spreading activity of the MSL complex for the X chromosome, this study offers a plausible explanation behind the advantages of HAS localization. HAS are enriched at the X chromosomal boundaries and not at autosomal boundaries, where all other boundary factors will bind indiscriminately. Furthermore, it was found that the few HAS that are not near a boundary also occupy locations of an elevated number of long-range contacts, indicating that HAS form interaction hubs for the spreading of the MSL complex (Ramírez, 2015).

    Hi-C as well as in vivo immunofluorescence show that active roX genes have more contacts and are closer to each other than inactive regions. These observations are in line with previous reports showing that active chromatin compartments interact more often with each other and that active chromatin localizes to the interface of the chromosomal territory. The results imply that different transcriptional programs in each cell line or tissue are likely to be associated with a particular arrangement of long-range contacts, suggesting that the dosage compensation must be flexible to act over such diverse conformations without disturbing them. This idea is consistent with the observation that the chromosome conformation remains unchanged after knockdown of the MSL complex, and stays in contrast to mammalian X inactivation, which involves chromatin condensation, gene inactivation, and alterations in chromosome conformation (Ramírez, 2015).

    Dosage compensation mechanisms in flies and mammals lead to opposite outcomes; namely, gene activation versus gene repression. However, both systems use lncRNAs transcribed from the dosage-compensated X chromosome. roX1 and roX2 RNA are expressed from the male hyperactivated X chromosome in D. melanogaster, whereas Xist is expressed from the inactivated X chromosome in mammalian females. Recent work has shown that Xist spreads to distal sites on the X chromosome. Interestingly, this spreading is dependent on the spatial proximity of sites distal to the Xist gene. This is further exemplified by ectopic expression of Xist from chromosome 21, where Xist spread only in cis on this chromosome. In this study, ectopic insertion of roX transgenes on autosomes demonstrated that the roX/MSL complex can reach the X chromosome and rescue male lethality. Therefore, acting in trans is a special feature of roX RNAs (in conjunction with the MSL complex) not observed for Xist, indicating that the two systems utilize the respective lncRNAs differently. In both systems, however, the lncRNAs need to be functional because the stem loop structures of the roX RNAs are required for dosage compensation in D. melanogaster, whereas Xist needs the "A repeat domain" to induce mammalian X chromosome inactivation. The distinct mechanisms utilized by the Xist and roX RNAs exemplify the great versatility by which lncRNAs can be involved in the global regulation of single chromosomes and might reflect important differences between the two systems. In mammals, only one of the two X chromosomes needs to be inactivated. Therefore, a trans action of Xist RNA on the sister X chromosome would be detrimental to the organism. In contrast, the dosage-compensated X chromosome is present singularly in males in Drosophila. However, because the roX RNAs can act in trans, it may be disadvantageous to target the activating MSL complex to active genes on autosomes, hence the need for specific target regions (the HAS) unique to the X chromosome (Ramírez, 2015).

    To fully understand the occurrence of HAS at sites with extensive long-range interactions on the X chromosomes, it could be helpful to consider evolutionary models proposing that X chromosomes tend to evolve faster than autosomes (faster X effect). Under the faster X effect, traits only beneficial for males can introduce significant changes specific to the X chromosome on a short evolutionary timescale. Based on these and other observations suggesting that the X chromosome in flies is different from autosomes, it is assumed that selective pressures on males favored the occurrence of HAS at regions of increased interactions, like TAD boundaries. Future analyses of different Drosophila species will open exciting opportunities to study the evolutionary changes of HAS in the context of X chromosomal architecture. Moreover, conformation-based affinity could be a generic mechanism for other regulatory elements to exert their functions. It remains to be seen in which contexts the in cis versus in trans action of different lncRNAs is essential for their function and how chromosome conformation, long-range contacts, HAS, and regulation of transcription have co-evolved for dosage compensation (Ramírez, 2015).

