siRNA that participates in Drosophila dosage compensation is produced by many 1.688X and 359 bp repeats.

Organisms with differentiated sex chromosomes must accommodate unequal gene dosage in males and females. Male fruit flies increase X-linked gene expression to compensate for hemizygosity of their single X chromosome. Full compensation requires localization of the Male-Specific Lethal (MSL) complex to active genes on the male X, where it modulates chromatin to elevate expression. The mechanisms that identify X chromatin are poorly understood. The euchromatic X is enriched for AT-rich, ∼359 bp satellites termed the 1.688X repeats. Autosomal insertions of 1.688X DNA enable MSL recruitment to nearby genes. Ectopic expression of dsRNA from one of these repeats produces siRNA and partially restores X-localization of MSLs in males with defective X recognition. Surprisingly, expression of double stranded RNA from three other 1.688X repeats failed to rescue males. We reconstructed dsRNA-expressing transgenes with sequence from two of these repeats and identified phasing of repeat DNA, rather than sequence or orientation, as the factor that determines rescue of males with defective X recognition. Small RNA sequencing revealed that siRNA was produced in flies with a transgene that rescues, but not in those carrying a transgene with the same repeat but different phasing. We demonstrate that pericentromeric X heterochromatin promotes X-recognition through a maternal effect, potentially mediated by small RNA from closely related heterochromatic repeats. This suggests that the sources of siRNAs promoting X recognition are highly redundant. We propose that enrichment of satellite repeats on Drosophilid X chromosomes facilitates the rapid evolution of differentiated sex chromosomes by marking the X for compensation.

Identification of X chromatin is modulated by complementary pathways in Drosophila melanogaster.

Drosophila melanogaster males have one X chromosome while females have two. This creates an imbalance in X:A gene dosage between the sexes. This imbalance is corrected by increasing transcription from male X-linked genes approximately 2-fold. This process involves the Male-Specific Lethal (MSL) complex, which is recruited to Chromatin Entry Sites (CES) and transcribed X-linked genes, where it modifies chromatin to increase expression. Repetitive sequences strikingly enriched in X euchromatin, the 1.688X satellite repeats, also promote recruitment of the MSL complex to nearby genes. Unlike CES, the 1.688X repeats do not recruit the MSL complex directly. The genetic architecture of recruitment by these DNA elements remains speculative. To facilitate dissection of the mechanism of recruitment, we developed a luciferase reporter system for recruitment of compensation to an autosome. The system was validated by knock down of genes known to participate in compensation. Knock down of factors genetically linked to X recognition reveals that 1.688X repeats recruit through a different mechanism than the CES. Our findings suggest that 1.688X repeats play a larger role during embryogenesis, whereas the contribution of 1.688X repeats and CES is equivalent later in development. Our studies also reveal unexpected complexity and potential interdependence of recruiting elements.

When Down Is Up: Heterochromatin, Nuclear Organization and X Upregulation.

Organisms with highly differentiated sex chromosomes face an imbalance in X-linked gene dosage. Male Drosophila solve this problem by increasing expression from virtually every gene on their single X chromosome, a process known as dosage compensation. This involves a ribonucleoprotein complex that is recruited to active, X-linked genes to remodel chromatin and increase expression. Interestingly, the male X chromosome is also enriched for several proteins associated with heterochromatin. Furthermore, the polytenized male X is selectively disrupted by the loss of factors involved in repression, silencing, heterochromatin formation or chromatin remodeling. Mutations in many of these factors preferentially reduce male survival or enhance the lethality of mutations that prevent normal recognition of the X chromosome. The involvement of primarily repressive factors in a process that elevates expression has long been puzzling. Interestingly, recent work suggests that the siRNA pathway, often associated with heterochromatin formation and repression, also helps the dosage compensation machinery identify the X chromosome. In light of this finding, we revisit the evidence that links nuclear organization and heterochromatin to regulation of the male X chromosome.

Drosophila Satellite Repeats at the Intersection of Chromatin, Gene Regulation and Evolution.

