Victoria H. Meller

Epigenetic Spreading of the Drosophila Dosage Compensation Complex from roX RNA Genes into Flanking Chromatin

The multisubunit MSL dosage compensation complex binds to hundreds of sites along the Drosophila single male X chromosome, mediating its hypertranscription. The male X chromosome is also coated with noncoding roX RNAs. When either msl3, mle, or mof is mutant, a partial MSL complex is bound at only approximately 35 unusual sites distributed along the X. We show that two of these sites are the roX1 and roX2 genes and postulate that one of their functions is to provide entry sites for the MSL complex to recognize the X chromosome. The roX1 gene provides a nucleation site for extensive spreading of the MSL complex into flanking chromatin even when moved to an autosome. The spreading can occur in cis or in trans between paired homologs. We present a model for how the dosage compensation complex recognizes X chromatin.

roX1 RNA Paints the X Chromosome of Male Drosophila and Is Regulated by the Dosage Compensation System

The Drosophila roX1 gene is X-linked and produces RNAs that are male-specific, somatic, and preferentially expressed in the central nervous system. These RNAs are retained in the nucleus and lack any significant open reading frame. Although all sexually dimorphic characteristics in Drosophila were thought to be controlled by the sex determination pathway through the gene transformer (tra), the expression of roX1 is independent of tra activity. Instead, the dosage compensation system is necessary and sufficient for the expression of roX1. Consistent with a potential function in dosage compensation, roX1 RNAs localize specifically to the male X chromosome. This localization occurs even when roX1 RNAs are expressed from autosomal locations in X-to-autosome translocations. The novel regulation and subnuclear localization of roX1 RNAs makes them candidates for an RNA component of the dosage compensation machinery.

The roX genes encode redundant male‐specific lethal transcripts required for targeting of the MSL complex

The roX1 and roX2 genes of Drosophila produce male‐specific non‐coding RNAs that co‐localize with the Male‐Specific Lethal (MSL) protein complex. This complex mediates up‐regulation of the male X chromo some by increasing histone H4 acetylation, thus contributing to the equalization of X‐linked gene expression between the sexes. Both roX genes overlap two of ∼35 chromatin entry sites, DNA sequences proposed to act in cis to direct the MSL complex to the X chromosome. Although dosage compensation is essential in males, an intact roX1 gene is not required by either sex. We have generated flies lacking roX2 and find that this gene is also non‐essential. However, simultaneous removal of both roX RNAs causes a striking male‐specific reduction in viability accompanied by relocation of the MSL proteins and acetylated histone H4 from the X chromosome to autosomal sites and heterochromatin. Males can be rescued by roX cDNAs from autosomal transgenes, demonstrating the genetic separation of the chromatin entry and RNA‐encoding functions. Therefore, the roX1 and roX2 genes produce redundant, male‐specific lethal transcripts required for targeting the MSL complex.

Ordered assembly of roX RNAs into MSL complexes on the dosage-compensated X chromosome in Drosophila

BACKGROUND In the male Drosophila, the X chromosome is transcriptionally upregulated to achieve dosage compensation, in a process that depends on association of the MSL proteins with the X chromosome. A role for non-coding RNAs has been suggested in recent studies. The roX1 and roX2 RNAs are male-specific, non-coding RNAs that are produced by, and also found associated with, the dosage-compensated male X chromosome. Whether roX RNAs are physically part of the MSL complex has not been resolved. RESULTS We found that roX RNAs colocalize with the MSL proteins and are highly unstable unless the MSL complex is coexpressed, suggesting a physical interaction. We were able to immunoprecipitate roX2 RNA from male tissue-culture cells with antibodies to the proteins Msl1 and Mle, consistent with an integral association with MSL complexes. Localization of roX1 and roX2 RNAs in mutants indicated an order of MSL-complex assembly in which roX2 RNA is incorporated early in a process requiring the Mle helicase. We also found that the roX2 gene, like roX1, is a nucleation site for MSL complex spreading into flanking chromatin in cis. CONCLUSIONS Our results support a model in which MSL proteins assemble at specific chromatin entry sites (including the roX1 and roX2 genes); the roX RNAs join the complex at their sites of synthesis; and complete complexes spread in cis to dosage compensate most genes on the X chromosome.

Sequence-specific targeting of Drosophila roX genes by the MSL dosage compensation complex

MSL complexes bind the single male X chromosome in Drosophila to increase transcription approximately 2-fold. Complexes contain at least five proteins and two noncoding RNAs, roX1 and roX2. The mechanism of X chromosome binding is not known. Here, we identify a 110 bp sequence in roX2 characterized by high-affinity MSL binding, male-specific DNase I hypersensitivity, a shared consensus with the otherwise dissimilar roX1 gene, and conservation across species. Mutagenesis of evolutionarily conserved sequences diminishes MSL binding in vivo. MSL binding to these sites is roX RNA dependent, suggesting that complexes become competent for binding only after incorporation of roX RNAs. However, the roX RNA segments homologous to the DNA binding sites are not required, ruling out simple RNA-DNA complementarity as the primary targeting mechanism. Our results are consistent with a model in which nascent roX RNA assembly with MSL proteins is an early step in the initiation of dosage compensation.

roX RNAs Are Required for Increased Expression of X-Linked Genes in Drosophila melanogaster Males