    Modulation of heterochromatin by male specific lethal proteins and roX RNA in Drosophila melanogaster males

    The ribonucleoprotein Male Specific Lethal (MSL) complex is required for X chromosome dosage compensation in Drosophila males. Beginning at 3 h of development the MSL complex binds transcribed X-linked genes and modifies chromatin. A subset of MSL complex proteins, including MSL1 and MSL3, is also necessary for full expression of autosomal heterochromatic genes in males, but not females. Loss of the non-coding roX RNAs, essential components of the MSL complex, lowers the expression of heterochromatic genes and suppresses position effect variegation (PEV) only in males, revealing a sex-limited disruption of heterochromatin. MLE, but not Jil-1 kinase, was found to contribute to heterochromatic gene expression. To determine if identical regions of roX RNA are required for dosage compensation and heterochromatic silencing, a panel of roX1 transgenes and deletions was tested; the X chromosome and heterochromatin functions were found to be separable by some mutations. Widespread autosomal binding of MSL3 occurs before and after localization of the MSL complex to the X chromosome at 3 h AEL. Autosomal MSL3 binding was dependent on MSL1, supporting the idea that a subset of MSL proteins associates with chromatin throughout the genome during early development. It is postulated that this binding may contribute to the sex-specific differences in heterochromatin that have been noted (Koya, 2015).

    A central question raised by this study is how factors known for their role in X chromosome dosage compensation also modulate autosomal heterochromatin. Although the MSL proteins were first identified by their role in X chromosome compensation, homologues of these proteins participate in chromatin organization, DNA repair, gene expression, cell metabolism and neural function throughout the eukaryotes. Furthermore, flies contain a distinct complex, the Non-Sex specific Lethal (NSL) complex, containing MOF and the MSL orthologs NSL1, NSL2 and NSL3. The essential NSL complex is broadly associated with promoters throughout the fly genome, where it acetylates multiple H4 residues. In light of the discovery that the MSL proteins represent an ancient lineage of chromatin regulators, it is unsurprising that members of this complex fulfill additional functions (Koya, 2015).

    An alternative hypothesis for the dosage compensation of male X-linked genes proposes that the MSL proteins are general transcription regulators, and recruitment of these factors to the male X chromosome reduces autosomal gene expression, thus equalizing the X:A expression ratio. Arguing against this idea are ChIP studies finding that the MSL complex, and engaged RNA polymerase II, are increased within the bodies of compensated X-linked genes. In agreement with this, a study that normalized expression to genomic DNA concluded that compensation increases the expression of male X-linked genes. The current study now reveals that autosomal heterochromatic genes are indeed dependent on a subset of MSL proteins for full expression. However, native heterochromatic genes make up only 4% of autosomal genes, and their misregulation is not expected to compromise genome-wide expression studies normalized to autosomal expression (Koya, 2015).

    Expression of heterochromatic genes is thought to involve mechanisms to overcome the repressive chromatin environment. It is possible that a complex composed of roX RNA and a subset of MSL proteins participates in this process. This would explain why heterochromatic genes are particularly sensitive to the loss of these factors. Alternatively, it is possible that roX and MSL proteins participate in heterochromatin assembly. This would explain the simultaneous disruption of heterochromatic gene expression and suppression of PEV at transgene insertions (Koya, 2015).

    Heterochromatin assembly is first detected at 3-4 h AEL, a time when MSL3 is bound throughout the genome. Intriguingly, studies from yeast identify a role for H3K4 and H4K16 acetylation in formation of heterochromatin. Active deacetylation of H4K16ac is necessary for spreading of chromatin-based silencing in yeast, demonstrating the need for a sequential and ordered series of histone modifications (Koya, 2015).

    As MOF is responsible for the majority of H4K16ac in the fly, a MOF-containing complex could fulfill a similar role during heterochromatin formation. While this study found a significant effect of MOF in expression only on the X and 4th chromosomes, it is possible that examination of a larger number of genes would reveal a more widespread autosomal effect (Koya, 2015).

    In roX1 roX2 males the 4th chromosome displays stronger suppression of PEV and more profound gene misregulation than do other heterochromatic regions. This is consistent with the observation that heterochromatin on the 4th chromosome is genetically and biochemically different from that on other chromosomes. Loss of roX RNA leads to misregulation of genes in distinct genomic regions, the dosage compensated X chromosome and autosomal heterochromatin. This study found that the regulation of these two groups is, to some extent, genetically separable. MSL2, which binds roX1 RNA and is an essential member of the dosage compensation complex, is not required for full expression of heterochromatic genes in males. Ectopic expression of MSL2 in females induces formation of MSL complexes that localize to both X chromosomes, inducing inappropriate dosage compensation. As would be expected from the lack of a role for MSL2 in autosomal heterochromatin in males, ectopic expression of this protein in females has no effect on PEV (Koya, 2015).

