male-specific lethal 2


msl2 mRNA is bound by free nuclear MSL complex in Drosophila melanogaster

In Drosophila, the global increase in transcription from the male X chromosome to compensate for its monosomy is mediated by the male-specific lethal (MSL) complex consisting of five proteins and two non-coding RNAs, roX1 and roX2. After an initial sequence-dependent recognition by the MSL complex of 150-300 high affinity sites, the spread to the majority of the X-linked genes depends on local MSL-complex concentration and active transcription. This study have explored whether any additional RNA species are associated with the MSL complex. No additional roX RNA species were found, but a strong association was found between a spliced and poly-adenylated msl2 RNA and the MSL complex. Based on these results, a model is proposed in which a non-chromatin-associated partial or complete MSL-complex titrates newly transcribed msl2 mRNA and thus regulates the amount of available MSL complex by feedback. This represents a novel mechanism in chromatin structure regulation (Johansson, 2011).

For many chromatin-associated factors, the relative concentrations direct their binding. Thus, correct targeting will strictly depend on the concentration of these protein complexes. It is well established that the MSL complex is restricted to males by a translational block caused by Sxl protein binding to the msl2 RNAs. However, it has been suggested that Sxl binds not only all msl2 transcripts, but also to a subset of msl1 transcripts and prevents their translation in females. This reflects the potential of these two RNAs to be regulated through specific protein interactions. These two RNAs encode the core of the dosage compensation complex. This study provides evidence that a non-chromatin bound fraction of the MSL complex has high affinity for nuclear msl2 mRNA and also affinity for nuclear msl1 mRNA. Based on this, a novel mechanism is proposed for fine-tuning the MSL complex concentration by feedback (Johansson, 2011).

Both formaldehyde fixed nuclear extracts and native nuclear extracts were used in RNA-immunoprecipitation (RIP) experiments. In both these sample types, strong enrichments of roX1, roX2 and msl2 transcripts were detected. In the native extracts, no significant enrichment of RNA transcribed from genes targeted by the MSL complex nor any enrichment of RNA molecules transcribed from MREs could be detected. Although, robust enrichment of roX1 and roX2 was found, no additional roX RNA was detected that could explain the escape of male lethality seen in roX1 roX2 double mutants. It should be stressed that it is still possible that a roX-like encoding gene is harbored in the heterochromatic regions which are not fully covered by the tiling arrays. Alternatively, several different RNAs may substitute for roX1 and roX2 but with much lower affinity or the MSL complex can exert its function without an RNA molecule. In contrast, in the formaldehyde fixed sample enrichments of transcripts were detected from genes targeted by the MSL complex. It is concluded that RIP using formaldehyde cross-linked extracts will be enriched in RNA species directly linked to the targeted protein as well as nascent RNA molecules linked via chromatin and active transcription. This is important to consider when using formaldehyde fixation in RNA immunoprecipitation experiments (Johansson, 2011).

Two potential functions were described for the strong association of a nuclear MSL complex with the msl2 RNA. First, msl2 may aid the correct targeting of the MSL complex, by functioning similarly to roX. This could have been a function in a more ancient form of the MSL complex and may in part have been retained during evolution. In favour of this model is the fact that roX1 roX2 double mutations are not completely male lethal, in contrast to msl mutations. The elevation of cellular levels of MSL1 and MSL2 that partially suppress roX1 roX2 male lethality, also supports this model. Second, since the local concentration of MSL complex is important for spreading and, therefore, also for the correct regulation of most X-linked genes, it can be hypothesized that the non-chromatin bound fraction of the MSL complex titrates the msl2 RNA. Thus, this process would regulate the amount of MSL2 protein and assembled MSL complex, as a feedback mechanism (Johansson, 2011).

Since the msl2 RNA is highly enriched in a nuclear but not chromatin-associated MSL complex, it is hypothesized that this interaction functions as a feedback control that avoids elevated MSL-complex levels and compensates for overshooting. It has been observed that ectopic expression of an msl2-GFP transgene led to reduction of the endogenous MSL2 protein levels. This is in line with what would be predicted from a feedback mechanism as hypothesized in this study. The classical example of self-mRNA targeting of proteins is from Escherichia coli, in which the ribosomal proteins bind to their mRNA and inhibit translation. The ribosome biogenesis can thus be regulated solely through the amount of rRNA which synthesis rate becomes rate limiting for ribosome assembly. As long as the process of ribosome assembly requires ribosomal proteins, the corresponding mRNAs will escape translation inhibition. Intrinsic association of RNA in chromatin regulation has been reported for the yeast SET1C histone methyl transferase complex. In this case, the association is linked to co-translational assembly of the complex (Johansson, 2011).

The dosage compensation system is under strong evolutionary pressure to respond as new genes, chromosome regions or chromosome arms join the X chromosome and it has been shown that the MSL proteins are under adaptive evolution. Interestingly, strong positive selection was detected in MSL1 and MSL2 protein domains shown to be responsible for their specific targeting to the X chromosome. Positive selection has also been shown to act on the MSL binding sites. This means that the dosage compensation system is under constant pressure to adapt to emerging changes. The target sequences are under constant selection as are the targeting proteins, in particular MSL1 and MSL2. Thus, the concentration requirements will also need constant adjustments. A feedback module based on association of a rate limiting mRNA would provide an optimal target for evolution to act on in order to provide a dynamic fine-tuning of complex concentration, and therefore, correct targeting. This may explain the affinity of the MSL complex to both msl1 and msl2 RNAs which encode the core proteins for a functional complex. At limiting concentrations of freely diffusible proteins and protein complexes only the sites with highest affinity will bind while at high concentrations even sites with low affinity will bind. It should be stressed that chromatin-associated factors are often described to be highly sensitive to correct dose. This is observed for the Polycomb-group proteins involved in maintaining repression of homeotic genes as well as for suppressors of variegation, important for heterochromatin formation. In both these cases many loci exhibit dosage effects, indicating strict dosage needs. As more RNA immunoprecipitation data are produced it is likely that the msl2 titration reported in this study is just one example of a general self-affinity module important for feedback regulation (Johansson, 2011).

Autoregulation of the Drosophila Noncoding roX1 RNA Gene

Most genes along the male single X chromosome in Drosophila are hypertranscribed about two-fold relative to each of the two female X chromosomes. This is accomplished by the MSL (male-specific lethal) complex that acetylates histone H4 at lysine 16. The MSL complex contains two large noncoding RNAs, roX1 (RNA on X) and roX2, that help target chromatin modifying enzymes to the X. The roX RNAs are functionally redundant but differ in size, sequence, and transcriptional control. This study examined how roX1 production is regulated. Ectopic DC can be induced in wild-type (roX1+) roX2+) females if a heterologous source of MSL2 is provided. However, in the absence of roX2, it was found that roX1 expression failed to come on reliably. Using an in situ hybridization probe that is specific only to endogenous roX1, it was found that expression was restored if either roX2 or a truncated but functional version of roX1 were introduced. This shows that pre-existing roX RNA is required to positively autoregulate roX1 expression. Massive cis spreading of the MSL complex was also observed from the site of roX1 transcription at its endogenous location on the X chromosome. It is proposed that retention of newly assembled MSL complex around the roX gene is needed to drive sustained transcription and that spreading into flanking chromatin contributes to the X chromosome targeting specificity. Finally, it was found that the gene encoding the key male-limited protein subunit, msl2, is transcribed predominantly during DNA replication. This suggests that new MSL complex is made as the chromatin template doubles. A model is offered describing how the production of roX1 and msl2, two key components of the MSL complex, are coordinated to meet the dosage compensation demands of the male cell (Lim, 2012).

Previous studies of roX1 transcriptional control argued that either MSL2 alone or with a full set of MSL proteins was sufficient to drive male-specific expression. This study presents evidence that the expression of roX1 gene is instead controlled through an autoregulatory loop. Pre-existing roX RNA, presumably in mature MSL complex, is required to drive new transcription. The reason this study reached a different conclusion is largely attributed to removing the functionally redundant roX2 in most of the experiments and assaying transcription only from the wild type roX1 locus at its normal location on the X (Lim, 2012).

A model is presented for how such an autoregulatory loop might operate (see Autoregulation model). Because roX RNA is not maternally deposited into embryos, one problem is how male embryos could build their first MSL complex needed to initiate the cycle. Earlier studies have shown that roX1 transcription switches on in both sexes around blastoderm, just as general zygotic transcription begins. This suggests that an embryonic roX1 promoter is active without MSL complex and could supply the first roX1 RNA molecules to males, but these RNAs are eventually degraded in females. Early RNA assembles with MSL proteins and then drive roX1 transcription from the known male-specific MSL-dependent promoters, setting up a positive autoregulatory loop necessary for the future maintenance of roX1 expression in males. In the current experiments, it was shown that roX2 RNA or truncated roX1 RNA can also initiate endogenous roX1 expression late in development after the early roX1 transcripts are gone (Lim, 2012).

This model requires that male embryos preferentially sequester newly assembled MSL complex at the roX1 gene to drive sustained transcription instead of allowing it to diffuse away to the vastly larger pool of ordinary X linked genes that must be dosage compensated. Only after roX1 transcription is successfully upregulated can MSL complexes be released to the target genes along the X chromosome. Such behavior has been previously documented in cells containing abundant free MSL subunits and low levels of roX1 transcription, exactly the conditions believed to occur as young male embryos initiate dosage compensation. Examining cells shortly after MSL2 first turned on roX1 transcription showed the earliest roX1 transcripts remained near the site of synthesis consistent with the idea that newly formed MSL complexes preferentially act on the roX1 gene. At later times, such as seen in H83roX1-Δ39 animals that have had five days to drive endogenous roX1 expression, every cell painted the entire X with roX1 RNA. While massive local cis spreading of the MSL complex has been reported for roX1 and roX2 transgenes inserted into autosomes, the physiological relevance of this is not widely accepted. This study instead reports striking local cis spreading of newly made MSL complex from the wild type roX1 gene in its normal X chromosome environment. The data agree well with previous reports of local MSL spreading along the X and support a role for cis spreading in the normal process of dosage compensation (Lim, 2012).

It has not been directly determined what region of the roX1 gene is necessary for autoregulation. However, a strong candidate is the ~200 bp male-specific DNase I hypersensitive site (DHS) found about 1.5 kb downstream of the adult roX1 promoters. The DHS is sufficient to recruit the MSL complex to ectopic sites when moved to the autosomes in a sequence specific manner. While partial complexes lacking MOF or MSL3 remain bound to the DHS, incomplete complexes lacking either roX RNA or the MLE RNA helicase postulated to fold roX RNA bind the DHS poorly. This element stimulates roX1 transcription when MSL complex is bound and represses basal transcription when MSL complex is absent. Also, deleting the DHS greatly reduces transcription of roX1 transgenes. Together, these findings support a model where transcription from the roX1 gene requires pre-existing roX RNA within MSL complexes bound to the internal DHS enhancer. However, this view is likely an oversimplification because very large internal deletions such as roX1ex7B, comparable to the H83roX1-Δ39 transgene, remain transcriptionally active despite the loss of the DHS enhancer (Lim, 2012).

Translation of msl2 mRNA is normally subject to elaborate controls acting through the 5'and 3' UTRs. Little attention has been given to its transcription control, although recently anti-MSL2 antibodies were found to precipitate msl2 mRNA (Johansson, 2011). msl2 transcription is associated with replication, and this is likely important for its normal control. Without MSL2 protein, naked roX1 RNA is rapidly destroyed (Meller, 2000). This study has found the converse; cells rapidly clear any MSL2 protein not bound to roX RNA. When free MSL2 subunits are artificially stabilized with proteosome inhibitors, they coat all the chromosomes indiscriminately. This implies that the synthesis of MSL2 and roX RNAs are closely coordinated so each component stabilizes the other ensuring that only correctly targeted molecules survive. Although MSL2 lacks known RNA binding motifs, previous work of others is consistent with an intimate interaction between MSL2 and roX RNA (Li, 2008). It is suspected the loss of replication-coupled transcription may contribute to the failure of dosage compensation in some cells relying exclusively on H83M2 for MSL2 protein and roX1 for roX RNA. This defect is corrected when both roX1 and MSL2 are coordinately made from the same hsp83 promoter. Cells in mosaic animals lose dosage compensation sometime between the end of embryogenesis and third instar larvae. Many tissues undergo significant changes in cell cycle near the end of embryogenesis, particularly the introduction of G1, and it is suspected that this shift contributes to loss of dosage compensation in mosaic animals. If MSL complex should ever drop below the level needed to sustain the autoregulatory roX1 loop, it could never recover regardless of later MSL2 production. The details remain unclear because H83M2, the construct used to drive MSL2, is vigorously transcribed at the developmental times examined, including S phase. It is not known whether the regulatory msl2 UTR sequences removed from H83M2 disrupt additional posttranscriptional controls that might promote efficient translation during replication (Lim, 2012).

A second issue is that the X is painted with MSL complex throughout the cell cycle. This might drive continuous rather than cyclic roX1 synthesis. While it was not possible to directly determine if roX1 transcription is cell cycle regulated, it is noted that the replication machinery components ORC2 and MCM are bound specifically to the roX1 DHS enhancer only in male cells. The significance of this is not known, but it is tempting to speculate that components of the pre-initiation complex bound to DHS compete with MSL complex thus inhibiting roX1 transcription in G1. Firing of the replication origins removes ORC2 and MCM possibly allowing MSL complex access to the DHS and so switch on transcription shortly after the onset of S phase. The replication machinery is commonly found near the promoters of many genes, so further experiments will be needed to determine if such binding actually plays any regulatory role here (Lim, 2012).

While this study did not specifically address transcriptional control of roX2, it must differ from roX1 in several important ways. Meller (2003) has previously shown that roX2 transcription lags roX1 by a few hours during early embryonic development and is always limited to males. This study found that roX2 transcription differs by not requiring pre-existing roX RNA and can be switched on several days later during larval development simply by ectopically expressing MSL2. MSL2 protein made by the constitutive H83M2 transgene only sporadically activates roX1, but robustly drives roX2. The roX2 gene also carries a DHS enhancer similar to that found in roX1 (Park, 2003), but if it plays a comparable role in roX2 regulation, it presumably would not require complete MSL complex. The region near the proline rich domain towards the C-terminus of MSL2 is essential for this regulation (Lim, 2012).

The roX1 autoregulation loop described above shares parallels to the SXL autoregulatory loop controlling all aspects of sex determination and dosage compensation in Drosophila. X:A counting elements act upon an early establishment Sxl promoter to make SXL in early female embryos. These first SXL proteins stimulate productive splicing of Sxl mRNAs transcribed from a distinct maintenance promoter ensuring further SXL production. MSL1, MSL3, MOF, and MLE are unable to package early roX1 RNA made from the embryonic promoter in female embryos to form a fully functional mature MSL complex. To do that, MSL2 protein made only in males is required. These earliest MSL complexes are thought to sustain roX1 transcription as embryos switch to the MSL-dependent promoters. Recently a new role for MSL2 in females has been described during the brief window when Sxl autoregulation is established (Gladstein, 2010). Perhaps females also fleetingly utilize the early burst of roX1 before abundant SXL represses msl2 translation (Lim, 2012).

Thousands of large noncoding RNAs have recently been discovered in vertebrates, many of which are associated with chromatin remodeling enzymes. It is likely that some of these will face similar regulatory and functional demands as the roX RNAs and may have evolved comparable strategies to control their production. For instance, the short RepA sequence at the 5′ end of mammalian Xist RNA may influence production of full length transcripts (Lim, 2012).

Targets of Activity

Expression of RNA on the X-1 is controlled by the Male specific lethal-2 directed dosage compensation system. Absence of any one of the four genes responsible for dosage compensation, completely eliminates roX1 expression. Interestingly, fading of the roX1 RNA in females takes place long after the association of MSL proteins (Meller, 1997 and Amrein, 1997).

Dosage compensation in Drosophila is controlled by a complex (DCC) of proteins and noncoding RNA that binds specifically to the male X chromosome and leads to fine-tuning of transcription. Male SL2 cells were used to characterize DCC function and dynamics during steady state of dosage compensation. Knocking down the key regulator of dosage compensation, male-specific-lethal 2 (MSL2), leads to loss of propagation of histone H4 lysine 16 acetylation and of the twofold elevation of transcription characteristic of the compensated male X chromosome. Surprisingly, lack of dosage compensation does not impair cell viability. Targeting of MSL2 to a reporter gene suffices to initiate dosage compensation in the cell model. Using photobleaching techniques in living cells, the association of MSL2 with the X chromosome was found to be exceptionally stable, essentially excluding dynamic redistribution of the DCC during interphase. This immobility distinguishes MSL2 from most other chromosomal proteins. These findings have profound implications for the mechanism underlying dosage compensation and furthermore provide a new, conceptual reference of stability in an otherwise highly dynamic nuclear environment (Straub, 2005a).

The mechanism through which gene expression originating from the single male or the two female X chromosomes in Drosophila is adjusted to autosomal gene expression has remained controversial. According to the prevalent model, transcription of the male X is increased twofold by the male-specific-lethal (MSL) complex. However, a significant body of data supports an alternative model, whereby compensation involves a global repression of autosomal gene expression in males by sequestration and neutralization of an activator onto the X chromosome. In order to rigorously discriminate between these models direct target genes for the MSL complex were identified and transcription was quantified in absolute terms after knockdown of MSL2. The results unequivocally document an approximate twofold activation of target genes by the MSL complex (Straub, 2005b ).

A long-standing model postulates that X-chromosome dosage compensation in Drosophila occurs by twofold up-regulation of the single male X, but previous data cannot exclude an alternative model, in which male autosomes are down-regulated to balance gene expression. To distinguish between the two models, RNA interference was used to deplete Male-Specific Lethal (MSL) complexes from male-like tissue culture cells. Expression of many genes from the X chromosome was found to be decreased, while expression from the autosomes was largely unchanged. It is concluded that the primary role of the MSL complex is to up-regulate the male X chromosome (Hamada, 2005).

Msl2 was chosen as a target for RNAi because it is the key limiting factor in the MSL complex. Msl2 protein is expressed in males but is normally repressed in females by the female-specific factor Sex Lethal (SXL). Since females lack Msl2, they do not form MSL complexes. However, when Msl2 is ectopically expressed in females, full complexes form on both X chromosomes, leading to delayed development and substantial lethality. Therefore, the absence of Msl2 is normally sufficient to completely block dosage compensation by the MSL complex (Hamada, 2005).

Assays were performed in SL2 tissue culture cells, which display potent RNAi activity. Sxl is absent in SL2 cells, while all of the known MSL protein components are expressed, form complexes, and are localized to the X chromosome, strongly suggesting a male identity (Hamada, 2005).

The effect of msl2 RNAi, or RNAi for an irrelevant gene (GFP), was assayed on the localization of MSL complexes to a nuclear subdomain presumed to be the X chromosome. After 4 d it was found that RNAi against msl2 largely eliminated the localization of MSL complexes. More than 90% of treated cells lacked Msl2 staining on the X chromosome and showed diminished overall signal within 4 d of double-stranded RNA (dsRNA) treatment. Treatment with msl2 dsRNA also resulted in a similar loss of Msl1 and Msl3 from the X chromosome. Immunostaining varied from cell to cell, but in general Mle and Mof appeared to be released from the X and redistributed throughout the nucleoplasm (Hamada, 2005).

One of the known consequences of MSL action on the X chromosome is the site- specific acetylation of histone H4 at Lys 16 by the MOF acetyltransferase. Therefore, SL2 cells were immunostained after RNAi treatment with antibodies that specifically recognize histone H4K16ac. In control cells, H4K16ac is concentrated on the X chromosome and also is variably detected throughout the nucleus, presumably on all chromosomes at a lower level. After msl2 RNAi treatment, tight foci of H4K16ac disappeared. H4K16ac staining was no longer detectable in ~25% of cells, while ~75% retained weak staining throughout the nucleus (Hamada, 2005).

To confirm that the RNAi effects correlated with a loss of msl2 mRNA, quantitative real-time RT-PCR analysis was performed. Four days after RNAi treatment, it was found that msl2 RNA levels were reduced ~92% in the experimental cells compared with control cells. To determine the consequence of decreased msl2 RNA levels on protein levels of Msl2 and the other MSL proteins, Western analyses was performed. It was found that lack of Msl2 protein resulted in lower levels of MSL1 and MSL3, similar to what is seen in wild-type females or msl2 mutants. In contrast, MOF and MLE levels appeared unchanged in the absence of Msl2. Therefore, the RNAi conditions appear to mimic the female state, in which Msl2 is absent, MSL1 and MSL3 are present only at low levels, and MOF and MLE show an abundance similar to males but appear delocalized (Hamada, 2005).

msl2 mutant cells appear to have a severe growth disadvantage during Drosophila development, based on the failure to recover homozygous msl2 mutant clones in most adult tissues of males following induction of mitotic recombination in heterozygous individuals. msl2 mutant males do survive to the larval stages, but they are severely delayed in development. Therefore, it was asked whether depletion of Msl2 from cells in culture would lead to changes in their doubling time. It was found that cell doubling was indistinguishable comparing mock-treated cells with cells treated with msl2 dsRNA through the 4-d course of this experiment. It is proposed that the lack of a requirement to execute sensitive developmental pathways allows cells without dosage compensation to remain healthy, at least within this limited time frame, and thus has allowed the separation of the direct from the majority of indirect effects caused by depletion of Msl2 (Hamada, 2005).

Total RNA was extracted after msl2 or GFP RNAi treatment of cells, and fluorescently labeled cDNA was produced and hybridized in parallel to Affymetrix Drosophila Genome 2.0 microarrays. Experiments were performed three times, with RNAi and GFP control in each case, for a total of six arrays (Hamada, 2005).

A key step in microarray analysis is to identify the genes that have a sufficiently consistent signal to be measured accurately. Genes with undetectable or low expression were removed from the analysis. The 18,000 genes on the array were filtered using Affymetrix Present/Absent calls, resulting in 7923 genes counted as present. A second filter was employed to ensure the consistency of fold ratios in the three experiments. After the second filter, ~5400 genes with reliable fold ratios were divided into those on the X chromosome (897) and those on the autosomes (4484), and the distribution of the ratios were plotted for each case in the log 2 scale. The two distributions were estimated using the median ratio among the triplicates by a smoothing technique. Genes on the X chromosome are down-regulated compared with autosomal genes. The microarray data were processed in many different ways but the results remained the same (Hamada, 2005).

To determine whether such a large difference in the distributions could have arisen by chance, the p-value was calculated for the null hypothesis that the underlying distributions are the same. Using either the t-test or Kolmogorov-Smirnov test, the p-value is <10-15. This is the probability of observing the difference in the distributions if they were in fact the same (Hamada, 2005).

Overall, using a threshold change of 1.4-fold, it was found that many X chromosomal genes (~29%) exhibited a decrease in transcript levels (261 out of 897), while only a small number of autosomal transcripts (~1.2%) decreased (56 out of 4484). Both X and autosomes had some transcripts that increased slightly under these conditions (~0.2% and 1.6%, respectively). Another way to examine the changes in expression is to locate the genes with differential expression by their chromosomal location. The strongly down-regulated genes are concentrated on the X chromosome. The 1.4 threshold for this figure but other threshold values result in a similar picture (Hamada, 2005).

Individual X-linked genes, CG14804, mRpL16, and Arm, showed a 1.3- to 1.5-fold decrease in these experiments. In addition, the autosomal RpII140 gene showed no change in either study. The Affymetrix values for five X and one autosomal gene were further validated by quantitative real-time RT–PCR (Hamada, 2005).