Satellite repeats make up a large fraction of the genomes of many higher eukaryotes. Until recently these sequences were viewed as molecular parasites with few functions. Drosophila melanogaster and related species have a wealth of diverse satellite repeats. Comparative studies of Drosophilids have been instrumental in understanding how these rapidly evolving sequences change and move. Remarkably, satellite repeats have been found to modulate gene expression and mediate genetic conflicts between chromosomes and between closely related fly species. This suggests that satellites play a key role in speciation. We have taken advantage of the depth of research on satellite repeats in flies to review the known functions of these sequences and consider their central role in evolution and gene expression.

Chromatin That Guides Dosage Compensation Is Modulated by the siRNA Pathway in Drosophila melanogaster.

Many heterogametic organisms adjust sex chromosome expression to accommodate differences in gene dosage. This requires selective recruitment of regulatory factors to the modulated chromosome. How these factors are localized to a chromosome with requisite accuracy is poorly understood. Drosophila melanogaster males increase expression from their single X chromosome. Identification of this chromosome involves cooperation between different classes of X-identity elements. The chromatin entry sites (CES) recruit a chromatin-modifying complex that spreads into nearby genes and increases expression. In addition, a family of satellite repeats that is enriched on the X chromosome, the 1.688X repeats, promotes recruitment of the complex to nearby genes. The 1.688X repeats and CES are dissimilar, and appear to operate through different mechanisms. Interestingly, the siRNA pathway and siRNA from a 1.688X repeat also promote X recognition. We postulate that siRNA-dependent modification of 1.688X chromatin contributes to recognition of nearby genes. In accord with this, we found enrichment of the siRNA effector Argonaute2 (Ago2) at some 1.688X repeats. Mutations in several proteins that physically interact with Ago2, including the histone methyltransferase Su(var)3-9, enhance the lethality of males with defective X recognition. Su(var)3-9 deposits H3K9me2 on some 1.688X repeats, and this mark is disrupted upon ectopic expression of 1.688X siRNA. Furthermore, integration of 1.688X DNA on an autosome induces local H3K9me2 deposition, but enhances expression of nearby genes in a siRNA-dependent manner. Our findings are consistent with a model in which siRNA-directed modification of 1.688X chromatin contributes to recognition of the male X chromosome for dosage compensation.

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. We previously demonstrated that the small interfering RNA (siRNA) pathway and siRNA from a family of X-linked satellite repeats (1.688X repeats) promote X recognition. Now, we test the ability of 1.688X DNA to attract compensation to genes nearby and report that autosomal integration of 1.688X repeats increases 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. We propose 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.

Host-Symbiont Interactions: Male-Killers Exposed.

Male-killing is one strategy used by maternally transmitted bacterial symbionts to boost transmission and spread in populations. In Drosophila melanogaster, Spiroplasma target males by hijacking an essential, male-limited epigenetic process. A new study reveals clues to the mode of killing.

Modulation of Chromatin by Noncoding RNA.

Noncoding RNAs (ncRNAs) are remarkably powerful, flexible, and pervasive cellular regulators. The involvement of these molecules in virtually all aspects of eukaryotic chromatin function is notable. Long and short ncRNAs play broadly complementary roles in these processes. Short ncRNAs underlie a programmable system of chromatin modification that silences mobile elements, identifies boundaries, and initiates the formation of constitutive heterochromatin in yeast. In contrast, long noncoding RNAs (lncRNAs) enforce developmentally appropriate expression and switch gene expression programs. lncRNAs accomplish this through diverse mechanisms, but often by modulating the activity or localization of chromatin regulatory complexes. Both long and short ncRNAs play key roles in organization of complex genomes of higher eukaryotes, and their coordinated actions appear to underlie some of the more dramatic examples of epigenetic regulation. This review contrasts well-studied examples of chromatin regulation by RNA and introduces examples of coordination between these systems.

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 melanogaster 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. To explore the molecular basis of this observation we examined additional proteins that participate in compensation and found that MLE, but not Jil-1 kinase, contributes to heterochromatic gene expression. To determine if identical regions of roX RNA are required for dosage compensation and heterochromatic silencing, we tested a panel of roX1 transgenes and deletions and find that the X chromosome and heterochromatin functions are separable by some mutations. Chromatin immunoprecipitation of staged embryos revealed widespread autosomal binding of MSL3 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. The broad localization of these proteins early in embryogenesis supports the idea of direct action at autosomal sites. We postulate that this may contribute to the sex-specific differences in heterochromatin that we, and others, have noted.