    Elegant, high-resolution studies reveal that MLE and MSL2 bind essentially indistinguishable regions of roX1. Three prominent regions of MLE/MSL2 binding have been identified, one overlapping the 3' stem loop. This stem loop incorporates a short 'roX box' consensus sequence that is present in D. melanogaster roX1 and roX2, and conserved in roX RNAs in related species (Koya, 2015).

    An experimentally supported explanation for the concurrence of MLE and MSL2 binding at the 3' stem loop is that MLE, an ATP-dependent RNA/DNA helicase, remodels this structure to permit MSL2 binding. The finding that disruption of this stem blocks dosage compensation but does not influence heterochromatic integrity is consistent with participation of roX1 in two processes that differ in MSL2 involvement. However, a region surrounding the stem loop is required for the heterochromatic function of roX1, as roX1Δ10, removing the stem loop and upstream regions, is deficient in both dosage compensation and heterochromatic silencing. Further differentiating these processes is the finding that low levels of roX RNA from a repressed transgene fully rescue heterochromatic silencing, but not dosage compensation. An intriguing question raised by this study is why the sexes display differences in autosomal heterochromatin (Koya, 2015).

    The chromatin content of males and females are substantially different as XY males have a single X and a large, heterochromatic Y chromosome. It is speculated that this has driven changes in how heterochromatin is established or maintained in one sex. A search for the genetic regulators of the sex difference in autosomal heterochromatin eliminated the Y chromosome and the conventional sex determination pathway, suggesting that the number of X chromosomes determines the sensitivity of autosomal heterochromatin to loss of roX activity. Interestingly, the amount of pericentromeric X heterochromatin, rather than the euchromatic 'numerator' elements, appears to be the critical factor. The recognition that heterochromatin displays differences in the sexes, and that a specific set of proteins are required for normal function of autosomal heterochromatin in males suggests a useful paradigm for the evolution of chromatin in response to genomic content (Koya, 2015).

    Male-killing spiroplasma alters behavior of the dosage compensation complex during Drosophila melanogaster embryogenesis

    Numerous arthropods harbor maternally transmitted bacteria that induce the preferential death of males. This sex-specific lethality benefits the bacteria because males are "dead ends" regarding bacterial transmission, and their absence may result in additional resources for their viable female siblings who can thereby more successfully transmit the bacteria. Although these symbionts disrupt a range of developmental processes, the underlying cellular mechanisms are largely unknown. It has previously been shown that mutations in genes of the dosage compensation pathway of Drosophila melanogaster suppress male killing caused by the bacterium Spiroplasma. This suggests that dosage compensation is a target of Spiroplasma. However, it remains unclear how this pathway is affected, and whether the underlying interactions require the male-specific cellular environment. This study investigated the cellular basis of male embryonic lethality in D. melanogaster induced by Spiroplasma. It was found that the dosage compensation complex (DCC), which acetylates X chromatin in males, becomes mis-localized to ectopic regions of the nucleus immediately prior to the killing phase. This effect is accompanied by inappropriate histone acetylation and genome-wide mis-regulation of gene expression. Artificially induced formation of the DCC in infected females, through transgenic expression of the DCC-specific gene msl-2, results in mis-localization of this complex to non-X regions and early Spiroplasma-induced death, mirroring the killing effects in males. These findings strongly suggest that Spiroplasma initiates male killing by targeting the dosage compensation machinery directly and independently of other cellular features characteristic of the male sex (Cheng, 2016)

    Sex chromosome-wide transcriptional suppression and compensatory cis-regulatory evolution mediate gene expression in the Drosophila male germline