In each replicate of the experiment, changes were detected in mRNA levels from many but not all X-linked genes, depending on the threshold value that was set. For example, ~19% of the expressed genes on the X changed expression <1.1-fold, suggesting that not all X-linked genes are controlled by the MSL complex. However, the partial dosage compensation seen here may also be an inevitable result of the methodologies combined with the difficulties in measuring small changes in gene expression. For example, RNAi often results in partial loss-of-function phenotypes, and in these experiments, msl2 RNA was reduced but not completely eliminated (on average to 8% of wild type). Therefore, the technology implemented does not currently allow definition of the precise number of genes affected by the MSL complex (Hamada, 2005).

It has been previously proposed that a significant subset of X-linked genes, carrying at least three Sxl-binding sites in their 3' UTRs, might be regulated by a distinct dosage compensation pathway. This Sxl-dependent, MSL-independent pathway was proposed to operate by down-regulation of target transcript stability or translation in females, rather than by up-regulation in males. It was found that nine of the previously identified X-linked genes with Sxl-binding sites were expressed in SL2 cells. Surprisingly, four of the nine showed a 1.4-fold or greater reduction in expression after RNAi for msl2, suggesting that they are normally up-regulated by the MSL complex. This was substantiated by analysis of a more comprehensive list of genes with Sxl-binding sites generated from the annotated Drosophila genomic sequence. Therefore, if MSL-independent regulation of this subset of genes does occur, it may be at a specific time or place during development not represented by SL2 cells grown in culture (Hamada, 2005).

In conclusion, current models were tested for the mechanism of dosage compensation in Drosophila by performing the first global analysis of gene expression after removal of the MSL complex in male cells. The results demonstrate that X-linked genes are widely affected as a group when Msl2 levels are reduced, consistent with the striking localization of the MSL complex to sites along the length of the male polytene X chromosome and the morphological changes to that chromosome when MSL proteins are depleted or overexpressed. The results clearly support a role for the MSL complex in up-regulation of X-linked genes in Drosophila males. The specific decrease in X-linked gene expression seen following msl2 RNAi is incompatible with the inverse dosage model (Hamada, 2005).

Male-specific lethal 2 protein controls sex-specific expression of the roX genes

The MSL complex of Drosophila upregulates transcription of the male X chromosome, equalizing male and female X-linked gene expression. Five male-specific lethal proteins and at least one of the two noncoding roX RNAs are essential for this process. The roX RNAs are required for the localization of MSL complexes to the X chromosome. Although the mechanisms directing targeting remain speculative, the ratio of MSL protein to roX RNA influences localization of the complex. The transcriptional regulation of the roX genes was studied; MSL2 controls male-specific roX expression in the absence of any other MSL protein. It is proposed that this mechanism maintains a stable MSL/roX ratio that is favorable for localization of the complex to the X chromosome (Rattner, 2004).

The roX RNAs play crucial roles in male dosage compensation and their regulation is likely to be an integral part of their normal function. Even though the stability of the roX transcripts and their accumulation along the X chromosome are tightly dependent on the presence of the five male-specific lethal genes, male-specific transcription also occurs and is dependent only on MSL2. None of the other MSL proteins is essential for this function; mutation in each of them does not prevent MSL2-driven transcription of the endogenous wild-type roX1 gene. Likewise, MOF-mediated acetylation of histone H4 at lysine 16 is not a prerequisite for roX1 transcription, nor is the activity of the RNA/DNA helicase, MLE. In contrast, these two activities are essential for the in cis spreading of MSL complexes from DHS and for the stability of roX RNA in males. The observation that MSL2 holds a function independent of MSL1 was unanticipated. MSL1 and MSL2 have been suggested to comprise the chromatin-binding activity of the MSL complex and to function together during the initiation of its association with the X chromosome. In addition, direct MSL2 interaction with MSL1 has been demonstrated in vitro. Ectopic expression of MSL2 in females appears to stabilize MSL1. These two proteins are mutually dependent for localization at ~35 CES on the X chromosome in the absence of MSL3, MLE, or MOF. The absence of an msl1 role in roX transcriptional regulation is supported by the demonstration that the MSL2 RING finger, a domain essential for dosage compensation and for the interaction between MSL1 and MSL2, is dispensable for roX1 transcription. This emphasizes that transcriptional regulation of the roX genes represents a novel role for MSL2 that is genetically and molecularly distinct from its function as an MSL complex subunit (Rattner, 2004).

Expression of MSL2 in an otherwise normal female allows roX transcription. These females deploy the male dosage compensation system, but they are not otherwise sexually transformed and are presumed to retain normal expression of SXL. Since SXL directs female gene expression patterns, this makes it unlikely that roX transcription is normally blocked in females by a sex-limited factor. However, it is possible that MSL2 acts by relieving a general transcriptional repression at the roX genes. Alternatively, MSL2 may control roX sex specificity by binding to nascent transcripts, thus relieving a transcriptional pause. The present results do not allow distinguishing between stimulation of transcription or a relief of an inhibition that occurs before transcriptional initiation or during early elongation (Rattner, 2004).

The male-specific roX1 DHS has been shown to recruit MSL complexes to autosomes and to support spreading of these complexes into flanking chromatin. In spite of the overall lack of similarity between the roX genes, roX2 also overlaps a male-specific DNase I hypersensitive site (DHS) that recruits MSL complexes. The presence of these regions in two genes that are each regulated by MSL2 was highly suggestive. Since the DHS is the only sequence within roX1 known to interact with MSL proteins, the DHS is the primary candidate for the MSL2-responsive enhancer governing roX1 transcription. Surprisingly, transcription from roX1 alleles lacking the DHS reveals that MSL2 does not require this sequence to drive roX1 transcription. Furthermore, these roX1 excisions do not derepress roX1 transcription in females. If MSL2 acts to relieve a general repression of roX transcription, repression does not require the presence of the DHS or other internal sequences that have been excised. The roX1 transcription assay used in these studies is likely to reflect the input of all regulatory elements, including distant enhancers and local chromatin context. For this reason it is expected that the transcriptional assay provides an accurate indication of the transcriptional status of roX1 in its native context (Rattner, 2004).

What could be the advantage of MSL2 having two roles in dosage compensation, one as a subunit of the MSL complex and another as the transcriptional regulator of RNAs in the same complex? A recent model proposes that the ratio between MSL proteins and roX RNA influences spreading from roX DHS. This model posits that when the MSL/roX ratio is high (for example, due to reduced roX RNA in the nucleus), complexes are fully assembled before the release of the nascent roX transcripts from the DNA templates. These complexes can immediately bind to chromatin and tend to accumulate in the vicinity of roX genes. By contrast, if the MSL/roX ratio is low, final assembly of the complex occurs in the nucleoplasm following release of the roX transcript. The assembled complex, no longer associated with a particular region, is free to move throughout the nucleus and may travel in trans to other chromosomes. Although the molecular interactions that promote in cis spreading remain obscure, this model is supported by experimental manipulations of MSL and roX levels. For example, when one of the two roX genes is mutated and MSL1 and MSL2 are increased, males display a dramatic enrichment of MSL complex surrounding the remaining roX gene. These findings suggest that the normal distribution of MSL proteins along the length of the male X chromosome is at least in part due to maintenance of MSL/roX ratios. Regulation of roX transcription by MSL2 suggests a mechanism by which the level of available MSL2 protein dictates the supply of roX transcripts, thus maintaining a constant ratio between these two molecules (Rattner, 2004).

A model is proposed in which MSL2 is in a dynamic equilibrium between two possible states. Most of the MSL2 in normal males is assembled into dosage compensation complexes. The amount of roX RNA in the nucleus will determine how much MSL2 can assemble into functional complexes and how much of the protein is available to drive transcription of more roX RNA. It is unknown if MSL2 that is assembled into complexes can stimulate roX transcription, but the vast majority of MSL2 in this form is bound along the length of the X chromosome and is not free to do so. Binding of partial complexes to the roX DHS has been shown to require MSL2 and MSL1. However, it is clear that MSL2 can stimulate roX transcription in the absence of any other MSL protein and that interaction with the roX1 DHS is not required for transcription of this gene. If roX transcription is driven only by free MSL2, transcription would keep pace with the available supply of precursor proteins, thus maintaining a stable MSL/roX ratio. This would hold some advantages for the fly. Small changes in the level of roX RNA could be rapidly corrected. Such an autoregulatory mechanism would ensure that the rates of both roX transcription and of MSL complex assembly onto nascent roX RNAs are optimal (Rattner, 2004).

Splicing and translational control of MSL-2 messenger RNA

Dosage compensation in Drosophila requires the male-specific lethal (msl) proteins (Msl) to make gene expression from the single male X chromosome equivalent to that from both female X chromosomes. Expression of ms12 is repressed post-transcriptionally by Sex lethal (Sxl). Although MSL2 mRNA is alternatively spliced in males and females, this does not alter its coding potential and splicing is not required for male-specific expression of Msl2 protein. Instead, these results suggest that the association of Sxl protein with multiple sites in the 5' and 3' untranslated regions of the MSL2 transcript represses its translation in females. Thus, this well characterized alternative splicing factor regulates at least one target transcript by a distinct mechanism (Kelley, 1997).

Sex-lethal acts synergistically through sequences in both the 5' and 3' untranslated regions of MSL-2 to mediate repression. There is a small intron in the 5' UTR of MSL-2 pre-messenger RNA that is retained in females and removed in males (the male specific intron). Additionally, there are poly (U) runs in both the 5' and 3' UTRs that resemble the SXL-binding sites in SXL and TRA. The male-specific intron present in the 5' UTR of female mRNA functions primarily to allow SXL binding. Removal of either the 3' or both the 3' and 5' UTRs result in expression of MSL-2 protein in females. Endogenous MSL-2 mRNA is not retained in the nucleus of wild-type animals. Two possible mechanisms of SXL-mediated posttranscriptional regulation have been proposed: (1) that SXL binds MSL-2 mRNA and prevents its export from the nucleus, and (2) that SXL binds MSL-2 mRNA and directly blocks its translation. The observation that MSL-2 mRNA is predominantly localized in the cytoplasm of both males and females argues in favor of a direct role for SXL in translational regulation. This direct role has been verified by establishing that the levels of mRNA in the cytoplasm are not influences by the presence or absence of SXL protein. This is the first characterized instance of an mRNA that has target sequences for the same translational regulatory factor in both the 5' and 3' UTRs (Bashaw, 1997).

MSL-2 transcripts are present in somatic tissues of both sexes, in larvae and adults, while no transcripts are detected in isolated female ovaries. The average size of MSL-2 transcripts in females (4.0kb) appears to be slightly larger than in males (3.8kb). The MSL-2 primary transcript has a 132 bp intron in its untranslated leader sequence that is retained in females and is spliced out in males (resulting in a functional mRNA in males). At the 5'-end and near the 3' end of this intron there are stretches of 10 and 17 thymine residues respectively, reminiscent of the eight thymine residue stretch found in the Sex Lethal consensus binding site (T8C) of TRA and SXL transcripts. If SXL is involved in excision of this MSL-2 intron, its mode of action would be the same as in the sex differentiation pathway (splicing of TRA message) or in its own autoregulation, but consequences would be different: rather than preventing an acceptor site from being used so that splicing occurs at an alternate acceptor site, SXL would block splicing from occurring at all by binding to the intron sequence (Zhou, 1995). In fact, MSL2 mRNA is not used as a messenger in females, as no MSL2 protein is detected in females (Kelley, 1995).

Male-specific expression of the protein Male-specific-lethal 2 (Msl-2) controls dosage compensation in Drosophila. Msl-2 gene expression is inhibited in females by Sex-lethal (Sxl), an RNA binding protein known to regulate pre-mRNA splicing. An intron present at the 5' untranslated region (UTR) of MSL-2 mRNA contains putative Sxl binding sites and is retained in female flies. Sxl plays a dual role in the inhibition of MSL-2 expression. Cotransfection of Drosophila Schneider cells with a Sxl expression vector and a reporter containing the 5' UTR of MSL-2 mRNA results in retention of the 5' UTR intron and efficient accumulation of the unspliced mRNA in the cytoplasm, where its translation is blocked by Sxl, but not by the intron per se. Both splicing and translation inhibition by Sxl were recapitulated in vitro and found to be dependent on Sxl binding to high-affinity sites within the intron, showing that Sxl directly regulates these events. These data reveal a coordinated mechanism for the regulation of MSL-2 expression by the same regulatory factor: Sxl enforces intron retention in the nucleus and subsequent translation inhibition in the cytoplasm (Gebauer, 1998).

The protein Sex-lethal (Sxl) controls dosage compensation in Drosophila by inhibiting the splicing and translation of male-specific-lethal-2 (msl-2) transcripts. Splicing inhibition of msl-2 requires a binding site for Sxl at the polypyrimidine (poly(Y)) tract associated with the 3' splice site, and an unusually long distance between the poly(Y) tract and the conserved AG dinucleotide at the 3' end of the intron. Only this combination allows efficient blockage of U2 small nuclear ribonucleoprotein particle binding and displacement of the large subunit of the U2 auxiliary factor (U2AF65) from the poly(Y) tract by Sxl. Crosslinking experiments with ultraviolet light indicate that the small subunit of U2AF (U2AF35: see U2 small nuclear riboprotein auxiliary factor 50) contacts the AG dinucleotide only when located in proximity to the poly(Y) tract. This interaction stabilizes U2AF65 binding such that Sxl can no longer displace it from the poly(Y) tract. These results reveal a novel function for U2AF35, a critical role for the 3' splice site AG at the earliest steps of spliceosome assembly and the need for a weakened U2AF35-AG interaction to regulate intron removal (Merendino, 1999).

Translational repression of male-specific-lethal 2 (MSL-2) mRNA by Sex-lethal (SXL) controls dosage compensation in Drosophila. In vivo regulation involves cooperativity between Sxl-binding sites in the 5' and 3' untranslated regions (UTRs). To investigate the mechanism of MSL-2 translational control, a novel cell-free translation system has been developed from Drosophila embryos that recapitulates the critical features of mRNA translation in eukaryotes: cap and poly(A) tail dependence. Importantly, tight regulation of MSL-2 translation in this system requires cooperation between the Sxl-binding sites in both the 5' and 3' UTRs, as seen in vivo. However, in contrast to numerous other developmentally regulated mRNAs, the regulation of MSL-2 mRNA occurs by a poly(A) tail-independent mechanism. The approach described here allows mechanistic analysis of translational control in early Drosophila development and has revealed insights into the regulation of dosage compensation by Sxl (Gebauer, 1999).

Translational regulation of MSL-2 mRNA in vitro primarily rests on the cooperation between Sxl-binding site B in the 5' UTR and the sites located in the 3' UTR. The presence of functional sites in only one of the UTRs results in a 2-fold inhibition by Sxl, while the presence of sites in both UTRs achieves greater than 10-fold inhibition. This suggests that Sxl action from the 5' and 3' UTR sites cooperates to prevent translation. One possible scenario is that Sxl-driven interactions between the 5' and 3' UTRs package the MSL-2 mRNP into a conformation that renders it poorly accessible to the translational machinery. An alternative explanation is that Sxl inhibits different processes at the 5' and 3' UTRs independently, but that these processes cooperate for translation. This model would not pose any a priori requirement for interactions between Sxl molecules bound to the two ends of MSL-2 mRNA. A major result of this work is the realization that Sxl action is independent of the presence of the poly(A) tail, at least in vitro. The cordycepin-treated mRNA contains a small fraction of messages with a poly(A) tail of 30 residues. These do not appear to contribute significantly either to overall translation or to regulation by Sxl, because the non-treated mRNA, which contains a higher proportion of A30 molecules, is translated and repressed by Sxl with comparable efficiency. Importantly, the Drosophila embryo system is principally poly(A) dependent. Since it so closely reflects the known aspects of MSL-2 regulation in vivo, it is predicted that poly(A) independence is an important feature of how Sxl regulates dosage compensation in the fly. The Drosophila cell-free translation system described here now allows the study of an important aspect of developmental biology in the test tube. Future experiments using this system should help to shed further light on the mechanism by which Sxl inhibits translation. Equally importantly, it provides a novel approach to dissect other examples of translational regulation during Drosophila embryogenesis, and to study the mechanism by which the poly(A) tail promotes translation in multicellular eukaryotes (Gebauer, 1999).

The gene virilizer is needed for dosage compensation and sex determination in females and for an unknown vital function in both sexes. In genetic mosaics, XX somatic cells mutant for vir differentiate male structures. One allele, vir2f, is lethal for XX, but not for XY animals. This female-specific lethality can be rescued by constitutive expression of Sxl or by mutations in msl (male-specific lethal) genes. Rescued animals develop as strongly masculinized intersexes or pseudomales. They have male-specifically spliced mRNA of tra, and when rescued by msl, also of Sxl. These data indicate that vir is a positive regulator of female-specific splicing of Sxl and of tra pre-mRNA (Hilfiker, 1995).

Animals with two X chromosomes and mutant for vir2f are rescued by loss of function of msl genes. In view of the sex-transforming effect of mutations in vir, and since mutations in msl genes do not interfere with sexual differentiation, it is not surprising that the rescued animals are transformed into sterile pseudo-males. It is concluded that vir2f allows the inappropriate activity of the msl genes in XX zygotes, and that the affected animals then die as a result of overexpression of X-linked genes. Elimination of any one of the msl genes lowers X-chromosomal transcription towards a level that is lethal for XY animals, but is appropriate for a single X in XX animals. However, the fact that the rescue of XX;vir2f animals by mutations in msl genes is weak, is a warning that the regulatory network of dosage compensation is more complex. Sxl controls an early female-specific vital function that is not dependent on msl gene activity (Hilfiker, 1995 and references therein).

How could vir participate in the regulation of the male specific lethal (msl) genes? Recent studies have shown that, of all the msl genes [mle; msl-1 and msl-3, only msl-2 is differentially expressed due to sex-specific splicing -- only males are capable of producing a functional protein; in females, the productive splice seems to be prevented by Sxl. Thus, it appears that the Sxl protein acts again, as in the regulation of its own mRNA and that of tra, by blocking a splice site in the msl-2 transcripts. Since the rescue of XX;vir2f animals by SxlM is not complete and since the SxlM1/Y males survive despite the presence of Sxl, it is concluded that the Sxl product requires vir function to efficiently prevent the male-specific processing of msl-2 transcripts (Hilfiker, 1995).

It was unexpected that XX;vir2f animals rescued by SxlM were transformed into pseudomales or strongly masculinized intersexes. A simple model in which vir acts above Sxl predicts that the rescued animals, due to the female-determining effect of Sxl, would be females; alternatively, if vir acted below Sxl, there would be no rescue of the lethality. The actual results are best interpreted by a model in which vir acts at both levels, upstream and downstream of Sxl, but ostensibly with differential effectiveness. Its function appears to be absolutely required for female-specific splicing of Sxl transcripts, but seems less important for the regulation of tra, and even less for msl-2 which is assumed to be the other target. This is inferred from the observation that the function provided by SxlM1 or SxlM4 in XX;vir2f animals is largely, but not completely, sufficient to prevent the inappropriate activity of the msl genes, but insufficient to make enough female-specific products of tra necessary for female sexual development (Hilfiker, 1995).

The protein Sex-lethal (Sxl) controls dosage compensation in Drosophila by inhibiting splicing and subsequently translation of male-specific-lethal-2 transcripts. Sxl blocks the binding of U2 auxiliary factor (U2AF) to the polypyrimidine (Py)-tract associated with the 3' splice site of the regulated intron. This study reports that a second pyrimidine-rich sequence containing 11 consecutive uridines immediately downstream from the 5' splice site is required for efficient splicing inhibition by Sxl. Psoralen-mediated crosslinking experiments suggest that Sxl binding to this uridine-rich sequence inhibits recognition of the 5' splice site by U1 snRNP (see sans fille)in HeLa nuclear extracts. Sxl interferes with the binding of the protein TIA-1 to the uridine-rich stretch. Because TIA-1 binding to this sequence is necessary for U1 snRNP recruitment to msl-2 5' splice site and for splicing of this pre-mRNA, it is proposed that Sxl antagonizes TIA-1 activity and thus prevents 5' splice site recognition by U1 snRNP. Taken together with previous data, it is concluded that efficient retention of msl-2 intron involves inhibition of early recognition of both splice sites by Sxl (Forch, 2001).

A co-repressor assembly nucleated by Sex-lethal in the 3'UTR mediates translational control of Drosophila msl-2 mRNA

Drosophila Sex-lethal-mediated translational repression of male-specific lethal 2 (msl-2) mRNA is essential for X-chromosome dosage compensation. Binding of Sxl to specific sites in both untranslated regions of msl-2 mRNA is necessary for inhibition of translation initiation. This study describes the organization of Sxl as a translational regulator and shows that the RNA binding and translational repressor functions are contained within the two RRM domains and a C-terminal heptapeptide extension. The repressor function is dormant unless Sxl binds to msl-2 mRNA with its own RRMs, because Sxl tethering via a heterologous RNA-binding peptide does not elicit translational inhibition. This study reveals proteins that crosslink to the msl-2 3' untranslated region (3'UTR) and co-immunoprecipitate with Sxl in a fashion that requires its intact repressor domain and correlates with translational regulation. Translation competition and UV-crosslink experiments show that the 3'UTR msl-2 sequences adjacent to Sxl-binding sites are necessary to recruit titratable co-repressors. These data support a model where Sxl binding to the 3'UTR of msl-2 mRNA activates the translational repressor domain, thereby enabling it to recruit co-repressors in a specific fashion (Grsckovic, 2003).

The architecture and function of Sxl as a translational repressor was analyzed. In female flies, translational repression of msl-2 mRNA by Sxl represents a critical regulatory step for dosage compensation. The results discriminate Sxl's function as a splicing regulator from its function in translational control. They also suggest that the translational repressor activity of Sxl is dormant, and activated upon binding to the msl-2 mRNA. Finally, functional evidence is provided that Sxl and the msl-2 3'UTR cooperate in the recruitment of proteins that appear to act as translational co-repressors (Grsckovic, 2003).

Modularity represents one of the most common principles of protein architecture. It permits the organization of biological functions into independent domains, and integration of these functions in a single molecule. This work shows that the mRNA binding and translational regulatory functions of Sxl are not organized into independent domains. Rather, both functions reside within aa 122-301 of the protein, a region that has been recognized as the RNA-binding domain of Sxl. The N-terminal domain of Sxl contributes an essential function only to alternative splicing, but is dispensable for translational control of msl-2 mRNA. The embedding of the translational regulatory function into the RNA-binding domain and a few additional C-terminal amino acids also explains why N-terminal truncations strongly impair Sxl function in sex determination, but not in dosage compensation in vivo (Grsckovic, 2003).

Based on the analysis of hybrid proteins between Drosophila and Musca Sxl, it is suggested that the region corresponding to RRM1 (aa 122-200) makes an important contribution to translational repression and represents a critical difference between Drosophila Sxl, which efficiently represses translation, and Musca Sxl, which does not. In addition to RRM1, an extension of 7 aa following the RRMs cooperates in translation inhibition. Interestingly, RRM1 has been shown to contact the snRNP component SNF, a protein thought to be involved in the autoregulatory splicing of Sxl pre-mRNA. RRM1 also binds SIN, a protein of unknown function. However, both SNF and SIN are much smaller than the crosslinked polypeptides that Sxl recruits to the 3'UTR of msl-2. The crystal structure of the Sxl RRMs bound to the RNA reveals that almost all of the residues that differ from those in mSXL are exposed on the accessible outer surface of the protein. None of them contacts the RNA or other parts of the RRMs. In accordance with these structural data, similar RNA-binding affinities of dRBD3 and mRBD are found in gel mobility-shift assays. Exposed amino acid residues on the outer surface of the RNA-binding domain of Sxl are ideally placed to mediate the interaction of Sxl with translational co-repressors (Grsckovic, 2003).