Identification of the Drosophila X chromosome: The long and short of it.

The different dose of X chromosomes in males and females produces a potentially fatal imbalance in X-linked gene products. This imbalance is addressed by dosage compensation, a process that modulates expression from an entire X chromosome in one sex. Dosage compensation acts on thousands of genes with disparate expression patterns. Both flies and mammals accomplish this with remarkable specificity by targeting epigenetic chromatin modifications to a single chromosome. Long noncoding RNAs that are expressed from the X chromosome are essential elements of the targeting mechanism in both lineages. We recently discovered that the siRNA pathway, as well as small RNA from satellite repeats that are strikingly enriched on the fly X chromosome, also promote X recognition. In this article we review the current understanding of X recognition in flies and discuss potential mechanisms by which the siRNA pathway, repetitive elements and long noncoding RNAs might cooperate to promote X recognition.

Sex Differences in Drosophila melanogaster Heterochromatin Are Regulated by Non-Sex Specific Factors.

The eukaryotic genome is assembled into distinct types of chromatin. Gene-rich euchromatin has active chromatin marks, while heterochromatin is gene-poor and enriched for silencing marks. In spite of this, genes native to heterochromatic regions are dependent on their normal environment for full expression. Expression of genes in autosomal heterochromatin is reduced in male flies mutated for the noncoding roX RNAs, but not in females. roX mutations also disrupt silencing of reporter genes in male, but not female, heterochromatin, revealing a sex difference in heterochromatin. We adopted a genetic approach to determine how this difference is regulated, and found no evidence that known X chromosome counting elements, or the sex determination pathway that these control, are involved. This suggested that the sex chromosome karyotype regulates autosomal heterochromatin by a different mechanism. To address this, candidate genes that regulate chromosome organization were examined. In XX flies mutation of Topoisomerase II (Top2), a gene involved in chromatin organization and homolog pairing, made heterochromatic silencing dependent on roX, and thus male-like. Interestingly, Top2 also binds to a large block of pericentromeric satellite repeats (359 bp repeats) that are unique to the X chromosome. Deletion of X heterochromatin also makes autosomal heterochromatin in XX flies dependent on roX and enhances the effect of Top2 mutations, suggesting a combinatorial action. We postulate that Top2 and X heterochromatin in Drosophila comprise a novel karyotype-sensing pathway that determines the sensitivity of autosomal heterochromatin to loss of roX RNA.

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. We recently reported that the siRNA pathway contributes to localization of the MSL complex, raising questions about the source of the siRNAs involved. The X-linked 1.688 g/cm(3) satellite related repeats (1.688(X) repeats) are restricted to the X chromosome and produce small RNA, making them an attractive candidate. We tested RNA from these repeats for a role in dosage compensation and found that ectopic expression of single-stranded RNAs from 1.688(X) repeats enhanced the male lethality of mutants with defective X recognition. In contrast, expression of double-stranded hairpin RNA from a 1.688(X) 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 1.688(X) repeats, which are nearly exclusive to the X chromosome, suggests that these are cis-acting elements contributing to identification of X chromatin.

A strategy for generation and balancing of autosome: Y chromosome translocations.

We describe a method for generation and maintenance of translocations that move large autosomal segments onto the Y chromosome. Using this strategy we produced ( 2;Y) translocations that relocate between 1.5 and 4.8 Mb of the 2nd chromosome.. All translocations were easily balanced over a male-specific lethal 1 (msl-1) mutant chromosome. Both halves of the translocation carry visible markers, as well as P-element ends that enable molecular confirmation. Halves of these translocations can be separated to produce offspring with duplications and with lethal second chromosome deficiencies . Such large deficiencies are otherwise tedious to generate and maintain.

Generation of a useful roX1 allele by targeted gene conversion.