    In the male germline of Drosophila melanogaster, a novel but poorly understood form of sex chromosome-specific transcriptional regulation occurs that is distinct from canonical sex chromosome dosage compensation or meiotic inactivation. Previous work shows that expression of reporter genes driven by testis-specific promoters is considerably lower-approximately 3-fold or more-for transgenes inserted into X chromosome versus autosome locations. This study characterized transcriptional suppression of X-linked genes in the male germline and its evolutionary consequences. Using transgenes and transpositions, most endogenous X-linked genes, not just testis-specific ones, were shown to be transcriptionally suppressed several-fold specifically in the Drosophila male germline. In wild-type testes, this sex chromosome-wide transcriptional suppression is generally undetectable, being effectively compensated by the gene-by-gene evolutionary recruitment of strong promoters on the X chromosome. A promoter element sequence motif was was identified and experimentally validated that is enriched upstream of the transcription start sites of hundreds of testis-expressed genes; evolutionarily conserved across species; associated with strong gene expression levels in testes; and overrepresented on the X chromosome. These findings show that the expression of X-linked genes in the Drosophila testes reflects a balance between chromosome-wide epigenetic transcriptional suppression and long-term compensatory adaptation by sex-linked genes (Landeen, 2016).

    The essential Drosophila CLAMP protein differentially regulates non-coding roX RNAs in male and females

    Heterogametic species require chromosome-wide gene regulation to compensate for differences in sex chromosome gene dosage. In Drosophila melanogaster, transcriptional output from the single male X-chromosome is equalized to that of XX females by recruitment of the male-specific lethal (MSL) complex, which increases transcript levels of active genes 2-fold. The MSL complex contains several protein components and two non-coding RNA on the X (roX) RNAs that are transcriptionally activated by the MSL complex. Targeting of the MSL complex to the X-chromosome has been shown to be dependent on the chromatin-linked adapter for MSL proteins (CLAMP) zinc finger protein. To better understand CLAMP function, the CRISPR/Cas9 genome editing system was used to generate a frameshift mutation in the clamp gene that eliminates expression of the CLAMP protein. clamp null females were found to die at the third instar larval stage, while almost all clamp null males die at earlier developmental stages. Moreover, it was found that in clamp null females roX gene expression is activated, whereas in clamp null males roX gene expression is reduced. Therefore, CLAMP regulates roX abundance in a sex-specific manner. These results provide new insights into sex-specific gene regulation by an essential transcription factor (Urban, 2017).

    Many species employ a sex determination system that generates an inherent imbalance in sex chromosome copy number, such as the XX/XY system in most mammals and some insects. In this system, one sex has twice the number of X-chromosome-encoded genes compared to the other. Therefore, a mechanism of dosage compensation is required to equalize levels of X-linked transcripts, both between the sexes and between the X-chromosome and autosomes. Dosage compensation is an essential mechanism that corrects for this imbalance by coordinately regulating the gene expression of most X-linked genes (Urban, 2017).

    In Drosophila melanogaster, transcription from the single male X-chromosome is increased 2-fold by recruitment of the male-specific lethal (MSL) complex. The MSL complex is composed of two structural proteins, MSL1 and MSL2, three accessory proteins, MSL3, males absent on the first (MOF), and maleless (MLE), and two functionally redundant non-coding RNAs, RNA on the X (roX1) and roX2. Previous work has shown that recruitment of the MSL complex to the X-chromosome requires the zinc finger protein chromatin-linked adapter for MSL proteins (CLAMP) (Soruco, 2013; Urban, 2017 and references therein).

    In addition to its role in male MSL complex recruitment, it was suggested that CLAMP has an additional non-sex-specific essential function because targeting of the clamp transcript by RNA interference results in a pupal lethal phenotype in both males and females (Soruco, 2013). Further understanding of CLAMP function in the context of the whole organism required a null mutant. However, due to the pericentric location of the clamp gene, no deficiencies or null mutations were available. Using the CRISPR/Cas9 system, a frameshift mutation was introduced in the clamp gene, leading to an early termination codon before the major zinc finger binding domain. This frameshift mutation generated the clamp2 allele, which eliminates detectable CLAMP protein production and is therefore a protein null allele. The majority of clamp2 mutant males die prior to the third instar stage. On the other hand, females die at the third instar stage, suggesting sex-specific functions for CLAMP. Furthermore, CLAMP regulates the roX genes in a sex-specific manner, activating their accumulation in males and repressing their accumulation in females. Overall, we present a new tool for studying dosage compensation and suggest that CLAMP functions to assure that roX RNA accumulation is sex specific (Urban, 2017).