Regulatory proteins that bind to specific mRNAs to control their translation employ different modes of action. In all cases, specificity demands that the regulatory protein should affect the translation of the mRNAs to which it binds, but not perturb the general translation machinery in a non-specific way. One solution to this problem is steric control, where the mRNA binding event is both necessary and sufficient for translational repression. In these cases, no translational effector domains per se are required, which could potentially act in trans and interfere with the function of a translation factor while the regulatory protein is not bound to its target mRNA. Steric control has been shown for the iron regulatory proteins, and is also evident in the translational inhibition of the bacteriophage lambda -- the lambda anti-terminator protein. msl-2 mRNA regulation by Sxl does not operate by such a steric mechanism, and the specificity problem must be solved differently (Grsckovic, 2003).

The binding of Sxl to its physiological binding sites in the 5' and 3'UTRs of WT or BLEF mRNA (a construct that harbours all msl-2 mRNA sequences required for full regulation by Sxl) cannot be substituted by tethered Sxl, suggesting that Sxl must bind the RNA with its own RRMs to be active as a translational repressor. This contrasts with other RNA-binding proteins, such as the poly(A)-binding protein (PABP) or proteins involved in nonsense-mediated mRNA decay, which retain their respective functions when being tethered to an mRNA. Importantly, the lambdaSXL protein (a hybrid protein containing the RNA-binding domain of the lambda phage anti-terminator protein, a 22 aa oligomer referred to as the lambda peptide) binds to the indicator mRNAs, as evidenced by its steric regulatory function and gel mobility-shift assays, and it is fully functional as a bona fide msl-2 regulatory protein when binding through its own RRMs. This excludes trivial misfolding effects as an explanation for the lack of tethered function (Grsckovic, 2003).

It seems most likely that the dormant translational repressor domain, which is embedded within the RNA-binding region of Sxl, is only activated when the protein binds to msl-2 mRNA, solving the specificity problem. Sxl binding to the EF region of the msl-2 3'UTR induces the binding of additional proteins to this site, which cannot be crosslinked in the absence of Sxl. It is possible that two molecules of Sxl binding to the adjacent E and F sites, respectively, cooperate in recruiting the co-repressor(s) by creating a binding surface partially lacking from monomeric Sxl. The recruitment of these additional proteins has two requirements. (1) Sxl but not musca SXL can mediate the crosslinking of these factors. This suggests that specific amino acids that are exposed on Sxl (or Sxl molecules binding closely to each other) contribute to the interaction. (2) Sxl binding to the 3'UTR probe but not the 5'UTR or mutated 3'UTR probes is required for crosslinking. This suggests that the 3'UTR RNA sequences make an additional specific contribution. Indeed, in addition to Sxl-binding sites E and F, sequences adjacent to these sites are essential for recruitment of the additional proteins. These factors that bind to the 3'UTR regulatory region are functional co-repressors, because only the 3'UTR WT RNA competes for translational inhibition, while the mut2456 and 5' RNAs (which bind Sxl but not the co-repressors) fail to do so (Grsckovic, 2003).

The proposed model for Sxl bears interesting parallels with the function of Pumilio (Pum) and other members of the Puf family of translational regulators. Pum inhibits the translation of maternal hunchback mRNA as part of a complex that is sequentially built on the Nanos-response element (NRE) in the 3'UTR of the message, and the translational repressor domain of Pum is embedded within its RNA binding region, the so-called Puf repeat. Binding of Pum to the NRE triggers the recruitment of Nanos and this event, in turn, stimulates the binding of Brain Tumor. Hence, the stepwise assembly of co-repressor complexes, which requires multiple specific protein-protein and RNA-protein interactions, emerges as a theme in translational control. The msl-2 regulatory complex inhibits translation initiation by blocking the stable binding of the small ribosomal subunit to the mRNA in a cap-independent fashion. Future work will aim to define the composition of this regulatory complex and reveal its molecular interactions with the machinery that executes early steps in translation initiation (Grsckovic, 2003).

A dual inhibitory mechanism restricts msl-2 mRNA translation for dosage compensation in Drosophila

Drosophila MSL-2 is the limiting component of the dosage compensation complex (DCC). Female flies must inhibit msl-2 mRNA translation for survival, and this inhibition is mediated by Sex-lethal (Sxl) binding to sites in both the 5' and the 3' untranslated regions (UTRs). This study uncovers the mechanism by which Sxl achieves tight control of translation initiation. Sxl binding to the 3'UTR regulatory region inhibits the recruitment of 43S ribosomal preinitiation complexes to the mRNA. Ribosomal complexes escaping this block and binding to the 5' end of the mRNA are challenged by Sxl bound to the 5'UTR, which interferes with scanning to the downstream initiation codon of the mRNA. This failsafe mechanism thus forms the molecular basis of a critical step in dosage compensation. The results also elucidate a two step principle of translational control via multiple regulatory sites within an mRNA (Beckmann, 2005).

This study investigates how Sxl silences translation of the msl-2 mRNA. In female flies, this regulation prevents the formation of the dosage compensation complex and thus deleterious hypertranscription of the two female X chromosomes. The results define the molecular basis of this critical regulatory step. They also reveal a mechanism of translational control that is based on the integration of two separable components. Individually, each of the two components provides insights into functional properties of 5′ and 3′ regulatory complexes to interfere with translation initiation, and each of these two components appears to be mechanistically unprecedented (Beckmann, 2005).

In male flies, MSL-1 and MSL-2 mediate the assembly of the DCC on the single X chromosome, which is thought to spread along the entire chromosome promoting an ~2-fold increase in transcription levels. The absence of MSL-2 in females does not allow DCC formation, while transgenic female flies expressing MSL-2 assemble the complex, showing that MSL-2 is the limiting subunit of the DCC. Although the msl-2 transcript levels in females are reduced to 20%-30% of those in male flies, translational control mediated by Sxl is crucial to block MSL-2 expression (Beckmann, 2005).

The msl-2 mRNA contains two SXL binding sites within the 5'UTR and four binding sites within the 3'UTR (sites A-F). Sxl bound to either UTR of msl-2 mRNA inhibits translation by interfering with initiation prior to 48S complex formation. Furthermore, the two regulatory regions independently interfere with translation initiation by different means. Earlier evidence suggested that the roles of Sxl bound to the 5′UTR and the 3′UTR, respectively, are noninterchangeable: (1) the 5′UTR Sxl binding site B cannot substitute for sites E and F when introduced into the 3′UTR; (2) UV-crosslinking experiments identified at least one protein that is recruited by Sxl specifically to the 3′UTR (Grskovic, 2003). The crosslinks require the sequences that flank the E and F sites, and RNA competition experiments functionally implicated this 3′UTR binding protein in Sxl-mediated translational control; by contrast, crosslinks to the 5′UTR are limited to Sxl itself. The simplest interpretation of this earlier work was that both UTRs engage in a functional and perhaps physical interaction to block the stable engagement of the small ribosomal subunit with the mRNA at a single step (Beckmann, 2005).

This work now reveals that such a simple assumption is incorrect. Rather than forming a single repressor complex that targets one step in the initiation pathway, Sxl acts as a bifunctional regulator: 3′UTR bound Sxl inhibits translation from cap-proximal and cap-distal AUGs identically well, while 5′UTR bound Sxl can only function when it binds upstream of the initiation codon. Based on these data and direct physical evidence provided by the toeprint experiments (to identify translation complexes that bind to the mRNA), it is concluded that Sxl bound to the 3′UTR impedes the initial recruitment of 43S complexes to the mRNA while 5′UTR bound Sxl stalls scanning 43S complexes upstream of its binding site (Beckmann, 2005).

How does Sxl binding to the B site achieve a scanning block? Apparently, Sxl hinders the transit of 43S complexes across site B. Steric hindrance of ribosomal scanning has been proposed for iron-regulatory proteins/iron-responsive element complexes that were introduced ~100 nucleotides downstream from the cap structure of a reporter mRNA. However, scanning inhibition by Sxl bound to the B site does not appear to follow a simple steric mechanism, i.e., to be imposed solely by high affinity mRNA binding. mRBD a Musca domestica Sxl derivative (Musca SXL does not function in X chromosome dosage compensation and mRBD does not repress msl-2) fails to repress translation or stall scanning 43S complexes, although it binds to site B with an affinity as high as that of Sxl. Furthermore, tethering of a λ peptide-Sxl fusion protein to a BoxB element replacing Sxl binding site B in the 5′UTR of msl-2 mRNA does not inhibit translation despite the high affinity of the λ peptide-BoxB interaction (Beckmann, 2005).

Therefore, it is proposed that Sxl regulates scanning either by altering the 5′UTR secondary structure and/or promoting the formation of a higher-order assembly on the B site. Such a complex could then act as a (steric) roadblock to scanning, without being able to halt elongating 80S ribosomes. Alternatively, Sxl and potential interacting proteins may specifically interfere with the function of a translation initiation factor or the small ribosomal subunit which is required for scanning but not for translation elongation. Interestingly, site B is composed of 16 uridine residues that could be bound by a Sxl dimer. Since Sxl engages in protein-protein interactions through its RNA binding domains, Sxl dimerization and additional factors recruited by Sxl may promote the formation of a higher-order repressor complex that blocks scanning, in as much as a stalled elongating ribosome can function as a block to scanning (Beckmann, 2005).

How does Sxl bound to the 3′UTR inhibit 43S preinitiation complex recruitment? The recruitment of the 43S complex represents a previously identified target of 3′UTR binding translational regulators. For example, Maskin, Cup, and Bicoid block the recruitment of the 43S complex by interfering with the assembly of eIF4F at the cap structure. However, Sxl-mediated inhibition is independent of the cap structure (Gebauer, 2003), implying that the 43S complex recruitment block imposed by Sxl is different from that mediated by these regulators (Beckmann, 2005).

It is noticed that 3′UTR-mediated translational inhibition by Sxl involves the accumulation of the repressed mRNA within unusually heavy RNP particles. Interestingly, Sxl has previously been found in large, RNase-sensitive complexes sedimenting faster than bulk mRNPs in sucrose density gradient experiments. An attractive possibility is that Sxl in association with the 3′UTR corepressor nucleates a large repressor complex or that the 3′UTR repressor complex promotes the multimerization of mRNPs leading to the formation of mRNP clusters. Multimerization of mRNPs has been observed during the localization of translationally silent bicoid mRNA to the anterior pole of the Drosophila oocyte. Clustered mRNAs may be less accessible to the translation initiation machinery, providing a possible mechanism of 3′UTR-mediated inhibition of 43S complex recruitment independent of mRNA-specific 3′ to 5′ end communication (Beckmann, 2005).

What are the biological advantages of the duality of translational control by Sxl? Such an integrated failsafe mechanism allows efficient repression of translation in situations where the leakiness of a single mechanism could be deleterious for the cell and/or the organism. Indeed, forced expression of the MSL-2 protein in female flies enables the loading of the dosage compensation complex onto the two X chromosomes and causes lethality. Therefore, the translational repression of female msl-2 mRNA must be robust, which is achieved by the combination of the two mechanisms that cooperate to prevent 43S complexes from reaching the initiation codon (Beckmann, 2005).

oskar (osk) mRNA translation in Drosophila oocytes also appears to be regulated at multiple steps. The synthesis of the posterior determinant Osk must be strictly restricted to the posterior pole of the embryo. This is achieved by the posterior accumulation of osk mRNA and the translational repression of unlocalized osk mRNA. The protein Bruno binds to Bruno-response elements (BREs) in the 3′UTR of the osk mRNA and is important for inhibition of translation. The fact that Bruno interacts with the repressor Cup suggested that the responsible mechanism targets translation initiation, although this has not been shown directly. A recent report identified a significant fraction of unlocalized osk mRNA in association with ribosomes, indicating that translation of the osk mRNA may be regulated (in addition) at a postinitiation step. Similar observations implicating multiple levels of translational control have also been made for the spatially and temporally controlled nanos mRNA in Drosophila embryos. In this case, both mechanisms may not be simultaneously active at all stages of development. Translational failsafe mechanisms like the one described here may become recognized as a more widespread principle of robust control over protein synthesis (Beckmann, 2005).

eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs

In female fruit flies, Sex-lethal (Sxl) turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2) mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors -- the U1/U2 snRNP protein Sans-fils (Snf), the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2)d--that have been directly implicated in Sxl splicing regulation (Graham, 2011).

Translation initiation is mediated by the binding of a pre-initiation complex to the 5' cap of the mRNA (reviewed in (Merrick, 1996 ; Gingras, 1999) that in turn recruits the small subunit of the 40S ribosome to the mRNA. The pre-initiation complex consists of the cap binding protein, eIF4E, and a scaffolding protein, eIF4G, which mediates interactions with various components of the 40S initiation complex. In many organisms there is also a third protein in the complex, eIF4A, an ATP dependent RNA helicase. Modulating eIF4E activity appears to be a key control point for regulating translation. One of the most common mechanisms of regulation is by controlling the association eIF4E with eIF4G. Factors such as poly-A binding protein that promote the association between eIF4E and eIF4G activate translation initiation, while factors such as the 4E-binding proteins (4E-BPs; see Drosophila 4E-BP) that block their association, inhibit initiation (Graham, 2011 and references therein).

Although eIF4E's primary function in the cell is in regulating translation initiation, studies over the past decade have revealed unexpected activities for eIF4E at steps prior to translation. Among the more surprising findings is that there are substantial amounts of eIF4E in eukaryotic nuclei. One role for eIF4E in the nucleus is the transport of specific mRNAs, like cyclin D1, to the cytoplasm (Rousseau, 1996). This eIF4E activity is distinct from translation initiation since an eIF4E mutation that prevents it from forming an active translation complex still allows cyclin D1 mRNA transport. The transport function of eIF4E is modulated by at least two other proteins, PML and PRH (Topisirovic, 2002; Topisirovic, 2003). While PML seems to be ubiquitously expressed, PRH is found only in specific tissues. In addition, the intracellular distribution of eIF4E exhibits dynamic changes during Xenopus development (Strudwick, 2002). These observation raise the possibility that eIF4E might have additional functions in the nucleus during development. Consistent with this idea, this study shows that eIF4E plays a novel role in the process of sex determination in Drosophila (Graham, 2011).

Sex determination in the fly is controlled by the master regulatory switch gene Sex-lethal (Sxl). The activity state of the Sxl gene is selected early in development by an X chromosome counting system. The target for the X/A signaling system is the Sxl establishment promoter, Sxl-Pe. When there are two X chromosomes, Sxl-Pe is turned on, while it remains off when there is a single X chromosome. Sxl-Pe mRNAs encode RRM type RNA binding proteins which mediate the transition from the initiation to the maintenance mode of Sxl regulation by directing the female-specific splicing of the first pre-mRNAs produced from a second, upstream promoter, the maintenance promoter, Sxl-Pm. Sxl-Pm is turned on before the blastoderm cellularizes, just as Sxl-Pe is being shut off. In the presence of Sxl-Pe proteins, the first Sxl-Pm transcripts are spliced in the female-specific pattern in which exon 2 is joined to exon 4 (see Model of the alternatively spliced region of Sxl ). The resulting Sxl-Pm mRNAs encode Sxl proteins that direct the female specific splicing of new Sxl-Pm pre-mRNAs and this establishes a positive autoregulatory feedback loop that maintains the Sxl gene in the 'on' state for the remainder of development. In male embryos, which lack the Sxl-Pe proteins, the Sxl-Pm pre-mRNAs are spliced in the default pattern, incorporating the male specific exon 3. This exon has several in-frame stop codons that prematurely truncate the open reading frame so that male specific Sxl-Pm mRNAs produce only small non-functional polypeptides. As a consequence the Sxl gene remains off throughout development in males (Graham, 2011).

In females, Sxl orchestrates sexual development by regulating the alternative splicing of transformer (tra) pre-mRNAs. Like Sxl, functional Tra protein is only produced by female-specific tra mRNAs, while mRNAs spliced in the default, male pattern encode non-functional polypeptides. Sxl also negatively regulates the dosage compensation system, which is responsible for hyperactivating X-linked transcription in males, by repressing male-specific lethal-2 (msl-2). Sxl represses msl-2 by first blocking the splicing of an intron in the 5' UTR of the msl-2 pre-mRNA, and then by inhibiting the translation of the mature mRNA. In addition, there are two other known targets for Sxl translational repression. One is the Sxl mRNA itself. Sxl binds to target sequences in the Sxl 5' and 3' UTRs and downregulates translation. It is thought that this negative autoregulatory activity provides a critical homeostasis mechanism that prevents the accumulation of excess Sxl protein. This is important as too much Sxl can disrupt development and have female lethal effects. The other known target is the Notch (N) mRNA (Penn, 2007). Sxl-dependent repression of N mRNA translation is important for the elaboration of sexually dimorphic traits in females. Like msl-2 and Sxl, translational repression appears to be mediated by Sxl binding to sites in the N UTRs (Graham, 2011).

Translational repression of msl-2 mRNA by Sxl is thought to involve two separate mechanisms acting coordinately. Binding sites for Sxl in the unspliced intron in the 5' UTR and in the 3'UTR of msl-2 are required for complete repression. Sxl binding to the 5'UTR blocks recruitment of the 40S pre-initiation complex (. While factors that act with Sxl at the 5'UTR of msl-2 have yet to be identified, repression by the 3'UTR requires Sxl, PABP and a co-repressor UNR. Somewhat unexpectedly, this complex does not affect recruitment of eIF4E or eIF4G to the 5' end. Instead it prevents ribosomes that do manage to attach to the msl-2 mRNA from scanning (Graham, 2011).

Although eIF4E does not appear to be a key player in the translational repression of msl-2 mRNAs, this study reports that it has an important role in the process of sex determination in Drosophila. eIF4E activity is required in females to stably activate and maintain the Sxl positive autoregulatory feedback loop and to efficiently repress msl-2. Surprisingly, this requirement for eIF4E activity in fly sex determination is in promoting the female-specific splicing of the Sxl and msl-2 transcripts, not in translational regulation (Graham, 2011).

The RNA binding protein Sxl orchestrates sexual development by controlling gene expression post-transcriptionally at the level of splicing and translation. To exert its different regulatory functions Sxl must collaborate with sex-non-specific components of the general splicing and translational machinery. In this study evidence is presented that one of the splicing co-factors is the cap binding protein eIF4E. eif4e was initially identified in a screen for mutations that dominantly suppress the male lethal effects induced by ectopic expression of a mutant Sxl protein, Sx-N, which lacks part of the N-terminal domain. The Sx-N protein is substantially compromised in its splicing activity, but appears to have closer to wild type function in blocking the translation of the Sxl targets msl-2 and Sxl-Pm. As the male lethal effects of Sx-N (in an Sxl- background) are due to its inhibition of Msl-2 expression, it is anticipated that general translation factors needed to help Sxl repress msl-2 mRNA would be recovered as suppressors in the screen. Indeed, one of the suppressors identified was eif4e. However, consistent with in vitro experiments, which have shown that Sxl dependent repression of msl-2 mRNA translation is cap independent, this study found that eif4e does not function in Sxl mediated translational repression of at least one target mRNA in vivo. Instead, the results indicate that eif4e is needed for Sxl dependent alternative splicing, and it is argued that it is this splicing activity that accounts for the suppression of male lethality by eif4e mutations. In wild type females, Sxl protein blocks the splicing of a small intron in the 5' UTR of the msl-2 pre-mRNA. This is an important step in msl-2 regulation because the intron contains two Sxl binding sites that are needed by Sxl to efficiently repress translation of the processed msl-2 mRNA. When this intron is removed repression of msl-2 translation by Sxl is incomplete and this would enable eif4e/+ males to escape the lethal effects of the Sx-N transgene (Graham, 2011).

Several lines of evidence support the conclusion that eif4e is required for Sxl dependent alternative splicing. One comes from the analysis of the dominant maternal effect female lethal interactions between eif4e and Sxl. The initial activation of the Sxl positive autoregulatory feedback loop in early embryos can be compromised by a reduction in the activity of splicing factors like Snf, Fl(2)d, and U1-70K, and mutations in genes encoding these proteins often show dose sensitive maternal effect, female lethal interactions with Sxl. Like these splicing factors, maternal effect female lethal interactions with Sxl are observed for several eif4e alleles. Moreover, these female lethal interactions can be exacerbated when the mothers are trans-heterozygous for mutations in eif4e and the splicing factors snf or fl(2)d. Genetic and molecular experiments indicate that female lethality is due to a failure in the female specific splicing of Sxl-Pm mRNAs. First, female lethality can be rescued by gain-of-function Sxl mutations that are constitutively spliced in the female mode. Second, transcripts expressed from a Sxl-Pm splicing reporter in the female Sxl-/+ progeny of eif4e/+ mothers are inappropriately spliced in a male pattern at the time when the Sxl positive autoregulatory loop is being activated by the Sxl-Pe proteins. While splicing defects are evident in these embryos at the blastoderm/early gastrula stage, obvious abnormalities in expression of Sxl protein are not observed until several hours later in development (Graham, 2011).

Though this difference in timing would favor the idea that eif4e is required for splicing of Sxl-Pm transcripts rather than for the export or translation of the processed Sxl-Pm mRNAs, the possibility cannot be excluded that there are subtle defects in the expression of Sxl protein at the blastoderm/early gastrula stage that are sufficient to disrupt splicing regulation during the critical activation phase yet aren't detectable in the antibody staining experiments. However, evidence from two different experimental paradigms using adult females indicates that this is likely not the case. In the first, it was found that reducing eif4e activity in a sensitized snf1621 Sxlf1/++ background can compromise Sxl dependent alternative splicing even though there is no apparent reduction in Sxl protein accumulation. In this experiment advantage was taken of the fact that once the positive autoregulatory feedback loop is fully activated a homeostasis mechanism (in which Sxl negatively regulates the translation of Sxl-Pm mRNAs) ensures that Sxl protein is maintained at the same level even if there are fluctuations in the amount of female spliced mRNA. While only a small amount of male spliced Sxl-Pm mRNAs can be detected in snf1621 Sxlf1/++ females, the level increases substantially when eif4e activity is reduced. Since these synergistic effects occur even though Sxl levels in the triply heterozygous mutant females are the same as in the control snf1621 Sxlf1/++ females, it is concluded that the disruption in Sxl dependent alternative splicing of Sxl-Pm transcripts in this context (and presumably also in early embryos) can not be due to a requirement for eif4e in either the export of Sxl mRNAs or in their translation. Instead, eif4e activity must be needed specifically for Sxl dependent alternative splicing of Sxl-Pm pre-mRNAs. Consistent with a more general role in Sxl dependent alternative splicing, there is a substantial increase in msl-2 mRNAs lacking the first intron when eif4e activity is reduced in snf1621 Sxlf1/++ females. In the second experiment the splicing was examined of pre-mRNAs from the endogenous Sxl gene and from a Sxl splicing reporter in females heterozygous for two hypomorphic eif4e alleles. Male spliced mRNAs from the endogenous gene and from the splicing reporter are detected the eif4e/+ females, but not in wild type females. Moreover, the effects on sex-specific alternative splicing seem to be specific for transcripts regulated by Sxl as no male spliced dsx mRNAs were seen in eif4e/+ females (Graham, 2011).