The male-specific lethal (MSL) ribonucleoprotein complex is necessary for equalization of X:A expression levels in Drosophila males, which have a single X chromosome. It binds selectively to the male X chromosome and directs acetylation of histone H4 at lysine 16 (H4Ac16), a modification linked to elevated transcription. roX1 and roX2 noncoding RNAs are essential but redundant components of this complex. Simultaneous removal of both roX RNAs reduces X localization of the MSL proteins and permits their ectopic binding to autosomal sites and the chromocenter. However, the MSL proteins still colocalize, and low levels of H4Ac16 are detected at ectopic sites of MSL binding and residual sites on the X chromosome of roX1− roX2− males. Microarray analysis was performed to reveal the effect of roX1 and roX2 elimination on X-linked and autosomal gene expression. Expression of the X chromosome is decreased by 26% in roX1− roX2− male larvae. Enhanced expression could not be detected at autosomal sites of MSL binding in roX1− roX2− males. These results implicate failure to compensate X-linked genes, rather than inappropriate upregulation of autosomal genes at ectopic sites of MSL binding, as the primary cause of male lethality upon loss of roX RNAs.

Dosage compensation: making 1X equal 2X

Animals that have XX females and XY or XO males have differing doses of X-linked genes in each sex. Overcoming this is the most immediate and vital aspect of sexual differentiation. A number of systems that accurately compensate for sex-chromosome dosage have evolved independently: silencing a single X chromosome in female mammals, downregulating both X chromosomes in hermaphrodite Caenorhabditis elegans and upregulating the X chromosome in male Drosophila all equalize X-linked gene expression. Each organism uses a largely non-overlapping set of molecules to achieve the same outcome: 1X = 2X.

Dosage compensation, the origin and the afterlife of sex chromosomes

Over the past 100 years Drosophila has been developed into an outstanding model system for the study of evolutionary processes. A fascinating aspect of evolution is the differentiation of sex chromosomes. Organisms with highly differentiated sex chromosomes, such as the mammalian X and Y, must compensate for the imbalance in gene dosage that this creates. The need to adjust the expression of sex-linked genes is a potent force driving the rise of regulatory mechanisms that act on an entire chromosome. This review will contrast the process of dosage compensation in Drosophila with the divergent strategies adopted by other model organisms. While the machinery of sex chromosome compensation is different in each instance, all share the ability to direct chromatin modifications to an entire chromosome. This review will also explore the idea that chromosome-targeting systems are sometimes adapted for other purposes. This appears the likely source of a chromosome-wide targeting system displayed by the Drosophila fourth chromosome.

A calmodulin-sensitive adenylate cyclase in the prothoracic glands of the tobacco hornworm, Manduca sexta

The Ca2+/calmodulin (CaM) dependence of adenylate cyclase activity in Manduca sexta prothoracic glands was investigated. Membrane fractions from two developmental stages were used, day 3 of the last larval instar and day 0 of the pupal stage, both of which respond to the neuropeptide prothoracicotropic hormone (PTTH) with increased cAMP production dependent on extracellular Ca2+. The data revealed that both larval and pupal prothoracic gland membrane fractions have a Ca2+/CaM-dependent adenylate cyclase which is inhibited by CaM antagonists and EGTA. The larval adenylate cyclase shows a multiphasic response to Ca2+/CaM, with a 2-fold stimulation between 0.02 and 0.01 microM, a further increase in adenylate cyclase activity at concentrations greater than 2 microM and a potentiation of NaF-stimulated activity at doses greater than 0.1 microM Ca2+/CaM. Pupal prothoracic gland membrane fractions exhibit only the second phase of stimulation. Stimulation by the GTP analogs GTP-gamma-S and Gpp(NH)p is dependent on CaM in larval, but not in pupal membrane fractions, suggesting a role for CaM in Gs protein-mediated regulation of adenylate cyclase. However, adenylate cyclase activity in glands from both stages is dependent on CaM, supporting our initial premise that Ca2+ is required for cAMP synthesis in the prothoracic glands.

The Drosophila brain revisited by enhancer detection

The patterns of gene expression in the Drosophila brain were studied by using the lacZ reporter gene carried on an enhancer detector element. From the analysis of serial sections of the heads of 6000 enhancer detector lines, reporter gene expression in some lines was found to generally follow boundaries established by cell type or anatomy, revealing distinct patterns of lacZ expression restricted to the lamina, the medulla, mushroom bodies, antennal lobes, or other anatomical subdivisions. About 15% of the lines showed ubiquitous expression in most or all head tissues and 25% of the lines showed expression throughout the CNS. Another quarter of the lines showed widespread expression in the CNS, with large regions of the brain showing expression. This suggests that the majority of detected genes are expressed with little spatial specificity. The expression patterns produced by 12 different insertions at the rutabaga locus were found to be extremely similar in the brain and offer strong evidence that the enhancer detector elements generally report the activity of an adjacent gene. Only 15% of the lines were judged to have relatively specific expression in one brain region, including those with preferential or specific expression in the mushroom bodies, antennal lobes, lamina, medulla, etc. The cytological insertion sites for elements showing preferential mushroom body expression were found to be dispersed in the genome at approximately 50 different chromosomal regions. In addition to providing a broad picture of the transcriptional activity in the Drosophila brain, these enhancer detector lines offer access to interesting new genes and form a novel collection of lines in which identifiable brain cells are marked in a reproducible way.