    Previous work demonstrated that CLAMP has an essential role in MSL complex recruitment to the male X-chromosome (Soruco, 2013). However, it was not possible to perform in vivo studies to further investigate CLAMP function because there was no available null mutant line. The current work present a CLAMP protein null mutant and determine that this protein is essential in both sexes. This allele will provide a key tool for future in vivo studies on the role of CLAMP in dosage compensation, as well as identification of the essential function of CLAMP in both sexes (Urban, 2017).

    The initial characterization of the clamp2 protein null allele revealed sexually dimorphic roles for CLAMP in regulation of the roX genes. CLAMP was seen to promotes roX2 transcription in males but represses transcription of both roX genes in females. It is likely that recruitment of the MSL complex to the roX2 locus by CLAMP promotes roX2 expression in males. In females, where the MSL complex is not present, CLAMP may function to repress these loci as an additional mechanism to ensure that dosage compensation is male-specific. Additionally, it was determined that most clamp2 homozygous males die earlier in development than clamp2 homozygous females. Earlier lethality in males is likely due to a misregulation of the dosage compensation process as a result of the loss of CLAMP-mediated MSL complex recruitment. However, CLAMP is enriched at the 5' regulatory regions of thousands of genes across the genome. Therefore, it is likely that other non-sex-specific regulatory pathways are disrupted resulting in female lethality (Urban, 2017).

    Furthermore, CLAMP is an essential protein because our CRISPR/Cas9-generated protein null clamp allele is homozygous lethal in both males and females. These results indicate that CLAMP has a previously unstudied non-sex-specific role that is essential to the viability of both males and females. An interesting observation that arose from this characterization is that polytene chromosome organization is disrupted in clamp2 mutant females, suggesting that CLAMP may play a role in regulation of genome-wide chromatin organization of interphase chromosomes. A function in regulating chromatin organization provides one possible explanation for how CLAMP performs sexually dimorphic functions. For example, CLAMP may repress roX expression in females by promoting the recruitment of a repressive chromatin-modifying factor in the absence of the MSL complex. In contrast, CLAMP may activate roX2 in males by creating a chromatin environment permissive for MSL complex recruitment in males. Although roX1 and roX2 are functionally redundant, the results suggest that CLAMP specifically activates roX2 but not roX1 in males. Interestingly, Villa (2016) recently reported that roX2, but not roX1, is likely to be an early site of MSL complex recruitment (Villa, 2016), suggesting that CLAMP may function early in the process of dosage compensation (Urban, 2017).

    Overall, the newly generated clamp2 protein null allele provides an important tool to study how the essential CLAMP protein regulates its many target genes in vivo. The generation of the clamp2 allele will facilitate future studies that will reveal a mechanistic understanding of how a single transcription factor can promote different sex-specific functions within an organism (Urban, 2017).

    Expansion of GA dinucleotide repeats increases the density of CLAMP binding sites on the X-Chromosome to promote Drosophila dosage compensation

    Dosage compensation is an essential process that equalizes transcript levels of X-linked genes between sexes by forming a domain of coordinated gene expression. Throughout the evolution of Diptera, many different X-chromosomes acquired the ability to be dosage compensated. Once each newly evolved X-chromosome is targeted for dosage compensation in XY males, its active genes are upregulated two-fold to equalize gene expression with XX females. In Drosophila melanogaster, the Chromatin-linked adaptor for MSL proteins (CLAMP) zinc finger protein links the dosage compensation complex to the X-chromosome. However, the mechanism for X-chromosome identification has remained unknown. This study combine biochemical, genomic and evolutionary approaches to reveal that expansion of GA-dinucleotide repeats likely accumulated on the X-chromosome over evolutionary time to increase the density of CLAMP binding sites, thereby driving the evolution of dosage compensation. Overall, this study presents new insight into how subtle changes in genomic architecture, such as expansions of a simple sequence repeat, promote the evolution of coordinated gene expression (Kuzu, 2016).

    Upon the evolution of heterogametic species, the process of dosage compensation became essential to ensure the appropriate balance of gene expression between males and females and the X and autosomes. Distinguishing the X-chromosome from autosomes is the key step in this process because MSL complex must be targeted to the correct chromosome to ensure the fidelity of dosage compensation. This study has demonstrate that in several species this process likely involved enriching the evolving X-chromosomes for long GA-repeat binding sites that can be recognized by the highly conserved CLAMP protein that recruits MSL complex (Kuzu, 2016).