Two models could potentially explain why eif4e is needed for Sxl dependent alternative splicing. In the first, eif4e would be required for the translation of some critical and limiting splicing co-factor. When eif4e activity is reduced, insufficient quantities of this splicing factor would be produced and this, in turn, would compromise the fidelity of Sxl dependent alternative splicing. In the second, the critical splicing co-factor would be eif4e itself. It is not possible to conclusively test whether there is a dose sensitive requirement for eif4e in the synthesis of a limiting splicing co-factor. Besides the fact that the reduction in the level of this co-factor in flies heterozygous for hypomorphic eif4e alleles is likely to be rather small, only a subset of the Sxl co-factors have as yet been identified. For these reasons, the first model must remain a viable, but unlikely possibility. As for the second model, the involvement of a translation factor like eif4e in alternative splicing is unexpected if not unprecedented. For this to be a viable model, a direct role for eif4e must be consistent with what is known about the dynamics of Sxl pre-mRNA splicing and the functioning of the Sxl protein. The evidence that the second model is plausible is detailed below (Graham, 2011).

Critical to the second model is both the nuclear localization of eIF4E and an association with incompletely spliced Sxl pre-mRNAs. Nuclear eIF4E has been observed in other systems, and this was confirmed for Drosophila embryos. It was also found that eIF4E is bound to Sxl transcripts in which the regulated exon2-exon3-exon4 cassette has not yet been spliced. In contrast, it is not associated with incompletely processed transcripts from the tango gene that are constitutively spliced. With the caveat that only one negative control is available, it is not surprising that Sxl transcripts might be unusual in this respect. There is growing body of evidence that splicing of constitutively spliced introns is co-transcriptional. However, recent in vivo imaging experiments have shown that the splicing of the regulated Sxl exon2-exon3-exon4 cassette is delayed until after the Sxl transcript is released from the gene locus in female, but not in male cells. These in vivo imaging studies also show that, like bulk pre-mRNAs, the 1st Sxl intron is spliced co-transcriptionally in both sexes. Consistent with a delay in the splicing of the regulated cassette, it has been previously reported that polyadenylated Sxl RNAs containing introns 2 and 3 can be readily detected by RNase protection, whereas other Sxl intron sequences are not observed. The delay in the splicing of the regulated Sxl cassette until after transcription is complete and the RNA polyadenylated could provide a window for exchanging eIF4E for the nuclear cap binding protein (Graham, 2011).

To function as an Sxl co-factor, eIF4E would have to be associated with the pre-mRNA-spliceosomal complex before or at the time of the Sxl dependent regulatory step. There is still a controversy as to exactly which step in the splicing pathway Sxl exerts its regulatory effects on Sxl-Pm pre-mRNAs and two very different scenarios have been suggested. The first is based on an in vitro analysis of Sxl-Pm splicing using a small hybrid substrate consisting of an Adenovirus 5' exon-intron fused to a short Sxl-Pm sequence spanning the male exon 3' splice site. These in vitro studies suggest that Sxl acts very late in the splicing pathway after the 1st catalytic step, which is the formation of the lariat intermediate in the intron between exon 2 and the male exon. According to these experiments Sxl blocks the 2nd catalytic step, the joining of the free exon 2 5' splice site (or Adeno 5' splice site) to the male exon 3' splice site. It is postulated that this forces the splicing machinery to skip the male exon altogether and instead join the free 5' splice site of exon 2 to the downstream 3' splice site of exon 4. Since this study has shown that eIF4E binds to Sxl-Pm pre-mRNAs that have not yet undergone the 1st catalytic step, it would be in place to influence the splicing reaction if this scenario were correct (Graham, 2011).

The second scenario is more demanding in that it proposes that Sxl acts during the initial assembly of the spliceosome. Evidence for Sxl regulation early in the pathway comes from the finding that Sxl and the Sxl co-factor Fl(2)d show physical and genetic interactions with spliceosomal proteins like U1-70K, Snf, U2AF38 and U2AF50 that are present in the early E and A complexes and are important for selecting the 5' and 3' splice sites. In addition to these proteins, Sxl can also be specifically cross-linked in nuclear extracts to the U1 and U2 snRNAs. Formation of the E complex depends upon interactions of the U1 snRNP with the 5' splice site, and this is thought to be one of the first steps in splicing. The other end of the intron is recognized by U2AF, which recruits the U2 snRNP to the 3' splice site. After the base pairing of the U2 snRNP with the branch-point to generate the A complex the next step is the addition of the U4/U5/U6 snRNPs to form the B complex. However, Sxl and Fl(2)d are not found associated with components of the splicing apparatus like U5-40K, U5-116K or SKIP that are specific for complexes B and B*, or the catalytic C complex. Nor can Sxl be cross-linked to the U4, U5 or U6 snRNAs. If Sxl and Fl(2)d dissociated from the spliceosome before U4/U5/U6 are incorporated into the B complex, then they must influence splice site selection during the formation/functioning of the E and/or A complex. (Since the transition from the E to the A complex has been shown to coincide with an irreversible commitment to a specific 5'—3' splice site pairing, Sxl would likely exerts its effects in the E complex when splice site pairing interactions are known to still be dynamic. If this is scenario is correct, eIF4E would have to be associated with factors present in the earlier complexes in order to be able to promote Sxl regulation. This is the case. Thus, eIF4E is found in complexes containing the U1 snRNP protein U1-70K, the U1/U2 snRNP protein Snf, and the two U2AF proteins, U2AF38 and U2AF50. With the exception of the Snf protein bound to the U2 snRNP, all of these eIF4 associated factors are present in the early E or A complexes, but are displaced from the spliceosome together with the U1 and U4 snRNPs when the B complex is rearranged to form the activated B* complex. This would imply that eIF4E is already in place either before or at the time of B complex assembly. Arguing that eIF4E associates with these E/A components prior to the assembly of the B complex is the finding that eIF4E is also in complexes with both Sxl and Fl(2)d. Thus, even in this more demanding scenario for Sxl dependent splicing, eIF4E would be present at a time when it could directly impact the regulatory activities of Sxl and its co-factor Fl(2)d (Graham, 2011).

Taken together these observations would be consistent with a Sxl co-factor model. While further studies will be required to explain how eIF4E helps promote female specific processing, an intriguing possibility is suggested by the fact that hastening the nuclear export of msl-2 in females would favor the female splice (which is no splicing at all). Hence, one idea is that eIF4E binding to the pre-mRNA provides a mechanism for preventing the Sxl regulated splice sites from re-entering the splicing pathway, perhaps by constituting a 'signal' that blocks the assembly of new E/A complexes. A similar post-transcriptional mechanism could apply to female-specific splicing of the regulated Sxl exon2-exon3-exon4 cassette. The binding of eIF4E (and PABP) to incompletely processed Sxl transcripts after transcription has terminated in females would prevent the re-assembly of E/A complexes on the two male exon splice sites, and thus promote the formation of an A complex linking splicing factors assembled on the 5' splice sites of exons 2 and on the 3' splice site of exon 4 (Graham, 2011).

Ordered assembly of the dosage compensation complex

The roX1 and roX2 genes of Drosophila produce non-coding transcripts that localize to the X-chromosome. In spite of their lack of sequence similarity, they are redundant components of an RNA/protein complex that up-regulates the male X-chromosome, contributing to the equalization of X-linked gene expression between males and females. roX1 is detected at 2 h AEL, prior to formation of the complex, and is present in both sexes. Maternally provided MLE (Maleless) is required for roX1 stability. By contrast, roX2 is male-specific and is first observed at 6 h. Either roX transcript can support X-localization of the complex, but localization is delayed in roX1 mutants until roX2 expression. These results support a model for the ordered assembly of the complex in embryos (Meller, 2003).

Localization of the MSL proteins to the salivary gland X-chromosome requires formation of the intact complex, and hence the presence of all the MSL proteins. At least one roX RNA is similarly required for localization in larvae, and a prior study using roX1mb710 combined with an embryonic lethal deletion removing roX2 suggests that one roX gene is also required for localization in male embryos. However, this study used deletions also removed the largest subunit of RNA polymerase II (RPII215), which is less than 10 kb distal to roX2. The resulting disruption in zygotic gene expression may affect dosage compensation non-specifically. The requirement for roX transcripts is therefore determined in embryos mutated for each of the roX genes, but otherwise fully viable (Meller, 2003).

Previous studies have documented the male-specificity of MSL2 immunoreactivity in embryos, and have established that MSL2 can first be detected localizing to the X-chromosome at the end of the blastoderm stage, about 3 h AEL. X-localization, which is dependent on the presence of all of the MSL proteins, precedes the onset of dosage compensation as detected by enrichment of H4Ac16. X-chromosome localization produces a characteristic punctate MSL immunostaining pattern that is readily distinguished from non-localized immunoreactivity. As expected, anti-MSL2 antibodies labeled ~50% of gastrulating wild type embryos, and the appearance of punctate staining indicative of X-chromosome localization could be clearly observed in males older than 3 h AEL. Populations of embryos in which all males were of identical genotype were produced, thus simplifying the scoring of stained preparations. One-third of the developing embryos from these collections are male, and MSL2 immunoreactivity is detected in the anticipated proportion of embryos. Embryos in which MSL2 was detected with HRP were scored for developmental stage and localized staining. When males carry wild type roX genes, the appearance of punctate MSL2 immunoreactivity is first detected at 3 h and is observed in approximately one-third of older embryos. Df(1)52 is a deficiency of less than 30 kb that removes roX2 and several closely linked essential genes, including RPII215. Male embryos carrying the Df(1)52 chromosome produce MSL2, but its localization is delayed by >2 h. Viability can be restored to Df(1)52 flies by supplying cosmid [w+4Delta4.3], which carries all essential genes removed by the deletion but lacks roX2. [w+4Delta4.3] restores the normal timing of MSL2 localization in Df(1)52 males. The roX2 gene is therefore unnecessary for the initial formation of the MSL complex or its localization. However, disruption of development by deletion of the region surrounding roX2 delays the onset of MSL2 localization in a manner unrelated to the presence of the roX2 gene (Meller, 2003).

The timing of MSL2 localization suggests that roX1 could be necessary for initial X-localization of the dosage compensation complex. This was tested by determining the timing of MSL2 localization in roX1ex6 males. The roX1ex6 allele was created by an imprecise excision removing 1.4 kb from the 5' end of the gene. roX1 RNA is not detected in larvae or adults carrying this allele. Although MSL2 could be detected in one-third of the developing embryos from these collections, it did not appear in a punctate pattern until 7 h. Therefore, in roX1ex6 males there is a 4 h lag in localization of the complex, which now follows shortly after roX2 expression. X-chromosomes mutated for both roX genes were used to determine the ability of MSL2 to localize in the absence of any wild type roX RNA. MSL2 expression is detected in these embryos, but the strong foci normally observed upon localization of the MSL complex to the X-chromosome are not observed in roX1ex6 roX2- males. However, weak foci of MSL2 staining could be detected in some mutant embryos during germ band retraction, and these are more apparent when detected by immunofluorescence. Although differing from the more consistent and intense foci observed in wild type males, the presence of weak foci in roX1ex6 roX2- embryos indicates that MSL2 retains some ability to localize within the nucleus, even in the absence of a wild type roX gene. This is consistent with the observation that, although strikingly reduced, some X-localization of MSL2 is retained on polytene preparations from roX1ex6 roX2- males (Meller, 2003).

During larval and adult stages, roX1 is highly unstable in the absence of an intact dosage compensation complex. The embryonic transcription of roX1 precedes expression of MSL2 and formation of the complex, yet roX1 appears stable in embryos. In addition, until 10 h AEL roX1 transcripts are detected in female embryos, which lack MSL2. With the exception of MSL2, the MSL proteins are maternally provisioned and support the formation of the initial dosage compensation complexes. To test the hypothesis that one or more of these maternal proteins is responsible for roX1 stability, roX1 was examined in embryos from mothers homozygous for mutations in each of the msl genes. As anticipated, the maternal genotype with respect to the missense msl21 mutation has no effect. Early roX1 expression is also unchanged in embryos from females homozygous for a null allele of male-specific lethal 1 (msl1L60, a 2 kb deletion removing most of the coding region), male-specific lethal 3 (msl32), and males absent on first (mof1 and mof2, missense and nonsense mutations, respectively). By contrast, although an initial burst of roX1 expression is detected in blastoderm stage embryos produced by mothers homozygous for the nonsense mle1 mutation, roX1 disappears upon gastrulation. Between 4 and 5 h, roX1 can once more be detected. MLE is a member of the DExH family of helicases, and has RNA and DNA helicase activity in vitro. Interestingly, a similar lack of roX1 stability was detected in embryos expressing the mutated MLEDQIH protein that lacks helicase activity, indicating that this activity is required for roX1 stability. MLE has also been linked to the ability of the roX transcripts to travel from their sites of synthesis on the polytene X-chromosome. In embryos from mle1 mothers, spots of transcription within blastoderm nuclei are particularly apparent, and embryos displaying either one or two sites of synthesis per nucleus are readily observed. This suggests that MLE is also required to move roX1 from its site of synthesis in embryos. These results demonstrate that none of the MSL proteins are required for initial roX1 transcription, but maternal stores of MLE contribute to roX1 RNA stability in early embryos (Meller, 2003).

The occasional observation of weak foci of MSL2 staining in older male embryos carrying X-chromosomes mutated for both of the roX genes suggests either that roX RNA is not absolutely essential for localization of the dosage compensation complex, or that the roX1ex6 allele, a partial excision, retains some ability to produce functional transcripts, or that other genes produce transcripts which can partially support complex assembly and localization. All of these theories have been previously raised to account for a low level of developmentally delayed adult male escapers that are roX1ex6 roX2- (Meller, 2003 and references therein).

The findings of this study will support a model for the assembly of dosage compensation complexes during embryogenesis. roX1, highly expressed in all blastoderm stage embryos, is transcribed in advance of MSL2 translation and is likely to form an initial complex with MLE. It is possible that several of the MSL proteins must contact roX1 during assembly of the complex. In all, three members of the dosage compensation complex, MLE, MSL3 and MOF, have been reported to have RNA binding activity in vitro, or to be removed from the X-chromosome by RNase A digestion. With the exception of MSL2, all of the MSL proteins are present upon initial transcription of roX1 at 2 h AEL. The onset of dosage compensation has been linked to the male-limited production of MSL2 about 3 h AEL. This sequence of events suggests that MSL2 may complete a complex that is already organized by several RNA-binding proteins and the roX1 transcript. The proposed primary association between roX1 and MLE could reflect a need for the MLE helicase activity to disrupt incorrect base pairing or RNA/protein interactions preventing the large roX1 transcripts from correctly assembling with the other RNA-binding proteins of the dosage compensation complex (Meller, 2003).

Binding of MSL-2 to the X-chromosome

MSL-2 protein is associated with the X chromosome in males and not in females. The male salivary gland chromosome banding pattern for MSL-2 protein is identical to that of MSL-1. Since MSL-1 colocalizes with MLE and MSL-3, all four MSL proteins bind to identical cytological sites on the male X chromosome (Kelley, 1995).

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. A 110 bp sequence in roX2 has been identified 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 diminish 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. These 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 (Park, 2003).

A strong MSL binding site within the roX1 gene has been mapped by a combination of male-specific DNase I hypersensitivity (DHS) assays and transgenic deletion analyses. The binding site was narrowed down to ~200 bp, centrally located within the roX1 transcription unit. Transposons carrying the 200 bp roX1 DHS segment can attract MSL complexes to ectopic sites on autosomes, and are sufficient, at least as a nine-copy multimer, to occasionally nucleate limited spreading of MSL complexes into flanking chromatin. Based on this information, the roX2 gene was checked for male-specific DNase I hypersensitivity and evidence was found for a DHS region at the 3' end of the gene. Although this site is not as prominent as its roX1 counterpart, this 270 bp segment is sufficient to attract MSL complexes to autosomes in multiple independent transgenic lines. Hereafter, 'DHS' and 'MSL binding site' will be used synonymously. Binding of MSL complexes to the ectopic roX2 DHS in polytene chromosomes is robust, but unlike the roX1 multimer, no detectable MSL spreading was observed from roX2 DHS multimer constructs inserted at six different autosomal locations. MSL binding at the roX2 DHS is msl3 independent, which is a hallmark of roX genes and ~33 other proposed 'chromatin entry sites' on the X chromosome (Park, 2003).

Searches for elements necessary to target dosage compensation to the X chromosome have failed to yield cis-acting DNA sequences. Although it has been theoretically possible that MSL complexes might recognize some sequence-independent structural characteristic in chromatin entry sites, a broad domain of small islands of consensus sequences has been found to be important for MSL binding at roX genes. Computer-based comparisons of the roX1 and roX2 sequences failed to identify this region. Only after male-specific DNase I hypersensitive sites were identified within each gene and assayed for MSL binding activity in vivo did the consensus target sequence become apparent. Candidates for the additional ~33 postulated chromatin entry sites could not be identified by searching for sequences similar to the consensus MSL binding sequence in roX genes. This may be due to a failure of the search parameters. Alternatively, this result is consistent with a model in which roX genes are thought to be fundamentally different from other entry sites (Park, 2003).

The most prominent feature of the two roX genes is that they produce noncoding RNA components of MSL complexes. When either is mutant, the other is sufficient for MSL function, but males mutant for both roX RNAs cannot localize their MSL complexes properly. This shows that if any of the other postulated entry sites produce an RNA component of MSL complexes, it is not sufficient to replace these two key components. Several additional lines of evidence now point to the existence of only two roX genes, rather than several dozen. (1) SAGE analysis for sex-specific transcripts in adult heads easily found roX1 and roX2 but no other candidate male-specific noncoding RNAs. (2) The roX genes differ from the other entry sites in being highly MLE dependent. Finally, if the conserved DHS sequence is a signature for roX genes, it occurs only twice in the genome (Park, 2003).

The locations of MSL proteins bound to the X chromosome have not been precisely mapped in roX1 roX2 mutant males due to the poor morphology of the chromosomes, but they resemble the pattern of chromatin entry sites. If true, this would indicate that MSL proteins can bind weakly to numerous sites on the X, but have a strict RNA requirement to bind the roX1 and roX2 genes. Such a roX RNA dependence is consistent with an earlier report that roX genes differ from other entry sites in that they are not bound by any MSL subunit unless the MLE helicase is present. In previous studies, it was found that the cytological locations of chromatin entry sites, visualized as partial MSL complex binding, are very similar in msl3, mle, or mof mutants. While the vast majority of sites are common in the three different genotypes, the roX2 site at 10C is specifically absent in the mle mutant. When a roX1 cDNA transgene was assayed in isolation, it was also found to require mle+ for binding. A requirement for roX RNA in complexes that bind these sites would be consistent with this mle requirement, since MLE was previously shown to be critical for roX RNA inclusion in partial MSL complexes (Park, 2003).

Based on these results, it is proposed that roX RNAs can assemble into complexes locally at their sites of transcription. roX RNAs are unstable in the absence of MSL proteins, suggesting that complex assembly must occur rapidly for the RNA to escape destruction. Although it had been considered that the MSL proteins might be prepositioned at roX genes to facilitate capture of nascent transcripts, it was instead found that MSL proteins become competent to bind roX genes only after roX RNA is incorporated into the complex. Only a few other chromatin binding proteins have been shown to require an RNA component for chromatin interaction. HP1, a major constituent of heterochromatin, was shown to require RNA for chromatin association. Heterochromatin silencing in fission yeast may also require an RNA component. Likewise, in plants, dsRNA can lead to gene silencing not only by destruction of cognate RNA through a standard RNAi mechanism, but also by methylating the gene producing the offending RNA. In this case, a large multisubunit complex with an RNA helicase subunit is thought to use a short ~22 nt RNA component to search the genome for sequence homology. The roX DHS sequence is found in the middle of all roX1 RNAs and at the 3' end of some roX2 transcripts. An initial test to determine if the MSL complex uses this segment of roX RNA as a template to search the genome for homology ruled this out. However, the possibility that other short elements within roX RNA might play such a role has not been ruled out. Alternatively, the roX RNA may play some structural role in positioning the MSL proteins so that they can make specific DNA contacts (Park, 2003).

An important clue leading to the identification of the MSL binding sequence was the discovery of male-specific DNase I hypersensitive sites within roX1 and roX2. Nuclease sensitivity is often attributed to mobile or absent nucleosomes exposing DNA to nuclear proteins. The most conspicuous feature within the DHS is three copies of the GAGA sequence separated by conserved distances. Several Drosophila proteins are known to bind similar sequences, including the GAGA factor encoded by the trithorax-like gene and the Pipsqueak protein. GAGA factor is thought to keep the chromatin of regulatory regions, such as promoters and Polycomb response elements, in an accessible DHS configuration, possibly by targeting the nucleosome remodeling factor NURF to these sites. However, the action of GAGA factor is not limited to male flies and hence cannot explain a male-specific DHS. Although the possibility cannot be excluded that the altered chromatin structure in this region precedes MSL binding, it seems more likely that MSL binding to this sequence in males induces the more exposed structure. Simple protein-DNA contacts often cover 10-20 bp, so finding essential MSL recognition elements distributed over several turns of the DNA helix suggests a requirement for multiple factors to create a context for MSL binding. It is not known whether any of the five characterized MSL proteins directly contact DNA, but it is interesting to note that in the absence of roX RNA, most MSL proteins are lost from the X and are instead ectopically bound to centric heterochromatin. Satellite IV sequences are located in the centric heterochromatin, making up over 1% of the diploid genome. It consists of the sequence (AAGAGAG)n, which resembles conserved elements in the roX DHS (Park, 2003).

Why does MSL binding at roX genes appear to differ from binding of the many MSL targets on the X chromosome? Two functions seem possible. (1) Although the isolated roX2 DHS transgenes do not support ectopic MSL spreading over flanking autosomal chromatin, in the context of complete roX genes, high-affinity MSL binding might facilitate epigenetic MSL spreading. (2) Little is known about the transcriptional control of the roX genes or how dosage compensation causes only a 2-fold upregulation of X-linked genes. Perhaps bound MSL complex contributes to regulation of roX RNA transcription, to provide a precise level of MSL complexes for hypertranscription of the X chromosome (Park, 2003).

The Drosophila GAGA factor is required for dosage compensation in males and for the formation of the male-specific-lethal complex chromatin entry site at 12DE

Drosophila males have one X chromosome, while females have two. To compensate for the resulting disparity in X-linked gene expression between the two sexes, most genes from the male X chromosome are hyperactivated by a special dosage compensation system. Dosage compensation is achieved by a complex of at least six proteins and two noncoding RNAs that specifically associate with the male X. A central question deals with how the X chromosome is recognized. According to a current model, complexes initially assemble at ~35 chromatin entry sites on the X and then spread bidirectionally along the chromosome where they occupy hundreds of sites. This study shows that mutations in Trithorax-like (Trl) lead to the loss of a single chromatin entry site on the X at 12DE, male lethality, and mislocalization of dosage compensation complexes (Greenberg, 2004).

Assembly of the dosage compensation complex depends most critically upon Msl1 and Msl2 and these two MSL proteins may provide some type of scaffold for recruiting/stabilizing other components of the complex. These other components include the two noncoding RNAs, roX1 and roX2, which have been implicated in targeting the complex to entry sites on the X chromosome. In addition, the chromatin-modifying enzymes themselves, Mof-1, a histone acetylase, and JIL-1, a tandem histone kinase, the putative helicase, Mle, and the Msl3 protein, are believed to associate with the complex through specific interactions. All but one of these factors appears to function primarily, if not exclusively, in the MSL dosage compensation system. The exception is the JIL-1 kinase that is required not only for proper dosage compensation, but also for other aspects of transcriptional regulation that are vital to both sexes. It would be reasonable to anticipate that other factors like JIL-1 that have crucial activities in dosage compensation, while at the same time functioning in other processes that are important for both sexes, will be identified. This seems to be true for the GAGA factor that is encoded by the Trl gene (Greenberg, 2004).