Methods for altering the sequence of endogenous Drosophila melanogaster genes remain labor-intensive. We have tested a relatively simple strategy that enables the introduction of engineered mutations in the vicinity of existing P-elements. This method was used to generate useful alleles of the roX1 gene, which produces a noncoding RNA involved in dosage compensation. The desired change was first introduced into a genomic clone of roX1 and transgenic flies were generated that carry this sequence in a P-element. Targeted transposition was then used to move the P-element into roX1. Remobilization of the targeted insertion produced large numbers of offspring carrying chromosomes that had precisely introduced the engineered sequences into roX1. We postulate that this occurred by gap repair, using the P-element on the sister chromatid as template. This strategy was used to introduce six MS2 loops into the roX1 gene (roX1(MS2-6)), enabling detection of roX1 RNA by a MCP-GFP fusion protein in embryos. The roX1(MS2-6) remains under the control of the authentic promoter and within the correct genomic context, features expected to contribute to normal roX1 function. The ability to replace relatively large blocks of sequence suggests that this method will be of general use.

Sex chromosome evolution: life, death and repetitive DNA.

Dimorphic sex chromosomes create problems. Males of many species, including Drosophila, are heterogametic, with dissimilar X and Y chromosomes. The essential process of dosage compensation modulates the expression of X-linked genes in one sex to maintain a constant ratio of X to autosomal expression. This involves the regulation of hundreds of dissimilar genes whose only shared property is chromosomal address. Drosophila males dosage compensate by up regulating X-linked genes 2 fold. This is achieved by the Male Specific Lethal (MSL) complex, which is recruited to genes on the X chromosome and modifies chromatin to increase expression. How the MSL complex is restricted to X-linked genes remains unknown. Recent studies of sex chromosome evolution have identified a central role for 2 types of repetitive elements in X recognition. Helitrons carrying sites that recruit the MSL complex have expanded across the X chromosome in at least one Drosophila species. (1) Our laboratory found that siRNA from an X-linked satellite repeat promotes X recognition by a yet unknown mechanism. (2) The recurring adoption of repetitive elements as X-identify elements suggests that the large and mysterious fraction of the genome called "junk" DNA is actually instrumental in the evolution of sex chromosomes.

Homologue pairing in flies and mammals: gene regulation when two are involved.

Chromosome pairing is usually discussed in the context of meiosis. Association of homologues in germ cells enables chromosome segregation and is necessary for fertility. A few organisms, such as flies, also pair their entire genomes in somatic cells. Most others, including mammals, display little homologue pairing outside of the germline. Experimental evidence from both flies and mammals suggests that communication between homologues contributes to normal genome regulation. This paper will contrast the role of pairing in transmitting information between homologues in flies and mammals. In mammals, somatic homologue pairing is tightly regulated, occurring at specific loci and in a developmentally regulated fashion. Inappropriate pairing, or loss of normal pairing, is associated with gene misregulation in some disease states. While homologue pairing in flies is capable of influencing gene expression, the significance of this for normal expression remains unknown. The sex chromosomes pose a particularly interesting situation, as females are able to pair X chromosomes, but males cannot. The contribution of homologue pairing to the biology of the X chromosome will also be discussed.

A role for siRNA in X-chromosome dosage compensation in Drosophila melanogaster.

Sex-chromosome dosage compensation requires selective identification of X chromatin. How this occurs is not fully understood. We show that small interfering RNA (siRNA) mutations enhance the lethality of Drosophila males deficient in X recognition and partially rescue females that inappropriately dosage-compensate. Our findings are consistent with a role for siRNA in selective recognition of X chromatin.

roX RNAs and Genome Regulation in Drosophila Melanogaster.

Organisms with dimorphic sex chromosomes suffer a potentially lethal imbalance in gene expression in one sex. Addressing this fundamental problem can be considered the first, and most essential, aspect of sexual differentiation. In the model organisms Drosophila, Caenorhabditis elegans, and mouse, expression from X-linked genes is modulated by selective recruitment of chromatin-modifying complexes to X chromatin. In both flies and mammals, large noncoding RNAs have a central role in recruitment and activity of these complexes. This review will summarize current knowledge of the function of the noncoding roX genes in this process in Drosophila. Identification of an autosomal function for the roX RNAs raises intriguing questions about the origin of the modern dosage compensation system in flies.