    CLAMP binding sites are not X-specific as the CLAMP protein binds to similar GA-rich sequences all over the genome. It is proposed that a higher density of sites within CES that contain longer GA-repeats evolved to optimize CLAMP binding on X to better target MSL complex for dosage compensation. Then, it is likely that the increased density of CLAMP at CES functions together with other cofactors with known roles in MSL complex recruitment such as H3K36me3 and roX RNAs. Once this initial process of X-chromosome identification occurs, synergistic interactions between maternally loaded CLAMP and the MSL complex [20] increase the X-enrichment of both factors (Kuzu, 2016).

    Interestingly, the CLAMP motif is much longer than most transcription factor binding sites. It is possible that the length of the CLAMP binding site ensures specificity by reducing the promiscuity of its binding and allowing it to compete with other similar proteins. In addition, recent work on transcriptional regulators in budding yeast has implicated the sequence context of transcription factor binding sites outside of the core binding site as critical for the recognition process. Therefore, current approaches to identifying transcription factor binding site motifs have likely underestimated their length due to the approaches used that often allow detection of only short motifs. In the future, it will be important to determine transcription factor recognition motifs using approaches like gcPBM that uses in vivo sequences to identify direct binding site motifs (Kuzu, 2016).

    There are several mechanisms by which the GA-repeat number could have been increased including expansions due to slippage of DNA polymerase. Helitron transposons containing GA-rich sequences have also been implicated in the X-enrichment of these sequences in D. miranda. It is possible that expansions of GA dinucleotides occurred within these transposons after they landed on the X-chromosome. These GA-repeat expansions could have been further propagated by gene conversion events that also occurred during the evolution of dosage compensation. Finally, long repeat sequences such as the 1.688 elements that produce siRNAs function during dosage compensation via an unknown mechanism (Menon, 2014). Therefore, it is possible that GA-repeat elements have been expanded over evolutionary time because of a general role in promoting dosage compensation. To support this hypothesis, a recent report identified GA-rich binding motifs almost identical to those that we characterized as CLAMP binding sites within the strongest MSL complex binding sites in three additional Drosophila species (Kuzu, 2016).

    Motifs that contain GA-repeats have been implicated in diverse processes that all involve generating open chromatin regions. GA-repeat containing motifs are highly enriched at sites that promote pausing of RNA Polymerase II and at developmentally regulated DNase I hypersensitivity sites. Furthermore, a GA-repeat motif is one of the two motifs that are enriched at genes that are activated first during the maternal to zygotic transition. The well-studied GAGA factor (GAF) protein also recognizes similar sequences to the CLAMP protein and has been implicated in pausing of RNA Polymerase II and opening of chromatin. Overall, it is likely that the dosage compensation machinery has evolved to take advantage of targeting GA-repeats that mark open chromatin regions to ensure that it only identifies active genes for further transcriptional upregulation by the MSL complex (Kuzu, 2016).

    It is possible that GA-rich sequences have roles in dosage compensation outside of Diptera. For example, it has been proposed that upregulation of the single active X occurs in mammals and this process is mediated by targeting the conserved MOF histone acetyltransferase component of MSL complex. Moreover, GA-repeats were found to be significantly enriched within regions of the X-chromosome that escape X-inactivation (X escape regions). There are no strong homologues of CLAMP in mammals but there are several possible functional orthologs such as the ETS family transcription factor GABP1 (GA binding protein-1). Furthermore, in C. elegans, there is an early upregulation of both X-chromosomes that is also mediated by the MOF histone acetyltransferase. One of the zinc finger proteins that targets the C. elegans dosage compensation machinery is SCC-2 (sister chromatid cohesion-2) which recognizes a GA-repeat sequence very similar to the CLAMP binding motif. Therefore, it is possible that GA-repeats are involved in dosage compensation beyond Diptera and this will be an exciting area for future investigation (Kuzu, 2016).