The importance of the GAGA factor in many aspects of gene regulation and chromatin dynamics has been extensively documented. Evidence presented in this study indicates that GAGA also has a more specialized role in X chromosome dosage compensation. (1) Heteroallelic combinations of weak and strong Trl mutations have much greater effects on male than on female viability. In fact, the differences in the viability of the sexes in the two heteroallelic Trl mutant combinations tested in this study are equivalent to, if not more pronounced than, those reported for mutations in jil-1. (2) As might be expected if the functioning of the dosage compensation system is compromised when Trl function is impaired, the male lethal effects of the two heteroallelic Trl mutant combinations are found to be exacerbated by reductions in the dose of either msl1 or msl2. Moreover, although the hypomorphic Trl13C allele by itself exhibits no sex-specific lethality when homozygous, male-specific lethality can be induced by reducing the dose of the MSL complex. Conversely, increasing the level of the Msl2 gene product using an hsp83 promoter to drive the expression of an msl2 cDNA partially rescues males carrying Trl mutations (Greenberg, 2004).

Although Trl appears to function primarily in chromatin remodeling rather than as a dedicated component of the transcriptional machinery, it is involved in the expression of a very large and diverse array of genes. Since males must upregulate transcription of X-linked genes to achieve the same level of expression as females, it would be reasonable to suppose that males are likely to be much more dependent upon the proper functioning of the general transcriptional machinery than are females. Accordingly, any reduction in the activity of a factor critical for transcription would be expected to have considerably more deleterious effects on males than on females. If this idea is correct, then the male-specific lethality of Trl mutations could simply be due to a decline in the overall activity or efficiency of the transcriptional machinery rather than to an effect specific to the process of dosage compensation itself. This 'impaired transcription' model would also explain why the male-specific lethal effects of Trl mutations are enhanced by a reduction in the dose of the msl genes and suppressed by increasing the dose of Msl2. Of course, this model predicts that hypomorphic mutations in components of the transcriptional apparatus should also exhibit male-specific lethality like mutations in Trl. Although some alleles of TAF250 do cause preferential male lethality, there is no evidence that compromising the activity of other general transcription factors affects males more than females. In fact, none of the many hypomorphic mutations in the gene coding for the 140-kD subunit of RNA polymerase II give rise to male lethality, and neither do mutations in the small subunit of TFIIA. An additional problem with the 'impaired transcription' model is that it would not account for the finding that Trl mutations enhance the female lethal effects of the hsp83:msl2 transgene. The opposite result would be expected, namely that Trl mutations would suppress the female lethal effects of ectopic Msl2 protein (Greenberg, 2004).

An alternative, more plausible explanation for the effects of Trl mutations on male viability is that the GAGA factor plays some important role in the functioning or activity of the msl-dependent dosage compensation system. In addition to accounting for both the male-specific lethality of Trl mutations and the genetic interactions between Trl and msl-complex genes, this suggestion would help explain two other findings. (1) Abnormalities in the distribution of the MSL complexes are observed in polytene chromosomes from Trl mutant males. These abnormalities include the presence of at least one ectopic site on the X chromosome and an increase in the number of autosomal sites. This redistribution of MSL complexes argues that the GAGA factor is important for correctly targeting the dosage compensation machinery to the X chromosome. A similar although more dramatic MSL redistribution is when roX1 and roX2 are simultaneously deleted in males. (2) One of the ~35 chromatin entry sites on the X chromosome, at 12DE, is found to be missing in Trl mutants. Unlike any of the other chromatin entry sites observed in polytene chromosomes, GAGA is localized to the 12DE site. This finding argues that the GAGA factor is important in the formation/maintenance of this particular chromatin entry site. Neither of these effects on the chromosomal association of MSL complexes would be explained by a model in which the male-specific lethality of Trl mutations is due to some general reduction in the activity of the transcriptional machinery (Greenberg, 2004).

While it would be reasonable to propose that there is a direct connection between the defects in the chromosomal association of Msl complexes and male lethality, the precise mechanism is not entirely clear. One possibility is that male lethality is due to the loss of the 12DE entry site. Supporting this idea, MSL complexes formed at ectopic entry sites on the autosomes usually spread only limited distances. This also appears to be true on the X chromosome. It is thus possible that the two entry sites flanking 12DE, at 12C and 12F, would be unable to compensate completely for the loss of the 12DE site. As a consequence, insufficient levels of the MSL complex would be recruited into the 12C-12F interval in Trl mutant males to fully upregulate gene expression, and this might result in male lethality (Greenberg, 2004).

Although the loss of the 12DE entry might significantly impair the upregulation of genes in the 12C-F interval, there are at least two potential complications with this simple model. (1) It seems unlikely that a reduction in the level of expression of genes in the 12C-F interval would in itself be sufficient to cause male lethality. Unless this chromosomal interval contains genes specifically required for male viability (e.g., encoding components of the dosage compensation machinery), this model would predict that this same interval is haplo-insufficient in females. However, there is no indication that deletions in this chromosomal interval have significant effects on female viability. (2) While the 12DE entry site is absent (in Trl; msl3 mutants), no obvious perturbation is seen in the distribution of MSL complexes in this region of the X chromosome in Trl mutants that are wild type for the MSL genes. One explanation for this discrepancy is that defects in MSL-complex distribution in the vicinity of the 12DE site are obscured because the recruitment and spreading of complexes is much more robust in polytene chromosomes (which consist of hundreds of chromosomes whose sequences are aligned in precise register) than in chromosomes from polyploid or diploid nuclei. In fact, in polytene chromosomes MSL complexes can spread from ectopic entry sites on the autosomes not only in cis but also in trans and can even skip over large chromosomal segments (Greenberg, 2004).

This simple model would also not explain why MSL complexes in polytenes of Trl mutants localize to many ectopic sites on the autosomes. The presence of these autosomal complexes indicates that there must be some defect in the loading of complexes onto the X chromosome. Since no obvious reduction is seen in the amount of complex in the 12C-F interval, it seems unlikely that the loss of the 12DE entry site alone could account for the presence of the autosomal complexes. Instead, this would suggest that the GAGA factor may be important in the loading or spreading of complexes from a number of entry sites located elsewhere on the X in addition to the 12DE entry site. In this respect, it is notable that the GAGA factor binds in close proximity to five MSL-complex entry sites, including roX1. If GAGA is important in the loading or spreading of complexes from a number of 'Trl-dependent' entry sites in addition to 12DE, the male lethal effects of the Trl would be explained by the cumulative effects of a reduction in the expression of genes located in several different chromosomal regions rather than in just the 12C-F interval (Greenberg, 2004).

Sex lethal and dosage compensation

Some aspects of dosage compensation are not carried out by Male-specific-lethals, including MSL-2. Early runt dosage compensation is directed by the product of the early promoter of Sex lethal. Thus the early transcripts of Sex lethal have a role in addition to splicing, that is, in directing the early stages of dosage compensation. runt dosage compensation is a consequence of early Sex lethal expression in females. Since MSL-1 and MSL-2 begin to associate with the X chromosome during the cellular blastoderm, it is likely that MSL-independent compensation of genes such as runt and MSL-mediated compensation of other early-acting X-linked genes could either be separated by a very short developmental period or could occur simultaneously. The mechanism of Sex lethal directed early dosage compensation is unknown (McDowell, 1996 and Bernstein, 1994).

The inhibition of male-specific lethal-2 (msl-2) mRNA translation in female flies is essential for X chromosome dosage compensation in Drosophila melanogaster. Translational repression of msl-2 requires Sex-lethal (Sxl) binding to uridine-rich sequences in both the 5' and 3' untranslated regions (UTRs) of the message. The msl-2 mRNA sequence elements that are important for regulation by Sxl have been delineated and functionally critical sequences have been identified adjacent to regulatory Sxl binding sites. Sxl inhibits translation initiation and prevents the stable association of the 40S ribosomal subunit with the mRNA in a manner that does not require the presence of a cap structure at the 5' end of the mRNA. These results elucidate a critical regulatory step for dosage compensation in Drosophila melanogaster (Gebauer, 2003).

Following the delineation of the mRNA elements that contribute to or are dispensable for the control of Msl-2 synthesis by Sxl, a 'minimal mRNA' (WTS) was generated that recapitulates the critical features of msl-2 mRNA regulation. The stepwise substitution of the 3992 nt msl-2 mRNA by the much shorter WTS mRNA (336 nt) proved critical to the success of the ribosome assembly assays because only this smaller mRNA yields a sufficiently high resolution between the different translation initiation intermediates in sucrose gradient assays (Gebauer, 2003).

The specific inhibition of 80S ribosomal complex formation in the presence of the translation elongation inhibitor cycloheximide shows that Sxl inhibits the initiation step of translation. This result does not exclude the possibility that an additional postinitiation step could also be affected. Importantly, the assembly of early initiation intermediates carrying only the small ribosomal subunit is clearly inhibited by Sxl. This observation is consistent with two possible scenarios: the regulatory mechanism may inhibit the initial, cap-mediated recruitment of the small ribosomal subunit to the mRNA, or it may interfere with the scanning of a recruited 43S complex to the translation initiation codon. The latter mechanism could prevent the stable association of the 43S complex with the mRNA because scanning intermediates are thought to readily dissociate from the mRNA (Gebauer, 2003).

Sxl must bind to both the 5' and 3' UTRs of msl-2 mRNA to inhibit translation efficiently. This requirement distinguishes Sxl from most other regulators, which inhibit translation by binding to only one of the untranslated regions, commonly the 3' UTR. The 3' UTR of msl-2 contributes a function in addition to Sxl binding. The critical sequence elements are located in close proximity to the 3' UTR Sxl binding sites E and F. It is suggested that Sxl and the regulatory sequences flanking its 3' UTR binding sites may cooperate in the recruitment of translational corepressors (Gebauer, 2003).

Both Sxl and the iron regulatory proteins (IRP) inhibit the stable association of the 40S ribosomal subunit with their target mRNAs. However, the regulatory mechanisms used by IRP and Sxl differ in several aspects. In contrast to Sxl, IRP must bind to a cap-proximal hairpin in ferritin mRNA to effectively block the binding of the 43S complex to the mRNA. Moreover, the IRP mechanism does not require contributions from the 3' UTR, as does Sxl. Furthermore, Sxl binding is not sufficient for translation inhibition of msl-2 mRNA, and a simple steric hindrance mechanism is hence unlikely to explain Sxl function. By contrast, the mere occupancy of a cap-proximal hairpin by a high-affinity RNA binding protein suffices for the IRP mechanism of translational control (Gebauer, 2003).

Translational repression of msl-2 mRNA by Sxl is independent of the presence of a cap structure and a poly(A) tail. This feature differs from other translational regulators such as CPEB. Because both the cap structure and the poly(A) tail play critical roles in promoting the recruitment of the small ribosomal subunit to mRNAs, these findings raise the interesting question of how Sxl regulates the interaction between the mRNA and the small ribosomal subunit independently of both of these mRNA end modifications. An intriguing possibility is that Sxl targets the scanning step of translation rather than the initial recruitment of the 43S complex. Translation inhibition may occur by formation of a higher order complex of repressors bound to the 5' and 3' UTRs of msl-2 mRNA. It is also possible that Sxl/corepressor interactions create a regulatory surface that targets specific translation initiation factors. Future experiments will aim to identify the responsible components and molecular interactions (Gebauer, 2003).

Local spreading of MSL complexes from roX genes on the Drosophila X chromosome

MSL proteins and noncoding roX RNAs form complexes to up-regulate hundreds of genes on the Drosophila male X chromosome, and make X-linked gene expression equal in males and females. Altering the ratio of MSL proteins to roX RNA dramatically changes X-chromosome morphology. In protein excess, the MSL complex concentrates near sites of roX transcription and is depleted elsewhere. These results support a model for distribution of MSL complexes, in which local spreading in cis from roX genes is balanced with diffusion of soluble complexes in trans. When overexpressed, MSL proteins can recognize the X chromosome, modify histones, and partially restore male viability even in the absence of roX RNAs. Thus, the protein components can carry out all essential functions of dosage compensation, but roX RNAs facilitate the correct targeting of MSL complexes, in part by nucleation of spreading from their sites of synthesis (Oh, 2003).

To date, all evidence for cis spreading comes from autosomal roX transgenes. MSL complexes do spread locally from the endogenous roX genes on the X chromosome, the natural target of dosage compensation. Wild-type males require a balance of MSL proteins and roX RNAs to evenly distribute MSL complexes both locally and at a distance along the X chromosome. When the amounts of MSL1 and MSL2, thought to be the limiting proteins, are artificially increased, MSL complexes spread predominantly over a local segment of the X chromosome surrounding a roX gene. More remote regions bind little MSL complex. This dramatically alters the morphology of polytene X chromosomes. Surprisingly, overexpressing MSL1 and MSL2 partially restores viability to males lacking roX RNA. This indicates that the MSL proteins have intrinsic affinity for the X chromosome that is enhanced or stabilized in wild-type males by the roX RNAs (Oh, 2003).

Earlier observations that the MSL complex could spread in cis from an autosomal roX transgene have lead to speculation that complexes normally spread from the endogenous roX loci on the X chromosome to paint the entire chromosome. However, the initial characterization of roX1 clearly demonstrates that soluble MSL complexes could diffuse between chromosomes. More recent work reveals that the ability of the MSL complex to spread from a site of roX transcription, or diffuse away, is highly sensitive to the balance between MSL proteins and roX transcripts in the nucleus. This study demonstrates that the wild-type pattern of MSL complexes along the male X chromosome is the result of a delicate interplay between two targeting strategies. Local spreading from roX loci operates in parallel with a second route where soluble MSL complexes diffuse and reattach to distant segments of the X chromosome. The proportion of MSL complexes entering each pathway can be altered by manipulating the amount of MSL proteins or roX RNAs present. It is speculated that the underlying mechanism controlling these two outcomes rests on how efficiently MSL subunits can assemble into functional complexes (Oh, 2003).

The Drosophila roX1 RNA gene can overcome silent chromatin by recruiting the male-specific lethal dosage compensation complex

The Drosophila MSL complex consists of at least six proteins and two noncoding roX RNAs that mediate dosage compensation. It acts to remodel the male's X chromatin by covalently modifying the amino terminal tails of histones. The roX1 and roX2 genes are thought to be nucleation sites for assembly and spreading of MSL complexes into surrounding chromatin where they roughly double the rates of transcription. Many transgenic stocks have been generated in which the roX1 gene was moved from its normal location on the X to new autosomal sites. Approximately 10% of such lines display unusual sexually dimorphic expression patterns of the transgene's mini-white eye-color marker. Males often displayed striking mosaic pigmentation patterns similar to those seen in position-effect variegation and yet most inserts were in euchromatic locations. In many of these stocks, female mini-white expression was very low or absent. The male-specific activation of mini-white depends upon the MSL complex. It is proposed that these transgenes are inserted in several different types of repressive chromatin environments that inhibit mini-white expression. Males are able to overcome this silencing through the action of the MSL complex spreading from the roX1 gene and remodeling the local chromatin to allow transcription. The potency with which an ectopic MSL complex overcomes silent chromatin suggests that its normal action on the X must be under strict regulation (Kelley, 2003).

The characteristics of mini-white marked transgenes are unique to the situation described in this paper. It is inferred that the roX1 gene is responsible for this unusual behavior. It is proposed that male-specific pigmented sectors reported in this study are a visible manifestation of ectopic dosage compensation occurring around autosomal GMroX1 transgenes, which landed in repressive chromatin environments. The MSL complex is active by midembryogenesis and stays on throughout development. The mosaic eye patterns seen here suggest that the MSL complex spreads a more 'open' chromatin architecture during embryonic development when the primordial eye disc has a small number of cells. This chromatin packaging competes with uncharacterized silencing factors and can be inherited through many mitotic divisions so that large clones of cells in the adult eye share the same on/off state. These results are similar to those reported for a w+ transgene lacking roX1 inserted at the heterochromatic base of the X. Such females suffered PEV but males had solid red eyes presumably due to the MSL complex spreading from flanking chromatin (Kelley, 2003).

This model is supported by the finding that ectopic expression of MSL2 in females is sufficient to overcome silencing. Thus the MSL complex is responsible for the activation, but must be targeted to the transgene. This could happen either by MSL proteins assembling on nascent roX1 transcripts or by mature MSL complex being recruited by DNA sequences within the roX1 gene (Kelley, 2003).

An alternative interpretation of dosage compensation in Drosophila, known as the inverse model, postulates that the MSL proteins normally have two key functions in wild-type males. (1) They sequester the MOF histone H4 acetyltransferase away from the autosomes by targeting it to the X chromosome. (2) The MSLs block overexpression of X-linked genes that might otherwise result from MOF-mediated nucleosome acetylation. In this model, histone acetylation by MOF has little effect on gene transcription in the wild-type male X chromosome, but a significant toxic effect in mle mutant males where MOF escapes from the X and hyperacetylates the entire genome. In contrast to expectations of the inverse model, histone H4 acetylation caused by MOF within complete MSL complexes is found to be a potent activator in wild-type males (Kelley, 2003).

The MSL complex can overcome different mechanisms of silencing. The GMroX1-80C line is subject to severe PEV. The surprising aspect of this insert is the strength of silencing in females where neither the presence of a Y chromosome nor the presence of Su(var) mutations allowed any mini-white expression. Yet in males, the MSL complex can spread from roX1 sequences through centric heterochromatin and into the euchromatic proximal arms of 3L and 3R, activating mini-white along the way. The insertion in the iroquois cluster demonstrates that the MSL complex can overcome Polycomb-mediated silencing in the ventral half of the eye. The MSL complex can also overcome silencing due to insertion in dispersed repeats (75C and 84E) (Kelley, 2003).

The insertion in one of the ~110 tandem copies of the histone gene cluster at 39DE is particularly interesting. Mini-white is sometimes poorly expressed within long repeats. A second explanation rests on close proximity of the histone cluster to centric heterochromatin. A high histone gene copy number had been thought necessary to supply cells with enough histone proteins during each replication cycle. However, the discovery that Drosophila hawaiiensis carries 20 copies of the histone genes and D. hydei carries only 5-10 copies called for a new explanation. In species with low copy number, the histone genes are located far from heterochromatin. However, the histone genes in D. melanogaster are adjacent to centric heterochromatin. Selection for increased copy number may have compensated for low expression per gene copy (Kelley, 2003).

In summary, the MSL complex is a versatile chromatin-remodeling machine able to act on many different chromatin substrates. This might be expected for a regulator that must normally act on several thousand unrelated genes expressed in different tissues throughout development. However, this behavior raises the question of how males can keep appropriate segments of the X silent in tissues in which a gene product is not needed and might even be harmful. Presumably the MSL complex is tightly regulated on the X so that only active genes are upregulated. Others have shown that the MSL complex can radically alter the morphology of the X when certain chromatin-modifying factors, such as ISWI or NURF, are mutated. Perhaps such proteins normally restrict the action of the MSL complex. In addition, chromosomes may be organized into loops or domains of activity in vivo so that the MSL complex can respect domain boundaries if it spreads along the chromosome. The roX1 transgenes studied here may subvert such regulation by placing a MSL-binding/assembly site internal to domain boundary elements (Kelley, 2003).

Because of its extreme sensitivity to a chromatin environment, mini-white-based P elements are being replaced with yellow+ or PAX6-EGFP marked vectors for mutagenesis screens. However, the GMroX1 transposon may be useful in screens to assay for repressive chromatin environments. Simply comparing the eye color of brothers and sisters from the same stock would quickly identify euchromatic inserts subject to subtle chromatin effects (Kelley, 2003).

Multiple classes of MSL binding sites target dosage compensation to the X chromosome of Drosophila

MSL complexes bind hundreds of sites along the single male X chromosome to achieve dosage compensation in Drosophila. It has been proposed that 35 'high-affinity' or 'chromatin entry' sites (CES) might nucleate spreading of MSL complexes in cis to paint the X chromosome. This was based on analysis of the first characterized sites roX1 and roX2. roX transgenes attract MSL complex to autosomal locations where MSL complexes can spread long distances into flanking chromatin. roX1 and roX2 also produce noncoding RNA components of the complex. A third site has been identified from the 18D10 region of the X chromosome. Like roX genes, 18D binds full and partial MSL complexes in vivo and encompasses a male-specific DNase I hypersensitive site (DHS). Unlike roX genes, the 510 bp 18D site is apparently not transcribed and shows high affinity for MSL complex and spreading only as a multimer. While mapping 18D, MSL binding to X cosmids that do not carry one of the 35 high-affinity sites was discovered. Based on additional analyses of chromosomal transpositions, it is concluded that spreading in cis from the roX genes or the 35 originally proposed 'entry sites' cannot be the sole mechanism for MSL targeting to the X chromosome (Oh, 2004).

To explore a model in which 35 high-affinity sites, including roX1 and roX2, initiate spreading of MSL complexes into flanking chromatin, an additional high-affinity site has been characterized at 18D10. An overlapping cosmid contig around 18D10 was constructed, transgenic lines for each of the cosmids were created, and they were tested for MSL binding at their new sites of insertion. In an msl3- genetic background in which the high-affinity sites are most easily monitored, only 18Dcos5 lines show a strong MSL signal, comparable to the endogenous 18D10 region on the X chromosome. However, all three of the 18D cosmids tested were able to recruit MSL complex in wild-type males. 18Dcos3 and 18Dcos4 do not contain a high-affinity site, but nevertheless MSL complex was recruited to their insertion sites. This result demonstrates that spreading in cis from high-affinity sites is not the sole mechanism for attracting MSL complexes to the X chromosome (Oh, 2004).

To determine whether the high-affinity site in 18Dcos5 has properties similar to roX genes, transgenic lines were assayed for MSL spreading. In wild-type, 18Dcos5 transgenes showed stronger MSL binding than 18Dcos3 or 18Dcos4 and infrequently (<5%) showed very limited spreading (usually two bands). The spreading frequency at one location (56C) increased up to 80% in roX1- or roX2- backgrounds. This behavior is typical of autosomal roX transgenes, which show markedly higher spreading frequency when the number of endogenous roX genes is decreased. Thus, 18D transgenes may face competition for MSL complexes from endogenous roX genes and perhaps other high-affinity sites on the X chromosome (Oh, 2004).

In the absence of roX RNA, MSL proteins bind to several regions on the X chromosome, which may be analogous to the previously mapped high-affinity sites. To see if 18Dcos5 recruits MSL proteins without roX RNA, polytene chromosome immunostaining was performed in roX-deficient male larvae, which showed consistent MSL protein binding to 18Dcos5 transgenes inserted at cytological positions 56C and 60C but not to 18Dcos3 or 18Dcos4 transgenes. This result demonstrates that the MSL binding site located within 18Dcos5 is different from the sites within roX genes, which require roX RNAs for binding (Oh, 2004).

To narrow down the genes or sequences functioning as a high-affinity site around 18D10, five overlapping subfragments from 18Dcos5 were tested for MSL binding in vivo. 18D-5B and 18D-5D showed significant binding and some modest spreading (<1%) in wild-type males. However, in the absence of MSL3, only 18D-5B showed MSL binding, which was significantly weaker than binding to the full-length 18Dcos5. This result indicates that 18D-5B (8.8 Kb) contains a high-affinity MSL binding site. Since 18D-5A did not interact with MSL complex, it seems that the 3' region of 18D-5B contains the binding activity. To test this, three more constructs, 18D-5B1 (4.5 Kb), 18D-5B2 (2.6 Kb), and 18D-5B3 (2.1 Kb), containing the 3' end of 18D-5B (8.8 Kb) were tested for MSL complex binding in transgenic flies. Although all three fragments still displayed the ability to recruit MSL complexes in wild-type males, they lost binding to partial MSL complexes lacking MSL3, suggesting the possibility that multiple sites are required for interaction with incomplete MSL complexes. However, the 18D-5B3 (2.1 Kb) subclone still showed modest but rare spreading (Oh, 2004).

