Comment on "Drosophila dosage compensation involves enhanced Pol II recruitment to male X-linked promoters".

Conrad et al. (Reports, 10 August 2012, p. 742) reported a doubling of RNA polymerase II (Pol II) occupancy at X-linked promoters to support 5' recruitment as the key mechanism for dosage compensation in Drosophila. However, they employed an erroneous data-processing step, overestimating Pol II differences. Reanalysis of the data fails to support the authors' model for dosage compensation.

Regional control of chromatin organization by noncoding roX RNAs and the NURF remodeling complex in Drosophila melanogaster.

Dosage compensation in Drosophila is mediated by a histone-modifying complex that upregulates transcription of genes on the single male X chromosome. The male-specific lethal (MSL) complex contains at least five proteins and two noncoding roX (RNA on X) RNAs. The mechanism by which the MSL complex targets the X chromosome is not understood. Here we use a sensitized system to examine the function of roX genes on the X chromosome. In mutants that lack the NURF nucleosome remodeling complex, the male polytene X chromosome is severely distorted, appearing decondensed. This aberrant morphology is dependent on the MSL complex. Strikingly, roX mutations suppress the Nurf mutant phenotype regionally on the male X chromosome. Furthermore, a roX transgene induces disruption of local flanking autosomal chromatin in Nurf mutants. Taken together, these results demonstrate the potent capability of roX genes to organize large chromatin domains in cis, on the X chromosome. In addition to interacting functions at the level of chromosome morphology, we also find that NURF complex and MSL proteins have opposing effects on roX RNA transcription. Together, these results demonstrate the importance of a local balance between modifying activities that promote and antagonize chromatin compaction within defined chromatin domains in higher organisms.

The Drosophila dosage compensation complex binds to polytene chromosomes independently of developmental changes in transcription.

In Drosophila, the dosage compensation complex (DCC) mediates upregulation of transcription from the single male X chromosome. Despite coating the polytene male X, the DCC pattern looks discontinuous and probably reflects DCC dynamic associations with genes active at a given moment of development in a salivary gland. To test this hypothesis, we compared binding patterns of the DCC and of the elongating form of RNA polymerase II (PolIIo). We found that, unlike PolIIo, the DCC demonstrates a stable banded pattern throughout larval development and escapes binding to a subset of transcriptionally active areas, including developmental puffs. Moreover, these proteins are not completely colocalized at the electron microscopy level. These data combined imply that simple recognition of PolII machinery or of general features of active chromatin is either insufficient or not involved in DCC recruitment to its targets. We propose that DCC-mediated site-specific upregulation of transcription is not the fate of all active X-linked genes in males. Additionally, we found that DCC subunit MLE associates dynamically with developmental and heat-shock-induced puffs and, surprisingly, with those developing within DCC-devoid regions of the male X, thus resembling the PolIIo pattern. These data imply that, independently of other MSL proteins, the RNA-helicase MLE might participate in general transcriptional regulation or RNA processing.

MSL complex associates with clusters of actively transcribed genes along the Drosophila male X chromosome.

Dosage compensation in Drosophila serves as a model system for understanding the targeting of chromatin-modifying complexes to their sites of action. The MSL (male-specific lethal) complex up-regulates transcription of the single male X chromosome, thereby equalizing levels of transcription of X-linked genes between the sexes. Recruitment of the MSL complex to its binding sites on the male X chromosome requires each of the MSL proteins and at least one of the two large noncoding roX RNAs. To better understand how the MSL complex specifically targets the X chromosome, we have defined the binding using high-resolution genomic tiling arrays. Our results indicate that the MSL complex largely associates with transcribed genes that are present in clusters along the X chromosome. We hypothesize that after initial recruitment of the MSL complex to the X chromosome by unknown mechanisms, nascent transcripts or chromatin marks associated with active transcription attract the MSL complex to its final targets. Defining MSL-complex-binding sites will provide a tool for understanding functions of large noncoding RNAs that have remained elusive.

Epigenetic aspects of X-chromosome dosage compensation.