    Effects of gene dose, chromatin, and network topology on expression in Drosophila melanogaster

    Deletions, commonly referred to as deficiencies by Drosophila geneticists, are valuable tools for mapping genes and for genetic pathway discovery via dose-dependent suppressor and enhancer screens. More recently, it has become clear that deviations from normal gene dosage are associated with multiple disorders in a range of species including humans. While some of the transcriptional effects brought about by gene dosage changes and the chromosome rearrangement breakpoints associated with them are beginning to be understood, much of this work relies on isolated examples. This study systematically examined deficiencies of the left arm of chromosome 2 and characterized gene-by-gene dosage responses that vary from collapsed expression through modest partial dosage compensation to full or even over compensation. Negligible long-range effects of creating novel chromosome domains at deletion breakpoints were found, suggesting that cases of gene regulation due to altered nuclear architecture are rare. These rare cases include trans de-repression when deficiencies delete chromatin characterized as repressive in other studies. Generally, effects of breakpoints on expression are promoter proximal (~100bp) or in the gene body. Effects of deficiencies genome-wide are in genes with regulatory relationships to genes within the deleted segments, highlighting the subtle expression network defects in these sensitized genetic backgrounds (Lee, 2016).

    PionX sites mark the X chromosome for dosage compensation

    The rules defining which small fraction of related DNA sequences can be selectively bound by a transcription factor are poorly understood. One of the most challenging tasks in DNA recognition is posed by dosage compensation systems that require the distinction between sex chromosomes and autosomes. In Drosophila melanogaster, the male-specific lethal dosage compensation complex (MSL-DCC) doubles the level of transcription from the single male X chromosome, but the nature of this selectivity is not known. Previous efforts to identify X-chromosome-specific target sequences were unsuccessful as the identified MSL recognition elements lacked discriminative power. Therefore, additional determinants such as co-factors, chromatin features, RNA and chromosome conformation have been proposed to refine targeting further. Using an in vitro genome-wide DNA binding assay this study shows that recognition of the X chromosome is an intrinsic feature of the MSL-DCC. MSL2, the male-specific organizer of the complex, uses two distinct DNA interaction surfaces-the CXC and proline/basic-residue-rich domains-to identify complex DNA elements on the X chromosome. Specificity is provided by the CXC domain, which binds a novel motif defined by DNA sequence and shape. This motif characterizes a subclass of MSL2-binding sites, which has been named PionX (pioneering sites on the X) as they appeared early during the recent evolution of an X chromosome in D. miranda and are the first chromosomal sites to be bound during de novo MSL-DCC assembly. These data provide the first documented molecular mechanism through which the dosage compensation machinery distinguishes the X chromosome from an autosome. They highlight fundamental principles in the recognition of complex DNA elements by protein that will have a strong impact on many aspects of chromosome biology (Villa, 2016).

    Satellite repeats identify X chromatin for dosage compensation in Drosophila melanogaster males

    A common feature of sex chromosomes is coordinated regulation of X-linked genes in one sex. Drosophila melanogaster males have one X chromosome, whereas females have two. The resulting imbalance in gene dosage is corrected by increased expression from the single X chromosome of males, a process known as dosage compensation. In flies, compensation involves recruitment of the male-specific lethal (MSL) complex to X-linked genes and modification of chromatin to increase expression. The extraordinary selectivity of the MSL complex for the X chromosome has never been explained. Previously work has demonstrated that the small interfering RNA (siRNA) pathway and siRNA from a family of X-linked satellite repeats (1.688X repeats) promote X recognition. This study now tests the ability of 1.688X DNA to attract compensation to genes nearby; autosomal integration of 1.688X repeats is shown to increase MSL recruitment and gene expression in surrounding regions. Placement of 1.688X repeats opposite a lethal autosomal deletion achieves partial rescue of males, demonstrating functional compensation of autosomal chromatin. Females block formation of the MSL complex and are not rescued. The 1.688X repeats are therefore cis-acting elements that guide dosage compensation. Furthermore, 1.688X siRNA enhances rescue of males with a lethal deletion but only when repeat DNA is present on the intact homolog. It is proposed that the siRNA pathway promotes X recognition by enhancing the ability of 1.688X DNA to attract compensation in cis. The dense and near-exclusive distribution of 1.688X sequences along the X chromosome suggests that they play a primary role in determining X identity during dosage compensation (Joshi, 2017).

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    Zygotically transcribed genes

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