Previously, a series of short blocks of conserved sequences associated with MSL binding to the roX1 and roX2 genes were identified. However, this configuration of consensus sequences was not found at other locations in the genome. The core of the consensus sequence within the roX genes, GAGAG and CTCTC, was not present within subclone 18D-5B, confirming that this MSL binding site is distinct. MSL binding sites in roX genes are coincident with male-specific DNase I hypersensitive sites (DHS). Therefore, 18D-5B was assayed for DNase I hypersensitivity and a male-specific site was found in the 3' part of the fragment, consistent with the location of MSL binding based on transgenic studies. To confirm that this male-specific DHS is caused by direct MSL complex interaction, the region was analyzed by chromatin immunoprecipitation (ChIP) using anti-MSL2 antibodies and salivary gland tissue. The larvae utilized had low MSL2 expression, in which complexes bind only to high-affinity sites and also carry an extra copy of 18D10 (18Dcos5 at 56C). To evaluate the ChIP experiment, roX1 (positive control) and pka (negative control) primers were used to measure enrichment of roX1 in the immunoprecipitated DNA. To locate MSL binding within 18D, subfragments of the 8.8 Kb 18D-5B subclone were analyzed by Southern blotting with probe prepared from the α-MSL2 IP. Compared to the control IP, the 3' end of 18D-5B was enriched in the α-MSL2 immunoprecipitation. This was further narrowed down to smaller subfragments, showing that MSL binding overlaps the male-specific DHS. The binding activity maps to intergenic DNA 3' of CG12237, whose function is unknown. These results show that the MSL complex interacts with 18D10 and modifies its chromatin structure as it does in the roX genes. However, unlike the roX genes, transcription of the 18D MSL binding site was not detected by Northern or RT-PCR using 18D DHS probes and primers (Oh, 2004).

To determine the importance of the male-specific DHS from 18D10 in MSL complex recruitment, a transgenic deletion analysis was performed. 128 bp (ΔS) or 618 bp (ΔL) of the DHS region were deleted from the 18D-5B3 transgene (2.1 Kb). All three ΔL lines and three of the four ΔS lines completely lost the ability to recruit MSL complexes, and the remaining ΔS transgene showed only a very weak signal. These data demonstrate that the 128 bp region deleted in the ΔS transgene, and perhaps additional elements in the ΔL 618 bp region, contain important cis-elements for MSL complex recruitment (Oh, 2004).

Previously it was shown that 200 bp of a roX DHS is sufficient for recruitment of the MSL complex even in the absence of MSL3. In addition, when the roX1 DHS is multimerized, it can show limited spreading into flanking chromatin. To determine whether the 18D10 DHS carries similar activities, at least four independent insertions of the following transgenes were analyzed: 510 bp (18D10-DHS-L), 271 bp (18D10-DHS-S), four tandem repeats of 510 bp (18D10-DHS-L4mer), or seven tandem repeats of 271 bp (18D10-DHS-S7mer). Unlike the roX1 DHS, 18D monomers of 510 bp and 271 bp were extremely weak for MSL complex binding. Seven tandem repeats of the 271 bp segment also showed very weak MSL complex recruiting activity. However, the transgene with four tandem copies of 510 bp showed a strong signal for MSL1 staining. Even in the absence of MSL3, the 18D10-DHS-L4mer was sufficient to recruit the incomplete MSL complex, with very strong and consistent signals of MSL1 staining, even stronger than that of 18D-5B (8.8 Kb) in an msl3- background. However, unlike 18Dcos5, the 18D10-L4mer did not recruit MSL proteins without roX RNA. These results indicate that a 510 bp 18D10 fragment carries key sequences for MSL complex targeting. However, despite the strong MSL binding observed, no sequence motifs common to the roX genes or otherwise enriched on the X chromosomes were detected (Oh, 2004).

Given the surprising finding that all 18D cosmids tested were able to recruit wild-type MSL complexes, analysis was extended to other regions of the X chromosome. Large X to autosome transpositions have been shown to retain both the ability to dosage compensate, as well as the characteristic diffuse appearance of the male X chromosome. Four fly lines containing large X-ray-induced X to autosome transpositions obtained from the Drosophila stock center were analyzed. Males hemizygous for the inserted chromosome segment contain an unpaired portion of the polytene chromosome protruding from the wild-type autosome. As observed in line Tp (1;3) rb+71g, the region of transposed X chromosome appears as wide as the paired autosomes that flank the insertion, suggesting that the transposed section of chromosome adopts a less compact chromatin structure similar to that of the intact male X chromosome. All four X to A transposition stocks that were tested showed MSL binding within the transposed fragments, including two that lack a mapped high-affinity site. These data provide further evidence that cis-acting sequences are present in large pieces of the X chromosome that enable them to recruit MSL complex regardless of whether they contain a putative chromatin entry site (Oh, 2004).

In contrast to the X to autosome transposition flies, an autosome to X transposition stock lacked MSL staining of a region of the third chromosome that was transposed to the X. MSL1 protein was detected at sites flanking the break points of the transposition, but no staining was observed within the transposed section of the third chromosome even though there is a nearby high-affinity site (4C12-16). These data indicate that linking autosomal sequences to the X chromosome is not sufficient to allow recruitment of the MSL complex, contradicting a key prediction of a simple spreading model (Oh, 2004).

To test the requirement of entry sites for recruitment of MSL complexes to smaller fragments (<39 Kb), transgenic lines harboring X-derived DNA fragments variable in size from 39 Kb to 0.3 Kb were immunostained. Interestingly, each cosmid showed strong MSL binding in wild-type, and in at least one case (cos13E) there was even some apparent spreading. These results demonstrate that even without a nearby high-affinity site, some X-derived fragments contain cis-acting sequences for MSL complex binding. These results raise the possibility that spreading in cis from the two roX genes may not be the major mechanism for MSL binding to the X chromosome (Oh, 2004).

The focus in this study was to identify additional putative chromatin entry sites and understand how they attract MSL complexes and whether they, like the roX genes, can nucleate MSL spreading. The site from cytological location 18D10 was sucessfully isolated, and its primary sequence, chromatin structure, MSL interaction, and ability to nucleate spreading was analyzed. The behavior of this site was significantly different from the behavior of roX genes in several ways. The current data can be interpreted in the following framework. Perhaps there are diverse DNA recognition elements on the X chromosome that have different affinities for MSL complex: high, intermediate, or weak. High-affinity cis-elements, such as within the roX genes, do not require additional cis-elements for recruiting MSL complexes and might be involved in multifold gene activation instead of 2-fold hypertranscription. This interaction might be strengthened by roX RNA. An intermediate-affinity cis-element, like the 18D10 site, might require additional intermediate- and/or weak-affinity elements for robust binding and would have the ability to attract partial MSL complexes with a minimal MSL1/MSL2 composition. Weak-affinity cis-elements might require interaction with several additional weak-affinity cis-elements, which might explain occasional autosomal MSL signals and how X fragments on the autosomes attract wild-type MSL complexes even without a CES (Oh, 2004).

The amino-terminal region of Drosophila MSL1 contains basic, glycine-rich, and leucine zipper-like motifs that promote X chromosome binding, self-association, and MSL2 binding, respectively

A version of MSL1 missing the first 84 amino acids with a FLAG tag at the amino end does not bind to the male X chromosome (Scott, 2000). Full-length MSL1 with an amino-terminal FLAG tag does bind to the male X chromosome, although binding is not strong. This indicates that the first 84 amino acids were important for X chromosome binding and that adding a FLAG tag at the amino end may interfer with binding. To determine if the amino-terminal domain is sufficient for X chromosome binding, transgenic Drosophila lines were made that expressed the domain with an HA epitope tag at the carboxyl end (MSL1NHA). The domain includes the conserved N-terminal basic region, the predicted coiled coil, and an acidic region (aa 179 to 186). It was predicted that this domain would be able to bind to the male X chromosome but only to the ~30 high-affinity sites. This is because the amino-terminal domain does not interact with MOF and MSL3, both of which are needed for the MSL1/MSL2 complex to bind to sites on the X chromosome other than the high-affinity sites (Gu, 1998, Palmer, 1994). However, it was found that the HA-tagged amino-terminal domain of MSL1 (MSL1NHA) bind to hundreds of sites on the male X chromosome. Identical results were obtained if MSL1NHA expression was controlled by either the strongly heat-inducible hsp70 promoter or the constitutive armadillo promoter. Further, it was found that with the hsp70 construct, basal-level expression at 25°C was sufficient to detect X chromosome binding of MSL1NHA. Heat shock treatment to overexpress MSL1NHA did not lead to a significant increase in binding to the autosomes, nor did it disrupt X chromosome binding by other components of the MSL complex. Since heat treatment was not necessary to detect X chromosome binding of MSL1NHA, all additional experiments in this study were performed with larvae raised at 25°C without heat shock. Surprisingly, daily heat-shock treatment of the progeny of an MSL1NHA line had little effect on male viability (85 male and 119 female progeny obtained), indicating that binding of MSL1NHA to the X chromosome did not significantly disrupt MSL complex activity. In contrast, overexpression of a truncated version of MSL1 missing the first 84 amino acids that does not bind to the X chromosome was lethal to males (Scott, 2000). Δ84HA, which is identical to MSL1NHA but lacks the first 84 amino acids, does not bind to the male X chromosome. The lack of binding could be because the Δ84HA protein lacks a nuclear localization sequence. However, staining of whole salivary glands showed that Δ84HA is localized to the nucleus. Thus, the first 84 amino acids of MSL1 appear to play an essential role in X chromosome binding (Li, 2005).

The observed binding of MSL1NHA to hundreds of sites on the male X chromosome could be because the domain recognizes all the sites or because it associates with the MSL complex bound to the X chromosome. To distinguish between these two possibilities, the appropriate crosses to generate larvae that carried the MSL1NHA transgene but lacked endogenous MSL1. In the absence of MSL1, none of the components of the MSL complex bound to the X chromosome. Since it can be difficult to obtain good-quality polytene chromosomes from dying msl1 mutant males, salivary glands were isolated from female larvae that constitutively expressed MSL2. In the absence of msl1, MSL1NHA bound to about 30 sites, which corresponded to the previously mapped high-affinity sites (Lyman, 1997). MSL2 colocalized with MSL1NHA to the high-affinity sites. MLE also colocalized to the high-affinity sites with MSL1NHA; however, MOF did not. The latter result was expected, since MOF binds to the carboxyl-terminal domain of MSL1 (Scott, 2000) and functional MOF is required for MSL complex binding to sites other than the high-affinity sites. In control sibling female larvae that were heterozygous for the msl1L60 null mutation, MSL1NHA bound to hundreds of sites on the X chromosomes. MSL2 and MOF colocalized with MSL1NHA. These results show the amino-terminal domain of MSL1 complexed with MSL2 can specifically recognize the high-affinity sites on the X chromosome. However, in the presence of native MSL complex, MSL1NHA binds to hundreds of sites, presumably via association with the complex (Li, 2005).

Since Δ84HA does not bind to the male X chromosome, three additional smaller deletion mutants were made to identify the region important for X chromosome binding. Like Δ84HA, Δ74HA did not bind to the male X chromosome. Δ50HA, however, bound very weakly to the male X chromosome in approximately 50% of the nuclei examined. In the other 50% of nuclei, no staining of the X chromosome with the anti-HA antibody could be detected above background levels. In contrast, Δ26HA bound more strongly to the X chromosome but with less intensity than MSL1NHA (Li, 2005).

Given that the binding of MSL1NHA to the X chromosome is restricted to the high-affinity sites in the absence of endogenous MSL1, it was next asked if Δ26HA could bind to the X chromosome in a msl1 null mutant background. It was found that there was no binding of Δ26HA to the X chromosomes in homozygous msl1L60 female larvae that expressed MSL2. This demonstrates that the first 26 amino acids of MSL1 are essential for binding to the high-affinity sites. This region contains several well-conserved basic and aromatic amino acid residues. To test the importance of some of these conserved amino acids in X chromosome binding, two mutant versions of MSL1NHA were made. In mut_bas1, three of the conserved basic amino acids, lysine 3, arginine 4, and lysine 6, were all replaced by alanine. In a wild-type genetic background, this mutant version of MSL1NHA bound to hundreds of sites on the male X chromosome. However, in the absence of endogenous MSL1, binding was restricted to only five of the high-affinity sites. Two of these sites mapped to the location of the roX genes, roX1 at 3F and roX2 at 10C. In the second mutation, mut_bas2, two of the conserved aromatic amino acids (phenylalanine 5 and tryptophan 7) were changed to alanine. This mutation did not appear to disrupt binding to the high-affinity sites in msl1L60 null female larvae that expressed MSL2. However, mut_bas2 bound to significantly more autosomal sites than MSL1NHA. Thus, it appears that three of the conserved basic amino acids are essential for binding to most of the high-affinity sites. In addition, two of the conserved aromatic amino acids appear to be important for distinguishing X from autosomes, that is, the specificity of binding (Li, 2005).

The binding of MSL1NHA to hundreds of sites on the male X chromosomes appears to be in part due to association with the native MSL complex. The observation that Δ26HA bound to these sites but Δ74HA did not indicated that the region between amino acids 26 and 74 is important for association with the MSL complex. This region is particularly rich in the amino acids glycine, proline, asparagine, and histidine in all Drosophila MSL1 proteins. Glycine-rich domains are a common feature of many proteins including RNA binding proteins and can mediate protein-protein interaction. The glycine-rich domain of the Drosophila Sex-lethal RNA binding protein, which is the master regulator of dosage compensation, promotes self-association. Therefore whether the MSL1 glycine-rich domain would facilitate MSL1 self-association was examined. It was found that MSL1 coimmunoprecipitates from whole-fly protein extracts with MSL1NHA and Δ26HA but not Δ84HA, Δ74HA, or Δ50HA. There was a small variation in immunoprecipitation efficiency of the HA-tagged proteins, which were also detected with the MSL1 antibody. However, this was not sufficient to account for the lack of coimmunoprecipitation of MSL1 with the more truncated versions of MSL1NHA. MSL2 was not required for MSL1 self-association, sicne protein extracts were prepared from adult females, which normally do not make MSL2 protein. Δ26HA did not coimmunoprecipitate with MSL3, showing the specificity of the interaction of Δ26HA with MSL1. Deletion of the first 84 amino acids did not, however, disrupt interaction with MSL2, confirming previous studies (Copps, 1998, Scott, 2000). Thus, MSL1NHA appears to interact with the native MSL complex via MSL1 self-association (Li, 2005).

It has been suggested that the predicted leucine zipper-like region of MSL1 may interact with an predicted amphipathic α-helix at the amino terminus of MSL2 to form a coiled-coil structure (Scott, 2000). Likely orthologs of MSL1 and MSL2 have been identified from invertebrate and vertebrate genome sequences. Amino acid sequence alignments of MSL1 and MSL2 orthologs showed a high degree of conservation of the predicted α-helical regions. Inspection of the alignments showed that both MSL1 and MSL2 proteins contained a highly conserved region that is largely apolar and precedes the coiled coil. For MSL1, a glutamine-rich spacer separated the apolar and coiled-coil regions. Alanine substitution mutations were made in the apolar, glutamine-rich, and leucine zipper-like regions of MSL1 to investigate the relative importance of these regions in dimerization with MSL2 (Li, 2005).

In vitro-translated [35S]methionine-labeled MSL1NHA coimmunoprecipitates with the FLAG-tagged amino-terminal domain of MSL2 (aa 1 to 193) (MSL2NFLAG) from transformed whole-fly extract. MSL1NHA does not coimmunoprecipitate with control extract prepared from untransformed wild-type flies. Immunoprecipitations were performed under stringent high-salt conditions (500 mM NaCl), and thus only specific interactions should be detected. This was confirmed by the lack of coimmunoprecipitation of the carboxyl-terminal domain of MSL1 (aa 705 to 1039) with MSL2NFLAG. A derivative of MSL1NHA with mutations in the apolar region (mut_apo) does not coimmunoprecipitate with MSL2NFLAG. In contrast, mutations in the glutamine-rich region (mut_QEQ) do not appear to disrupt the MSL1:MSL2 interaction. This cannot be due to differences in immunoprecipitation efficiency, since recovery of MSL2NFLAG was similar. Consistent with these in vitro binding results, mut_QEQ bind to hundreds of sites on the male X chromosome. Further, no binding of mut_apo to the male X chromosome could be detected. Thus, the apolar but not the glutamine-rich region of MSL1 appears to be important for interaction with MSL2 (Li, 2005). Dimerization of coiled-coil proteins is driven by interaction between apolar side chains in the a and d positions of the α-helix. The binding is enhanced by ionic interactions between charged amino acids in the e and g positions. Consequently alanine-substitution mutations were made in the a, d, e, and g positions in the leucine zipper-like motif that follows the glutamine-rich region. It was found that all of the mutant versions of MSL1NHA coimmunoprecipitate with MSL2NFLAG. However, there appeared to be significantly less coimmunoprecipitation of two of the mutations, mut_cc1 and mut_cc2, with MSL2NFLAG. The efficiency of immunoprecipitation of MSL2NFLAG was similar for all four coiled coil mutant preparations. These results suggest that the mut_cc1 and mut_cc2 alanine substitution mutations have weakened the interaction between MSL1 and MSL2 (Li, 2005).

Targeting determinants of dosage compensation in Drosophila

The dosage compensation complex (DCC) in Drosophila melanogasteris responsible for up-regulating transcription from the single male X chromosome to equal the transcription from the two X chromosomes in females. Visualization of the DCC, a large ribonucleoprotein complex, on male larval polytene chromosomes reveals that the complex binds selectively to many interbands on the X chromosome. The targeting of the DCC is thought to be in part determined by DNA sequences that are enriched on the X. So far, lack of knowledge about DCC binding sites has prevented the identification of sequence determinants. Only three binding sites have been identified to date, but analysis of their DNA sequence did not allow the prediction of further binding sites. Chromatin immunoprecipitation was used to identify a number of new DCC binding fragments and characterized them in vivo by visualizing DCC binding to autosomal insertions of these fragments, and it has been demonstrated that these fragments possess a wide range of potential to recruit the DCC. By varying the in vivo concentration of the DCC, evidence is provided that this range of recruitment potential is due to differences in affinity of the complex to these sites. It was also established that DCC binding to ectopic high-affinity sites can allow nearby low-affinity sites to recruit the complex. Using the sequences of the newly identified and previously characterized binding fragments, a number of short sequence motifs have been uncovered, that in combination may contribute to DCC recruitment. These findings suggest that the DCC is recruited to the X via a number of binding sites of decreasing affinities, and that the presence of high- and moderate-affinity sites on the X may ensure that lower-affinity sites are occupied in a context-dependent manner. Bioinformatics analysis suggests that DCC binding sites may be composed of variable combinations of degenerate motifs (Dahlsveen, 2006).

Using a ChIP strategy, several new DCC binding fragments have been identified and it has been demonstrated that they possess a wide range of potential to recruit the DCC. Because the majority of the isolated candidate fragments co-map with endogenous DCC binding sites at the resolution afforded by staining of polytene chromosomes, it is believed that the ChIP selection procedure is appropriate. By tuning DCC levels in vivo, it was concluded that the difference in recruitment ability is due to different affinity of the DCC for these fragments. At limiting concentrations of complex, only the sites of highest affinity are occupied. Conversely, at non-physiologically high concentrations of DCC, even 'cryptic' binding sites on autosomes are recognized by the complex. This suggests, in accord with previous observations, that selective interaction of the DCC with the X chromosome is a function of tightly controlled levels of complex components that are adjusted to assure interaction with binding sites of varying affinity clustered on the X, but insufficient to occupy cryptic sequences on autosomes. These data are also in broad agreement with observations that numerous sites on the X chromosomes contain DCC binding determinants. These determinants are not all equal, but represent a diverse set of DCC targets that differ by a wide range of affinities for the complex, as expected from a sequence determinant that during evolution became gradually enriched on the X chromosome (Dahlsveen, 2006).

The use of the term 'chromatin entry sites' for the subset of DCC binding sites that are still occupied by partial complexes in the absence of MSL3, implies that these sites were somehow qualitatively and perhaps functionally distinct from the remaining sites that only attract the intact complex. Although it is possible that not all DCC binding sites are functionally equivalent, the characterization of several new examples of both types of DCC binding sites suggests support for the 'affinities model'. According to this model, 'chromatin entry sites' are not qualitatively different from other sites, but only represent those sites with the highest affinity for the complex. A prediction from this model that is further substantiated by the results is that non-functional complexes that lack MSL3 or the acetyltransferase activity of MOF have lower affinity for target sites. Only those determinants with highest affinity for the DCC are able to recruit partial complexes in the absence of MSL3. Sites with slightly lower affinity are still able to recruit the complex in the mof1 mutant. Because the interaction of the DCC with the X chromosome is thought to be largely mediated by MSL1 and MSL2, it remains to be explored whether MSL3 and the acetylase activity of MOF affect the active concentration of MSL1 and MSL2 or lead instead to the adoption of a high-affinity conformation of the complex. Conversely, it remains to be seen if over-expression of MSL1 and MSL2 in the msl-31 and mof1 mutants would allow partial complexes to bind additional sites. In this respect it is intriguing that the mutation of both roX RNAs, which is presumed to lead to incomplete and non-functional complexes, can be partially rescued by the over-expression of MSL1 and MSL2 (Dahlsveen, 2006).

During analysis of DCC recruitment to high-affinity sites inserted into autosomes of wild-type males, an additional band of DCC binding was observed close to the insertion site in three independent cases (one insert each of DBF9, DBF5, and DBF7). Such minimal and rare 'spreading' has previously been observed for ectopic insertions of the 18D high-affinity site and from roX transgenes in the wild-type male background. This study now reveals that these additional DCC binding sites are not a result of random spreading, but are most likely due to interaction of the DCC with one of the low-affinity sites on autosomes that happened to reside close to the insertion site. These sites are usually observed only when the DCC concentrations are globally increased by over-expression of MSL1 and MSL2. Accordingly, it is suggested that the autosomal insertion of a high-affinity DCC binding site leads to a local rise in complex concentration, which allows these low-affinity sites to be recognized by the DCC even in wild-type males. However, additional requirements must clearly be met to allow low-affinity sites to profit from local increases in complex concentration, since not all ectopic high-affinity sites support the phenomenon. Permissive conditions may include active transcription or the presence of specific epigenetic marks (Dahlsveen, 2006).

It is envisioned that the clustering of DCC binding determinants of high and intermediate affinity on the X chromosome (combined with the transcription of the roX RNAs) elevates the concentration of the DCC within the X chromosomal territory and ensures the occupancy of lower-affinity sites in a context-dependent manner. This may explain the observation that autosomally derived transgenes often acquire dosage compensation. The transgenes may contain cryptic DCC binding determinants and may thus acquire binding if placed in the context of the X chromosomal territory. Conversely, an X chromosomal fragment that harbors only low-affinity sites may not be recognized if translocated to an autosomal context, and the fragment DBF3 may be an example for such a scenario. The presence of a large number of low-affinity sites may also contribute significantly to restricting the binding of the DCC to the X chromosome (Dahlsveen, 2006).