The X chromosomes of mammals and fruit flies exhibit unusual properties that have evolved to deal with the different dosages of X-linked genes in males (XY) and females (XX). The X chromosome dosage-compensation mechanisms discovered in these species are evolutionarily unrelated, but exhibit surprising parallels in their regulatory strategies. These features include the importance of noncoding RNAs, and epigenetic spreading of chromatin-modifying activities. Sex chromosomes have posed a fascinating puzzle for biologists. The dissimilar organization, gene content, and regulation of the X and Y chromosomes are thought to reflect selective forces acting on original pairs of identical chromosomes (1-3). The result in many organisms is a male-specific Y chromosome that has lost most of its original genetic content, and a difference in dosage of the X chromosome in males (XY) and females (XX).

Association and spreading of the Drosophila dosage compensation complex from a discrete roX1 chromatin entry site.

In Drosophila, dosage compensation is controlled by the male-specific lethal (MSL) complex consisting of MSL proteins and roX RNAs. The MSL complex is specifically localized on the male X chromosome to increase its expression approximately 2-fold. We recently proposed a model for the targeted assembly of the MSL complex, in which initial binding occurs at approximately 35 dispersed chromatin entry sites, followed by spreading in cis into flanking regions. Here, we analyze one of the chromatin entry sites, the roX1 gene, to determine which sequences are sufficient to recruit the MSL complex. We found association and spreading of the MSL complex from roX1 transgenes in the absence of detectable roX1 RNA synthesis from the transgene. We mapped the recruitment activity to a 217 bp roX1 fragment that shows male-specific DNase hypersensitivity and can be preferentially cross-linked in vivo to the MSL complex. When inserted on autosomes, this small roX1 segment is sufficient to produce an ectopic chromatin entry site that can nucleate binding and spreading of the MSL complex hundreds of kilobases into neighboring regions.

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA.

Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA, and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of approximately 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

The role of chromosomal RNAs in marking the X for dosage compensation.

Both flies and mammals remodel the architecture of the X chromosome to achieve dosage compensation. A novel class of noncoding RNAs that paint entire chromosomes are centrally involved in this process. The genes encoding these unusual RNAs are themselves located on the X, and are key sites that target the X for dosage compensation.

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.

Guilt by association: non-coding RNAs, chromosome-specific proteins and dosage compensation in Drosophila.

Dosage compensation is a striking example of the interplay between gene-specific regulation and chromosomal architecture. This process has evolved to make X-linked gene expression equivalent in males with one X chromosome and females with two. Examining species at the molecular level has shown that dosage compensation is mediated by sex-specific factors that decorate the X chromosomes to regulate chromatin structure and gene expression. In Drosophila, dosage compensation is achieved, at least in part, through site-specific histone H4 acetylation, which is modulated by a male- and X-specific protein complex. The discovery of non-coding RNAs that 'paint' dosage-compensated X chromosomes in mammals and in Drosophila suggests that RNAs play an intriguing, unexpected role in the regulation of chromatin structure and gene expression.

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.

Modulation of MSL1 abundance in female Drosophila contributes to the sex specificity of dosage compensation.

Dosage compensation in Drosophila is the mechanism by which X-linked gene expression is made equal in males and females. Proper regulation of this process is critical to the survival of both sexes. Males must turn the male-specific lethal (msl)-mediated pathway of dosage compensation on and females must keep it off. The msl2 gene is the primary target of negative regulation in females. Preventing production of MSL2 protein is sufficient to prevent dosage compensation; however, ectopic expression of MSL2 protein in females is not sufficient to induce an insurmountable level of dosage compensation, suggesting that an additional component is limiting in females. A candidate for this limiting factor is MSL1, because the amount of MSL1 protein in females is reduced compared to males. We have identified two levels of negative regulation of msl1 in females. The predominant regulation is at the level of protein stability, while a second regulatory mechanism functions at the level of protein synthesis. Overcoming these control mechanisms by overexpressing both MSL1 and MSL2 in females results in 100% female-specific lethality.

Complex formation by the Drosophila MSL proteins: role of the MSL2 RING finger in protein complex assembly.