The term 'spreading' has been used to describe the appearance of additional bands of DCC binding around autosomal insertions of roX cDNAs or fragments derived thereof. However, extensive, long-range spreading from roX transgenes, which leads to the appearance of many ectopic DCC bands at greater distances from the insertion sites, occurs only under unusual conditions and depends on the transcription of the roX RNA rather than the DCC binding sites on DNA. Long-range spreading of the complex also does not occur into autosomal chromatin translocated to the X chromosome. It is suggested that large translocations maintain their original chromosomal context (DCC enriched or not), and therefore no redistribution of DCC over the new chromosomal junction is observable at the resolution of the polytene chromosomes. Importantly, this study does not address the higher-resolution distribution of the DCC within a chromosomal band. It is possible that such a band contains many individual binding sites, also of varying affinity. At this resolution, the term 'spreading' may characterize the local diffusion of the DCC from high- to low-affinity sites. This study does not exclude this type of spreading, or indeed any other kind of complex distribution within a chromosomal band. High-resolution ChIP analyses will be necessary to resolve the detailed nature of DCC distribution (Dahlsveen, 2006).

Previously, only three high-affinity binding sites for DCC were known. This study identified nine more fragments, and this encouraged investigation of common features within a larger pool. Interestingly, all new DBFs were found to map to gene-rich regions and either overlap with or lie close to essential genes. Three high-affinity fragments (DBF12, DBF9, and DBF6) reside entirely within genes. It is possible that specific recruitment sites, such as those inferred to reside within the DBFs, have been enriched in and around genes that require dosage compensation during evolution, and consequently, high-affinity sites may represent loci that are particularly dosage sensitive. Previous experiments indicated that the DCC tends to bind to the coding regions of genes, and it was suggested that this was linked to transcriptional activity. Although recent observations suggest that transcriptional activity alone is not sufficient to attract DCC binding, it is possible that transcription influences DCC recruitment to specific sites. For example, high-affinity sites, which show consistent and strong recruitment of the DCC at many chromosomal positions, may not be influenced by transcription. However, sites with lower affinity and variable recruitment ability may profit from transcriptional activity. Developmental differences in transcriptional activity may therefore also explain the lack of DCC recruitment in salivary glands to fragments isolated by ChIP from embryos (Dahlsveen, 2006).

This study has attempted to identify common sequence elements within previously characterized and new high-affinity DCC binding fragments and have uncovered a number of short sequence elements, whose clustering in combinations could contribute to DCC recruitment. Clearly, the importance of these elements remains to be tested experimentally. Previous analysis of the roX DCC binding sites identified a 110 bp sequence containing several blocks of conservation between roX1 and roX2. DCC binding was affected by mutation in several of the conserved blocks, indicating that DCC binding sites may be made up of combinations of shorter elements. Such combinations have be sought by defining pairs of elements found within a 200 bp window in the high-affinity DCC binding fragments. Those pairs that are significantly enriched on the X chromosome compared to other chromosomes are presented. Importantly, these X-enriched pairs often occur in multiple copies in the high-affinity fragments and at higher frequencies compared to the lower-affinity fragments DBF9-A, DBF1, DBF11, DBF13, and DBF3. Nonetheless, there is no obvious correlation between the location of individual pairs on the X and any specific features such as predicted genes. It is hypothesized that the elements that define these pairs (and other such elements that may have escaped attention) correspond to building blocks of DCC binding sites. Accordingly, a DCC binding site of given affinity for the complex would not be determined by a unique DNA sequence, but by clustering of variable combinations of short, degenerate sequence motifs. Individual low-affinity binding sites may not be unique to the X, but their clustering on the X may contribute to high-affinity binding. There are already indications that the DCC binds to several sites in close proximity. The two parts of DBF9, DBF9-A and DBF9-B, are both able to recruit the DCC, albeit with different affinity. The analysis of the 18D high-affinity fragment also suggested that multiple elements over 8.8 kb contribute to the binding of the complex (Dahlsveen, 2006).

The pairs have been ordered according to sequence similarity. Interestingly, a large family of elements contain GAGA-related motifs. Mutation of GAGA or CTCT motifs in the 110 bp roX1/roX2 consensus severely affects DCC recruitment to that sequence, indicating that GAGA motifs are involved in DCC binding. The fact these elements enriched in several independently identified high-affinity fragments demonstrates the appropriateness of the algorithms used to find them. Besides elements with a clear relationship to GAGA motifs, several other element families were identified defined by sequence similarity. In order to visualize the element families, the related words may be aligned such that sequence logos representing degenerate motifs can be derived using the WebLogo software ( It is considered possible that some of these degenerate motifs may contribute to DCC binding sites. Evaluation of the contributions of these novel motifs to the targeting of the complex will require increased resolution analysis and systematic evaluation of candidate sequences in the in vivo recruitment assay (Dahlsveen, 2006).

This study suggests that high-affinity DCC binding sites are composed of variable combinations of clustered, degenerate sequence motifs. The degeneracy of the sequence motifs indicates that many individual elements may have low affinity. Therefore, the interaction of the DCC with each individual site should be in dynamic equilibrium. However, it was recently observed by photobleaching techniques that the DCC components most likely involved in chromatin binding, MSL2 and MSL1, interact with the X chromosomal territory in cultured cells in an unusually stable manner, which is not compatible with binding equilibria involving off-rates that commonly characterize protein-DNA interactions. Several hypotheses can be formulated, whose evaluation may lead to resolution of this apparent contradiction. (1) Formation of higher-order structures involving many DCC components engaged in numerous simultaneous DNA interactions may lead to a trapping of the DCC within the X chromosome territory. (2) An initial sequence-directed targeting event may be followed by a stabilization of the interaction through positive reinforcement involving additional principles, such as epigenetic marks or a topological linkage. (3) It is considered that the arrangement of the interphase genome in polytene chromosomes may differ in a relevant aspect from the more compact chromosomal territories of diploid cultured cells. Ultimately, the identification of the DNA-binding domains of DCC components and analysis of their mode of DNA interaction will be required to solve the targeting issue (Dahlsveen, 2006).

Long-range spreading of dosage compensation in Drosophila captures transcribed autosomal genes inserted on X

Dosage compensation in Drosophila males is achieved via targeting of male-specific lethal (MSL) complex to X-linked genes. This is proposed to involve sequence-specific recognition of the X at approximately 150-300 chromatin entry sites, and subsequent spreading to active genes. This study asked whether the spreading step requires transcription and is sequence-independent. It was found that MSL complex binds, acetylates, and up-regulates autosomal genes inserted on X, but only if transcriptionally active. It is concluded that a long-sought specific DNA sequence within X-linked genes is not obligatory for MSL binding. Instead, linkage and transcription play the pivotal roles in MSL targeting irrespective of gene origin and DNA sequence (Gorchakov, 2009).

To ask whether autosomal genes can effectively recruit MSL complex, a construct was created that was named TrojanHorse, consisting of a 14-kb fragment from chromosome arm 2L encompassing two small genes, Rpl40 and cg3702. These genes were chosen because they are expressed at all stages of development and are associated with H3K36me3, both hallmarks of typical MSL targets on the X. The sequence of the 14-kb fragment was altered by incorporating five 0.2-kb tags of non-Drosophila origin into the 3'-untranslated regions (UTRs) of Rpl40 and cg3702, and the nontranscribed regions of TrojanHorse. This design allowed clear discrimination between transcripts derived from the endogenous Rpl40 and cg3702 genes and their transposed copies on the X. Additionally, these tags enabled direct assessment of MSL protein and histone modification profiles across the TrojanHorse insertion on the X chromosome. The TrojanHorse construct was inserted in two precise genomic positions using the recently developed attB/attP phage phiC31 integration system. One insertion site (attP18 at cytological position 6C12) was in a gene-rich region encompassing numerous MSL gene targets. In contrast, the second landing site (attP3 at 19C4) was intentionally chosen to be distant from MSL targets and active genes, with the nearest MSL target 60 kb proximal to the TrojanHorse insertion. Thus, in the second case, the two constitutively expressed autosomal genes were inserted on X within a large, MSL-depleted region (Gorchakov, 2009).

If specific DNA sequences at each gene are required to complete the MSL pattern on X, then TrojanHorse genes, originating from an autosome, should not acquire MSL binding. Alternatively, if linkage to the X chromosome and the active state are sufficient, TrojanHorse genes should be bound and acquire H4K16ac. Therefore, ChIP assays were performed to determine the binding profiles for two MSL subunits, MSL2 and TAP-tagged MSL3, within TrojanHorse placed at either attP18 or attP3. Independent of the integration site, pronounced binding was observed of MSL proteins to the tags located at the 3' ends of both cg3702 and Rpl40 of TrojanHorse in mixed sex embryos and male larvae. In contrast, the untranscribed regions of TrojanHorse did not attract the MSL complex. Furthermore, MSL complex binding led to H4K16 acetylation of TrojanHorse chromatin. In agreement with the binding profiles for MSL2 and MSL3-TAP, H4K16ac was found to peak at the 3'-UTRs of cg3702 and Rpl40 in both embryos and male larvae. Notably, the histone H4 acetylation profile was broader than that of the MSL complex, consistent with recent studies. Finally, it was confirmed that the two genes within TrojanHorse were indeed transcribed in larvae and male and female embryos by mapping RNA polymerase II and histone methylation marks expected for active genes: dimethylated H3K4 (H3K4me2) at the 5' ends, and H3K36me3 biased toward the 3' ends. Taken together, these results demonstrate that transcribed and H3K36me3-marked autosomal genes can become typical MSL targets when placed on the male X, favoring a sequence-independent model for spreading. These data also indicate that targeting can occur in the apparent absence of a nearby CES, as the closest identified entry sites are located ~54 kb and 80 kb proximal to attP18 and attP3, respectively. Furthermore, attP3 is 60 kb away from the nearest active gene cluster or MSL-bound region, indicating that targeting can occur over long distances in cis (Gorchakov, 2009).

Despite the clear association of transposed autosomal genes with MSL protein in the male X environment, it remained to be determined whether their transcription or some other features were required for the observed complex binding. In order to address this question, a 'promoterless' version of TrojanHorse was created where the 1.2-kb region, including the promoters and 5' ends of both cg3702 and Rpl40, was deleted. This TrojanHorseδ, otherwise identical to TrojanHorse, was placed in the same attP18 site by attP/attB recombination. Quantitative PCR (qPCR) analysis confirmed that transcription of both genes in TrojanHorseδ was dramatically reduced (at least 25-fold), approaching the detection limit. In addition, the genes exhibited background levels of H3K36me3 in TrojanHorseδ. It was then asked whether transcriptionally inactive genes showed any changes in binding patterns of MSL and other proteins. Indeed, MSL3-TAP no longer bound nontranscribed cg3702 and Rpl40, resulting in decreased H4K16ac levels throughout TrojanHorseδ. This direct comparison clearly supports the importance of transcription for MSL recognition of its targets (Gorchakov, 2009).

To test whether the genes within the intact TrojanHorse were not only associated with MSL complex and marked with H4K16ac but also were subject to dosage compensation, the degree of up-regulation of cg3702 and Rpl40 was directly measured in the TrojanHorse context. These genes are transcribed and dosage-compensated in male larvae so that expression of one copy in males is approximately equal to two copies in females, and about twofold higher than in heterozygous females. Likewise, up-regulation is observed in male embryos. However, in this case the dosage compensation appears less complete, perhaps due to maternal deposition of the transcripts leading to an underestimate of the male/female zygotic transcription ratio. Taken together, these data indicate that the MSL complex bound to TrojanHorse retains all its functions: It acetylates the underlying chromatin and increases transcriptional output, resulting in dosage compensation of both Rpl40 and cg3702 (Gorchakov, 2009).

To exclude the possibility that the two genes in the TrojanHorse construct were somehow exceptional in their ability to attract MSL complex, a more extensive set of active autosomal genes was tested by engineering a larger A-to-X transposition. TrojanElephant was created by inserting a 65-kb region from chromosome 2L, containing 20 genes from cg13773 to snRNP70K into the attB-P[acman] vector via recombineering. None of the TrojanHorse or TrojanElephant genes attracted MSL complex when located in their endogenous positions on autosomes. Therefore, the TrojanElephant construct was inserted at the previously characterized attP3 site on the X, which is a gene-poor region. Several possible results were hypothesized: (1) Within the 65-kb segment, the MSL complex might target all H3K36me3-positive genes. (2) MSL binding might be detected over the active genes closest to the ends of the 65-kb TrojanElephant, with a gradual decrease in binding closer to the center. (3) The MSL complex might skip genes in TrojanElephant altogether (Gorchakov, 2009).

To determine which of these scenarios occurs in vivo, MSL binding across TrojanElephant was measured in third-instar larvae by ChIP followed by qPCR. The data obtained from both experiments clearly demonstrated the ability of the MSL complex to faithfully recognize the transcribed and H3K36me3-marked genes within the transposed material, with no evidence for skipping any of the active genes. Therefore, if the MSL complex spreads linearly from the closest identified flanking CES, it can travel at least 83 kb. The fact that all active autosome-derived genes were bound by MSL complex argues strongly against the idea that each X-chromosomal gene possesses special MSL recruiting signals. Instead, it is proposed that after the initial attraction of the MSL complex by a chromatin entry sites (CES) or roX RNA gene, transcription of active genes serves as the main guiding feature for MSL complex binding (Gorchakov, 2009).

These results are consistent with early transgenic studies, indicating that single genes could become dosage-compensated when inserted on X. However, the work still needs to be reconciled with results from established transposition stocks, in which much larger inserts of autosomal material onto X, failed to acquire dosage compensation or cytologically visible MSL binding, or showed binding only a few kilobases into the insertion by ChIP analysis. One possibility is that stable stocks carrying large spontaneous or X-ray-induced rearrangements may display exceptional behavior, as they have been preselected for viability and thus possibly for the maintenance of chromosome of origin regulation. Alternatively, these results may indicate that distance to the nearest functional CES is critical for MSL targeting, but that this critical distance cannot be reached in the current experiments. It may be that once a certain size is attained, any DNA insertion will tend to associate with its chromosome of origin rather than be generally localized within the X-chromosome three-dimensional territory, thus excluding spreading. Testing these possibilities may be within reach in the near future, as improvements in transgenic technology allow larger insertions at predefined breakpoints to be obtained (Gorchakov, 2009).

The data allow further refinement of the two-step model for MSL complex recruitment to the male X chromosome. In the first step, MSLs are thought to bind 150-300 CES containing MRE motifs on X, and ignore autosomes. In the second step, MSLs recognize the active genes on the X irrespectively of their sequence and origin through a transcription-dependent mechanism. Trimethylation of H3K36 is partially responsible for this sequence-independent step. Here it is speculated on the coexistence of two modes of MSL spreading on the X: (1) long-range spreading from the roX genes, and (2) local distribution of the MSL complex from the CES scattered throughout the X (with a median distance of ~100 kb). It is only in the context of the X chromosome that both roX genes and CES are found in cis to each other, making possible both correct chromosome identification and efficient spreading. The enigmatic nature of roX spreading remains to be understood, including whether features of active genes might be recognized directly by roX RNAs (Gorchakov, 2009).

Sex-lethal imparts a sex-specific function to UNR by recruiting it to the msl-2 mRNA 3' UTR: translational repression for dosage compensation

MSL-2 (male-specific lethal 2) is the limiting component of the Drosophila dosage compensation complex (DCC) that specifically increases transcription from the male X chromosome. Ectopic expression of MSL-2 protein in females causes DCC assembly on both X chromosomes and lethality. Inhibition of MSL-2 synthesis requires the female-specific protein sex-lethal (Sxl), which binds to the msl-2 mRNA 5' and 3' untranslated regions (UTRs) and blocks translation through distinct UTR-specific mechanisms. Translationally silenced msl-2 mRNPs has been purified and UNR (upstream of N-ras) has been identified as a protein recruited to the 3' UTR by Sxl. Sxl requires UNR as a corepressor for 3'-UTR-mediated regulation, imparting a female-specific function to the ubiquitously expressed UNR protein. These results reveal a novel functional role for UNR as a translational repressor and indicate that UNR is a key component of a 'fail-safe' dosage compensation regulatory system that prevents toxic MSL-2 synthesis in female cells (Duncan, 2006).

Sequence analysis revealed that the protein specifically associated with translationally silenced msl-2 mRNA exhibited significant similarity to the previously characterized mammalian protein UNR. This was surprising for a putative translational corepressor, because mammalian UNR, a cytoplasmically localized RNA-binding protein, stimulates translation of both viral and cellular internal ribosome entry site (IRES) containing mRNAs. UNR is also a major regulator of translationally coupled mRNA turnover mediated by the c-fos mCRD RNA element (Duncan, 2006).

UNR has five cold-shock nucleic acid-binding domains, each with the unique substitution of the sequence FFH for the canonical FVH in part of the RNA-binding surface. CG7015, coding for the identified protein, also has five cold-shock domains (CSDs) with the signature FFH motif. Overall sequence identity between CG7015 and human UNR is ~45%, and this is higher within the CSDs (70%, 56%, 51%, 53%, and 66% identity for CSD-1-CSD-5, respectively). The Drosophila genome encodes no other protein with similarly high sequence identity to mammalian UNR, and it is therefore concluded that ORF CG7015 is Drosophila UNR and it is referred to as 'UNR' hereafter (Duncan, 2006).

This study has identified a novel component of the dosage-compensation regulatory machinery that has eluded genetic methods. Using an mRNP purification approach and functional analysis, the Drosophila UNR protein has been demonstrated to be recruited to msl-2 mRNA 3' UTR by SXL for translational inhibition of msl-2 mRNA specifically in female cells. These data indicate that SXL imparts a female-specific translational repressor function to UNR, and imply that this novel function of UNR is critical for negative regulation of the dosage compensation machinery to prevent toxic effects in female cells (Duncan, 2006).

Previous results implied that region 2456 of the msl-2 mRNA, 3'-UTR sequences adjacent to the Sxl-binding sites, is important for translational regulation via the 3' UTR (Grskovic, 2003). Since this region flanks the Sxl-binding sites, it was hypothesized to bind a putative corepressor that acts in conjunction with Sxl. Glutathione RNA (GRNA) chromatography in combination with sucrose-density gradient centrifugation was used to purify this factor, identifying Drosophila UNR. Functional analyses of msl-2 reporter genes and endogenous msl-2 expression in female and male cell lines demonstrate that UNR is necessary for translational repression of msl-2 mRNA by Sxl via the 3' UTR, but does not affect msl-2 mRNA translation in the absence of Sxl. Taken together, these results show that UNR is a cofactor for translational repression of MSL-2 protein synthesis, specifically in female cells. These conclusions are strongly supported by the results of Abaza, (2006), who independently isolated UNR using a different approach and could demonstrate that direct interaction of UNR with Sxl helps recruit UNR to the msl-2 3' UTR and is critical for translational inhibition of msl-2 reporter mRNAs by Sxl in vitro. This is the first time that a translational corepressor has been identified by a combined strategy of gradient and specific mRNP purification, and it is anticipated that this method will prove useful as a general strategy (Duncan, 2006).

Analysis of msl-2-ß-gal reporters further supports the recently proposed dual-mechanism model for msl-2 mRNA translational inhibition, which predicts that 3'-UTR corepressors should be required exclusively for 3'-UTR-mediated inhibition. Indeed, UNR depletion significantly affects only msl-2 reporters with wild-type 3' UTRs, and the strongest effect is on the 5'mut reporter, where all regulation must occur through the 3' UTR. In this case, the quantitative effect of UNR depletion approaches the effect of Sxl depletion. Since RNAi produces a 'knockdown' effect that likely reflects a partial-loss-of-function rather than true null phenotype, differences in RNAi efficiency and/or differences in relative concentrations of UNR and Sxl necessary for inhibition may explain why the 5'mut reporter is still slightly repressed after UNR knockdown. In any case, the in vivo analysis presented here directly supports the concept of independent regulatory contributions of 5'- and 3'-UTR Sxl complexes, and implies that UNR is a critical component for 3'-UTR-mediated inhibition (Duncan, 2006).

How does UNR recruited by Sxl to the 3' UTR interfere with translation initiation at the mRNA 5' end? Presumably the Sxl/UNR corepressor complex interacts with factors that affect small ribosomal subunit recruitment. This interaction might require direct participation of Sxl, or Sxl might serve only to recruit UNR to the 3' UTR. Similarly, UNR might directly contact factors affecting small subunit recruitment, or may do so through additional bridging factors as part of a larger 'corepressor assembly'. The biochemical approach identified factors in addition to UNR that specifically copurify with the repressed mRNP. Interestingly, UNR is the only one of the copurified proteins that displays significant corepressor activity when assayed by RNAi in Kc cells (Duncan, 2006).

Since msl-2 mRNA repression functions in the absence of a 5' m7GpppN cap structure, translational regulatory proteins like Cup or d4EHP are unlikely to be the molecular targets of repression by the 3'-UTR complex. A candidate target is Drosophila PABP, since mammalian PABP interacts with UNR, and promotes small ribosomal subunit binding to the mRNA. Although msl-2 translational inhibition does not require a poly(A) tail, PABP appears to have a critical function in initiation that is independent of the poly(A) tail, raising the possibility that UNR might nevertheless interfere with PABP-mediated recruitment of the small ribosomal subunit to msl-2 mRNA. Future studies will aim to determine the mechanism by which Sxl and UNR bound at the 3' end of msl-2 mRNA block translation initiation at the 5' end (Duncan, 2006 and references therein).

Consistent with a general role as a regulator of dosage compensation, UNR mRNA expression is ubiquitous throughout Drosophila development. Interestingly, UNR protein is expressed at similar levels in both male and female cells in culture and in flies (Abaza, 2006), but interacts with msl-2 mRNA to modulate its translation only when Sxl is present. Thus, Sxl imparts a sex-specific, mRNA-specific translational repressor function to UNR. Sex-specific modulation of UNR function by Sxl is presumably crucial for dosage compensation, which would be compromised if the abundant UNR protein in males were able to inhibit msl-2 mRNA translation (Duncan, 2006).

Sex-specific function at the cellular and organismal level can also be viewed as context-specific function at the molecular level, with Sxl acting as a context-specific modulator of UNR function. The hypothesis that UNR function is modulated by molecular context is supported by the previously determined functions of mammalian UNR, which involve different protein-interaction partners, in the context of different RNA sequence elements. Indeed, the previously reported role for mammalian UNR as a translational activator of cellular and viral IRESes made it a rather unexpected candidate for a translational corepressor. These data identify the first function for UNR in Drosophila, and demonstrate the surprising finding that UNR can also be a critical component of translational repression complexes, underscoring the importance of both protein and RNA context in modulation of UNR function in post-transcriptional control of gene expression (Duncan, 2006).

Another notable difference between UNR-mediated translational repression and the UNR functions described previously is that in the former case UNR is the recruited protein, whereas in the latter cases, high-affinity interaction of UNR with an RNA element underlies subsequent recruitment of additional proteins by UNR. This distinction has two important implications for UNR function. First, UNR's potential regulatory targets are not confined to mRNAs with high-affinity binding sites for UNR. Second, context-specific modulators such as Sxl can be expected to be key determinants of how UNR affects regulation of a particular mRNA. Detailed mechanistic and structural analysis will be essential to answer the intriguing question of how UNR can function as a translational activator in one molecular context, and a repressor in another (Duncan, 2006).

UNR depletion in Kc cells causes a significant increase in MSL-2 protein to ~20% of that in male SL-2 cells or Sxl-depleted Kc cells. Clearly, Sxl-dependent, UNR-independent inhibition mediated by the msl-2 5' UTR contributes to repression of MSL-2 protein synthesis. It was also observed that Sxl promotes reduced endogenous msl-2 mRNA levels, but UNR does not. The results warrant interpretation in the context of previous studies of transgenic flies; females expressing msl-2 transgenes lacking the 3'-UTR regulatory sequences produce detectable MSL-2 protein, but at a significantly lower level than males or females expressing transgenes with both 5'- and 3'-UTR Sxl-binding sites deleted. The lower level of MSL-2 protein made in 3'-UTR mutant females is nevertheless sufficient to promote DCC loading onto female X chromosomes. Therefore, at the organismal level, UNR, acting through the msl-2 mRNA 3' UTR, can be expected to make a significant contribution to robust repression of MSL-2 protein synthesis and prevention of deleterious activation of the X-chromosome dosage-compensation machinery in females (Duncan, 2006).