Drosophila MSL proteins are thought to act within a complex to elevate transcription from the male X chromosome. We found that the MSL1, MSL2 and MSL3 proteins are associated in immunoprecipitations, chromatographic steps and in the yeast two-hybrid system, but that the MLE protein is not tightly complexed in these assays. We focused our analysis on the MSL2-MSL1 interaction, which is postulated to play a critical role in MSL complex association with the X chromosome. Using a modified two-hybrid assay, we isolated missense mutations in MSL2 that disrupt its interaction with MSL1. Eleven out of 12 mutated residues clustered around the first zinc-binding site of the RING finger domain were conserved in a Drosophila virilis MSL2 homolog. Two pre-existing msl2 alleles, which fail to support male viability in vivo, have lesions in the same region of the RING finger. We tested these in the two-hybrid system and found that they are also defective in interaction with MSL1. Mutation of the second zinc-binding site had little effect on MSL1 binding, suggesting that this portion of the RING finger may have a distinct function. Our data support a model in which MSL2-MSL1 interaction nucleates assembly of an MSL complex, with which MLE is weakly or transiently associated.

An analysis of maleless and histone H4 acetylation in Drosophila melanogaster spermatogenesis.

Maleless (mle) is essential in Drosophila melanogaster males both in somatic cells and in germ cells. In somatic cells mle is necessary for X-chromosome dosage compensation. The role of mle in the germline is unknown. We have analyzed the expression pattern and localization of MLE, the other MSLs and acetylated isoforms of histone H4 in male germ cells to address whether dosage compensation and/or X inactivation occur in the Drosophila germline. We observed that MLE is the only MSL expressed in the male germ cells and it is not localized to the X chromosome. We conclude that in the germline mle is not involved in chromosomal dosage compensation but may be involved in post-transcriptional gene regulation. We also observed that the acetylation pattern of histone H4 is very dynamic during spermatogenesis. While the pattern is not compatible with dosage compensation or X inactivation, it is consistent with all premeiotic chromosomes being in an active configuration that is replaced in post-meiotic stages with an inactive chromatin constitution.

Drosophila male-specific lethal-2 protein: structure/function analysis and dependence on MSL-1 for chromosome association.

MSL-2 is required for the male-specific assembly of a dosage compensation regulatory complex on the X chromosome of Drosophila melanogaster. We found that MSL-2 binds in a reproducible, partial pattern to the male X chromosome in the absence of MLE or MSL-3, or when ectopically expressed at a low level in females. Moreover, the pattern of MSL-2 binding corresponds precisely in each case to that of MSL-1, suggesting that the two proteins function together to associate with the X. Consistent with this hypothesis, we isolated EMS-induced loss of function msl-1 and msl-2 alleles in a screen for suppressors of the toxic effects of MSL-2 expression in females. We also used site-directed mutagenesis to determine the importance of the MSL-2 RING finger domain and second cysteine-rich motif. The mutations, including those in conserved zinc coordinating cysteines, confirm that the RING finger is essential for MSL-2 function, while suggesting a less stringent requirement for an intact second motif.

The NTPase/helicase activities of Drosophila maleless, an essential factor in dosage compensation.

Drosophila maleless (mle) is required for X chromosome dosage compensation and is essential for male viability. Maleless protein (MLE) is highly homologous to human RNA helicase A and the bovine counterpart of RNA helicase A, nuclear helicase II. In this report, we demonstrate that MLE protein, overexpressed and purified from Sf9 cells infected with recombinant baculovirus, possesses RNA/DNA helicase, adenosine triphosphatase (ATPase) and single-stranded (ss) RNA/ssDNA binding activities, properties identical to RNA helicase A. Using site-directed mutagenesis, we created a mutant of MLE (mle-GET) that contains a glutamic acid in place of lysine in the conserved ATP binding site A. In vitro biochemical analysis showed that this mutation abolished both NTPase and helicase activities of MLE but affected the ability of MLE to bind to ssRNA, ssDNA and guanosine triphosphate (GTP) less severely. In vivo, mle-GET protein could still localize to the male X chromosome coincidentally with the male-specific lethal-1 protein, MSL-1, but failed to complement mle1 mutant males. These results indicate that the NTPase/helicase activities are essential functions of MLE for dosage compensation, perhaps utilized for chromatin remodeling of X-linked genes.