The inhibition of male-specific lethal 2 (msl-2) mRNA translation by the RNA-binding protein sex-lethal (Sxl) is an essential regulatory step for X-chromosome dosage compensation in Drosophila. The mammalian upstream of N-ras (UNR) protein has been implicated in the regulation of mRNA stability and internal ribosome entry site (IRES)-dependent mRNA translation. The Drosophila homolog of mammalian UNR has been identified as a cofactor required for Sxl-mediated repression of msl-2 translation. UNR interacts with Sxl, a female-specific protein. Although UNR is present in both male and female flies, binding of Sxl to uridine-rich sequences in the 3' untranslated region (UTR) of msl-2 mRNA recruits UNR to adjacent regulatory sequences, thereby conferring a sex-specific function to UNR. These data identify a novel regulator of dosage compensation in Drosophila that acts coordinately with Sxl in translational control (Abaza, 2006).

Inhibition of msl-2 expression is essential for development of female flies; forced expression of MSL-2 causes the assembly of the DCC on both X chromosomes and lethality. The Drosophila homolog of mammalian UNR is necessary to inhibit msl-2 expression. Drosophila UNR is recruited to the 3' UTR of msl-2 mRNA by dSxl, a female-specific protein, and plays an essential role in repressing its translation. UNR associates to the 3' UTR of msl-2 mRNA in female cells and is necessary to repress msl-2 translation in vivo. Together, these data identify UNR as a regulator of dosage compensation (Abaza, 2006).

In vitro selection experiments (SELEX) indicate that human UNR binds to purine-rich regions in the mRNA, with the consensus sequences (A/G)5AAGUA/G or (A/G)8AACG and an apparent dissociation constant (Kd) of ~10 nM. Although Drosophila UNR also recognizes purine-rich sequences in the 3' UTR of msl-2 mRNA that fall within these consensus, it does so with a very poor affinity, a situation reminiscent to that of the bacterial cold-shock proteins. Binding of UNR to msl-2 mRNA requires the binding of Sxl. The observation that msl-2 RNA fragments containing mutated Sxl-binding sites, but wild-type UNR-binding sites, do not bind to either of the two proteins suggests that Sxl does not simply induce a conformational change in UNR that allows it to bind RNA. Rather, Sxl recruits UNR to bind in close proximity in the 3' UTR of msl-2 mRNA. Stable recruitment of UNR requires the interaction of UNR with both Sxl and msl-2 mRNA, as supported by the following evidence. First, UNR is not retained in the dRBD4 column unless this column is saturated with msl-2 mRNA. Second, dUNR is not retained in the mRBD column despite the presence of msl-2 mRNA. Third, no complex formation is observed in a gel mobility-shift assay when the UNR-binding sites are mutated (mut2456). Fourth, msl-2 mRNA and UNR do not interact in male flies, which lack Sxl. Nevertheless, UNR and Sxl can interact directly in vitro. Addition of EF RNA does not improve this interaction, and addition of embryo extract actually competes it. These results suggest that, although the interaction of Sxl and UNR can occur directly, the interaction with msl-2 mRNA stabilizes the complex in the competitive conditions of the extract (Abaza, 2006).

UNR protein and msl-2 mRNA do not interact in male flies despite their relative abundance. In addition, supplementing cytoplasmic embryo extracts with Sxl—a primarily nuclear protein—promotes UNR association with msl-2 mRNA, and translational repression by UNR is only observed in Sxl-containing cells. These data suggest that the interaction of UNR with msl-2 mRNA is mediated by Sxl in vivo, and imply that Sxl is the critical determinant for the formation of a repressive complex on the 3' UTR of msl-2 mRNA. In this scenario, Sxl conveys a sex-specific function to UNR. The stepwise assembly of a translation inhibitory complex on msl-2 mRNA is reminiscent of Drosophila hunchback. The 3' UTR of maternal hunchback mRNA is bound by Pumilio (Pum), and this event triggers the sequential recruitment of Nanos (Nos) and Brain tumor (Brat), which ultimately results in the translational repression of hunchback mRNA. Sequential binding of Sxl and UNR to msl-2 mRNA could result from their respective subcellular locations: While Sxl is nuclear and associates with msl-2 pre-mRNA, UNR is primarily, if not exclusively, cytoplasmic. Interestingly, UNR accumulates at the nuclear periphery, which perhaps reflects or ensures the rapid formation of repressive complexes as msl-2 and probably other mRNAs are exported to the cytoplasm. Additionally, accumulation around the nucleus could reflect the association of UNR with the endoplasmic reticulum, as reported for mammalian UNR (Abaza, 2006).

Dosage compensation is believed to function from the blastoderm stage. As expected for a protein involved in the regulation of dosage compensation, UNR is present throughout development. Curiously, although UNR mRNA is dramatically more abundant in female flies, this difference is compensated at the protein level, suggesting the existence of sex-specific mechanisms to modulate UNR expression. Indeed, the amount of UNR might need to be tightly controlled. Overexpression of mammalian UNR leads to cell death, and preliminary data suggest that substantial overexpression of UNR results in lethality of both male and female flies. Three forms of UNR mRNA can be detected in Drosophila. Several mRNAs have also been detected in mammals, consistent with the observation of three alternative polyadenylation sites of the hUNR gene and alternative splicing of the hUNR pre-mRNA. These data suggest that the different UNR mRNAs arise by alternative processing, although the significance of this observation remains to be explored (Abaza, 2006).

The role of UNR in Drosophila contrasts with the known functions of UNR in mammals. hUNR is part of a complex assembled on the coding region of c-fos mRNA that is involved in the deadenylation-dependent destabilization of this transcript. The interaction of hUNR with PABP within this complex is believed to bridge the complex to the poly(A) tail, although the mechanism by which the complex influences deadenylation is unknown. In Drosophila, the steady-state levels of msl-2 mRNA are, indeed, lower in females. However, no effect of UNR and Sxl on msl-2 mRNA stability was detected in translation assays. hUNR also binds to the IRES elements in the 5' UTRs of several transcripts and activates their translation. In the best understood example, that of Apaf-1 mRNA, hUNR induces a conformational change in the IRES that makes it accessible for binding of PTB, a positive regulator of Apaf-1 translation. Contrary to hUNR, Drosophila UNR binds to the 3' UTR of msl-2 mRNA and represses its translation. Nonetheless, the underlying effects of UNR binding may be similar if UNR acts as an RNA or RNP chaperone to facilitate an RNA conformation, or the assembly of repressive factors, that inhibit translation (Abaza, 2006).

Translation of msl-2 occurs via a cap-dependent mechanism. Cap-dependent translation initiation involves the recruitment of 43S ribosomal complexes (molecular assemblies of the 40S ribosomal subunit with a set of translation initiation factors and the initiator tRNA) to the cap structure at the 5' end of the mRNA. Translation inhibition mediated by the 3' UTR of msl-2 results from a block of 43S ribosomal recruitment. However, translational repression of msl-2 mRNA by Sxl can occur in the absence of a cap structure and a poly(A) tail. Understanding how 43S recruitment is affected by Sxl without the involvement of the cap is, indeed, intriguing. A possibility is that, as with mammalian UNR, UNR interacts with PABP. PABP could, in turn, exert a poly(A)- and cap-independent effect on translation. Certainly, the mapping of UNR domains relevant for translational control and the identification of dedicated factors that interact with UNR are likely to provide insights into this mechanism of translation regulation that is key to control dosage compensation in Drosophila (Abaza, 2006).

SU(VAR)3-7 links heterochromatin and dosage compensation in Drosophila

In Drosophila, dosage compensation augments X chromosome-linked transcription in males relative to females. This process is achieved by the Dosage Compensation Complex (DCC), which associates specifically with the male X chromosome. It has been found that the morphology of this chromosome is sensitive to the amounts of the heterochromatin-associated protein SU(VAR)3-7. This study examined the impact of change in levels of SU(VAR)3-7 on dosage compensation. It was first demonstrated that the DCC makes the X chromosome a preferential target for heterochromatic markers. In addition, reduced or increased amounts of SU(VAR)3-7 result in redistribution of the DCC proteins MSL1 and MSL2, and of Histone 4 acetylation of lysine 16, indicating that a wild-type dose of SU(VAR)3-7 is required for X-restricted DCC targeting. SU(VAR)3-7 is also involved in the dosage compensated expression of the X-linked white gene. Finally, it was shown that absence of maternally provided SU(VAR)3-7 renders dosage compensation toxic in males, and that global amounts of heterochromatin affect viability of ectopic MSL2-expressing females. Taken together, these results bring to light a link between heterochromatin and dosage compensation (Spierer, 2008).

Drosophila uses two systems of whole chromosome regulation: dosage compensation mediating the two fold up-regulation of male X-linked genes and the Painting of fourth, POF, regulating the mainly heterochromatic fourth chromosome. Binding of POF to the fourth chromosome is dependent on the heterochromatic protein HP1). POF and HP1 colocalize on fourth chromosome-linked genes and both are involved in the global regulation of the fourth chromosome. It has been proposed that POF stimulates and HP1 represses genes expression and that the interdependent binding of these two proteins precisely tunes output from the fourth chromosome (Spierer, 2008).

Dosage compensation targets the male X chromosome to correct the unbalance between the unique X chromosome of males and the two X chromosomes of females. To compensate for the resulting disparity in X chromosome-linked gene expression, most X-linked genes in males are hyperactivated. The Dosage Compensation Complex (DCC) consists of five proteins called the MSLs for Male Specific Lethal (MSL1, MSL2, MSL3, MLE and MOF) as well as two non-coding RNAs, roX1 and roX2. In males, the expression of MSL2 mediates the stabilization of the other proteins and the assembly of the DCC specifically on the X chromosome. This results in an enrichment of acetylation of histone H4 at lysine 16 (H4K16ac) on the male X chromosome, due to the MOF protein of the complex. The histone mark could in part explain the subsequent hypertranscription of X-linked genes in males. In females, the Sex-lethal gene turns off the dosage compensation system by repressing the Msl2 translation (Spierer, 2008).

One of the most intriguing issues of dosage compensation is the specific recognition of the male X chromosome by the DCC. Searches for X chromosomal DNA sequences determining DCC binding failed to identify a consensus sequence. Global mapping of MSL proteins on the X chromosome has demonstrated that the DCC associates primarily with genes rather than intergenic regions, displays a 3'- bias and correlates with transcription. Moreover, the MSL complex is attracted to genes marked by H3K36 trimethylation, a mark of active transcription. Furthermore, the levels of DCC proteins MSL1 and MSL2 are critical for correct targeting to the X chromosome. Over-expression of both msl1 and msl2 results in inappropriate MSLs binding to the chromocenter and chromosome 4. MSL2, deleted of its C-terminal part, binds as a complex with MSL1 to the heterochromatic chromocenter. roX RNAs are also key components for X chromosome targeting since roX1roX2 mutants cause relocation of MSLs complex to autosomal regions and the chromocenter. These data reveal an unpredicted physical association between the MSL complex and heterochromatic regions (Spierer, 2008 and references therein).

H4K16 acetylation is not the only chromatin mark distinguishing the Drosophila male X chromosome from the autosomes. Phosphorylation of H3 at serine 10, catalyzed by JIL-1, is a histone modification highly enriched on the male X chromosome. The JIL-1 kinase interacts with the DCC and is involved in dosage compensation of X-linked genes. Interestingly, Jil-1 mutant alleles affect both condensation of the male X chromosome and expansion of heterochromatic domains, providing evidence for a dynamic balance between heterochromatin and euchromatin. Other general modulators of chromatin state, as ISWI or NURF, are also required for normal X chromosome morphology in males. The NURF complex and MSL proteins have opposite effects on X chromosome morphology and on roX transcription (Spierer, 2008 and references therein).

An intriguing genetic interaction has been discovered between the heterochromatic proteins SU(VAR)3-7 and HP1, and dosage compensation (Spierer, 2005). Su(var)3-7 encodes a protein mainly associated with pericentromeric heterochromatin and telomeres, but also with a few euchromatic sites. Specific binding to pericentric heterochromatin requires the heterochromatic protein HP1 (Spierer, 2005). HP1 localizes to heterochromatin through an interaction with methylated K9 of histone H3 (H3K9me2), a heterochromatic mark mainly generated by the histone methyltransferase SU(VAR)3-9. SU(VAR)3-7 interacts genetically and physically with HP1 and with SU(VAR)3-9. Su(var)3-7, Su(var)205, encoding HP1, and Su(var)3-9 are modifiers of position effect variegation (PEV), the phenomenon of gene silencing induced by heterochromatin. These three genes enhance or suppress the PEV depending on their doses and thus are considered as encoding structural components of heterochromatin. Strikingly, the amounts of SU(VAR)3-7 and HP1 affect male X chromosome morphology more dramatically than other chromosomes. Reduced doses of SU(VAR)3-7 or HP1 result in bloating of the X chromosome specifically in males (Spierer, 2005). Increased doses of SU(VAR)3-7 cause the opposite phenotype, a spectacular condensation of the X chromosome associated with recruitment of other heterochromatin markers. Some unique feature of the male X chromosome makes it particularly sensitive to change in SU(VAR)3-7 amounts. In addition, knock-down of Su(var)3-7 results in reduced male viability leading to a 0.7 male/female ratio in the progeny of Su(var)3-7 homozygous mutant mothers (Delattre, 2004). The possibility of interaction between activating and repressive chromatin factors on the male X chromosome led to an analysis of the impact of SU(VAR)3-7 on dosage compensation (Spierer, 2008).

This study shows that wild-type levels of SU(VAR)3-7 are required for male X chromosome morphology, X chromosome-restricted DCC targeting, expression of P(white) transgenes in males and for coping with increased MSL1 and MSL2 levels. Evidence is provided for interplay between heterochromatin and dosage compensation in Drosophila (Spierer, 2008).

This work reveals a connection between heterochromatin and dosage compensation in Drosophila. SU(VAR)3-7 is implicated in male X chromosome morphology, in correct distribution of the DCC, in the expression of the dosage compensated white gene and in male viability. This study describes some of the complex interactions between SU(VAR)3-7 and the DCC and illustrates the ability of heterochromatin/DCC balance to affecting chromatin conformation and protein distribution. The results support a model whereby the activating dosage compensation system in Drosophila is influenced by chromatin silencing factors (Spierer, 2008).

Reduced levels of SU(VAR)3-7 induce bloating of the male X chromosome, whereas increased levels cause condensation of the male X chromosome. Moreover, at high dose, SU(VAR)3-7, normally restricted to heterochromatin, invades preferentially the male X chromosome and, to a lesser extent, the autosomes. These observations led to an investigation of the features rendering the male X chromosome particularly sensitive to SU(VAR)3-7. This paper examined the genetic interaction between a gene essential for dosage compensation, mle, and Su(var)3-7 on the morphology of the male X chromosome. Bloating and shrinking of the X chromosome both require the presence of the DCC, and assembly of the DCC in females is sufficient to make their X chromosomes preferential targets for SU(VAR)3-7, when in excess. The dosage compensation system is thus responsible for the sensitivity of the male X chromosome to changes in SU(VAR)3-7 amounts. One explanation for the X chromosome sensitivity is that increased levels of H4K16 acetylation induced by the DCC render chromatin of the male X chromosome more accessible to chromatin factors and more sensitive to disturbances than other chromosomes. The possibility cannot be excluded that SU(VAR)3-7-induced X chromosome defects are indicators of a more general effect of the protein on all chromosomes as described for ISWI: Null mutations in the gene encoding ISWI cause aberrant morphology of the male X chromosome but not of autosomes and females X chromosomes, but expression of a very strong dominant negative form of ISWI in vivo leads indeed to decondensation of all chromosomes in both sexes. Nevertheless other data in this study, to be described below, favour the hypothesis whereby X chromosome defects result from a specific interaction between SU(VAR)3-7 and dosage compensation (Spierer, 2008).

Male X chromosome sensitivity to SU(VAR)3-7 was rather unexpected, as in a wild-type context, in contrast to over-expression conditions, no preferential binding of SU(VAR)3-7 to the male X chromosome was detected. The absence of detectable SU(VAR)3-7 enrichment on the male X polytene chromosome from third instar larvae may be due either to lack of sensitivity of the immunostaining procedure or to observations made in inappropriate tissues or development stages. Similar puzzling observations have been made for HP1, which is not preferentially seen on the male X polytene chromosomes, although reduced HP1 induces bloating of the male X chromosome. In cultured cells however, a moderate HP1 enrichment was detected with the DamID technique on the male X chromosome and not on the female X chromosomes, suggesting that HP1 participates in the structure of the male X chromosome (Spierer, 2008).

A striking and novel result of this study is that precise wild-type amounts of the heterochromatic protein SU(VAR)3-7 are required to restrict MSLs binding to the X chromosome. In Su(var)3-7 mutants, it was observed that the MSL proteins are recruited to the chromocenter. Furthermore, when SU(VAR)3-7 is present in excess, MSLs are massively delocalized from the X chromosome to many sites on autosomes (Spierer, 2008).

Two hypotheses are proposed. First, the effect of SU(VAR)3-7 on the MSLs distribution is indirect and due to the regulation of the expression of a component of the DCC. Indeed, increased amounts of MSL1 and MSL2 lead to MSLs binding on autosomes and at chromocenter, and MSLs delocalization from the X chromosome to autosomes and chromocenter is detectable in roX1roX double mutants. A careful regulation of MSLs and roX RNAs concentration is therefore important to restrict DCC activity to appropriate targets. In addition, increased levels of MSL2, or of both MSL2 and MSL1, result in a diffuse morphology of the X chromosome. This phenotype resembles the bloated X chromosome of Su(var)3-7 and Su(var)2-5 mutants, suggesting that the amounts of MSL2 and MSL1 are downregulated by the heterochromatic proteins. Expression of many euchromatic genes are under the control of the HP1 protein, leading the idea of testing whether changes in SU(VAR)3-7 amounts modify the expression of roXs, msl1 and msl2 or the stability of MSL1 and MSL2. Quantitative RTPCR and Western blots did not detect significant changes in the amounts of DCC components. In HP1 mutant msl1 transcription is also not affected. These results speak against the hypothesis of regulation of expression of a DCC component by a SU(VAR)3-7/HP1 complex (Spierer, 2008).

The second hypothesis is that SU(VAR)3-7 modifies the MSLs distribution by changing the chromatin state of the X chromosome and of the pericentric heterochromatin. Changes in chromatin conformation or epigenetic marks could modify affinity of the DCC for entry sites. Numerous entry sites on the X chromosome have been described, and a hierarchy of entry sites has been suggested with different affinities for the DCC. Even cryptic binding sites on autosomes and at the chromocenter are recognized by the DCC in certain conditions. It is proposed that increasing SU(VAR)3-7 amounts on the X chromosome results in an enrichment of HP1 and H3K9 dimethylation, and leads to a more compact heterochromatic-like structure of the X chromosome which then blocks access to the high-affinity entry sites. The free DCC, chased from the X chromosome sites turns toward low-affinity sites present on autosomes, but not toward those embedded into the chromocenter. Indeed, cryptic chromocenter sites become more inaccessible by heterochromatin compaction, a phenomenon also responsible for the enhancement of variegation by increased SU(VAR)3-7 levels. Inversely, the absence of SU(VAR)3-7 induces a more relaxed chromatin state at the chromocenter (Spierer, 2005), thus increasing affinity of the entry sites embedded into heterochromatin, and allowing MSLs binding at the chromocenter. Similar recruitments of MSLs at heterochromatin have been described in the literature in three situations: (1) in roX1roX2 mutants, (2) in presence of excess of MSL2 and in (3) C-terminal truncated MSL2 mutants. This means that cryptic entry sites present in heterochromatin become more accessible to the MSLs either in a Su(var)3-7 mutants or if DCC composition is modified. The explanation of heterochromatin affinity for the MSLs remains obscure. On the X chromosome, the Su(var)3-7 mutation induces the bloated morphology resembling that described as a result of decreased levels of silencing factors as HP1, ISWI and NURF, or of increased MSLs levels. The current study and others suggest that male X chromosome morphology depends on the balance between silencing and activating complexes. The simultaneous existence of the repressive SU(VAR)3-7/HP1 proteins and the MSLs complex may provide a set of potential interactions that cumulatively regulate dosage compensation (Spierer, 2008).

Several arguments support a role for SU(VAR)3-7 in dosage compensation. Reduced male viability in the progeny of Su(var)3-7 homozygous females is a first argument for a function played by the protein specifically in males. The results also show that wild-type amounts of SU(VAR)3-7 are required to cope with increased MSL1 and MSL2 levels. In absence of maternal SU(VAR)3-7 product, the transgenes expressing MSL1 and MSL2 become toxic to males, whereas no lethality is observed with wild-type or half amounts of SU(VAR)3-7. This suggests that SU(VAR)3-7 is required very early in development to counteract an excess of MSL1 or MSL2 activity. Corroborating this effect, it was determined that the global amount of heterochromatin affects the viability of females engineered to expressing msl2. The presence of the highly heterochromatic Y chromosome kills half of the females expressing msl2. It has been proposed that the Y chromosome functions as a sink for heterochromatic factors as SU(VAR)3-7 and HP1). A Y chromosome added to XX females could sequester heterochromatic proteins, and induce lethality in a context of female dosage compensation. All these data lead to the conclusion that SU(VAR)3-7 is required for the viability of dosage-compensated flies. Two explanations are proposed: 1) Either SU(VAR)3-7 is required to restrict DCC on the X chromosome and the lethality induced by the lack of SU(VAR)3-7 is due to the MSLs ectopic activity outside of the X chromosome (at heterochromatin), or 2) SU(VAR)3-7 is required on the dosage compensated X chromosome and, in this case, the Su(var)3-7 mutant lethality results from malfunctioning of the DCC on the X (Spierer, 2008).

To discriminate between these two hypothesis, expression of X-linked genes was examined in Su(var)3-7 mutants. Although small changes are visible, the RT-PCR analysis did not sufficient to allow the concluion that the lack of SU(VAR)3-7 affects significantly the levels of transcripts of seven X-linked genes. If they exist, changes were indeed expected to be very small. For MSLs mutations, the magnitude of the decrease is very modest considering the severe failure of dosage compensation (around 1.5). Taking into account that the Su(var)3-7 mutation induces only 30% lethality among males, expected changes in transcript accumulation are predicted to be even smaller. Moreover, transcripts analysis was done in male larvae and some slight biological variations between the three samples cannot be avoided though great care was taken on samples homogeneity. Finally, normalizing to internal autosomal genes RNA could also introduce a bias. It is believed that in the case, quantitative RT-PCR experiment was not the appropriate method to detect very small changes of expression (Spierer, 2008).

In consequence, an alternative system was used to test the implication of SU(VAR)3-7 on dosage compensation. The effect of increased or decreased Su(var)3-7 expression on the dosage compensated expression of the white gene carried by P transgenes was determined. Strikingly, it was observed that lack and excess of SU(VAR)3-7 decreases the white expression specifically in males, and never in females. This is a strong indication that the wild type dose of SU(VAR)3-7 is required for correct dosage compensated expression of the white gene. Interestingly, Su(var)3-7 over-expression affects white expression when the gene is localized on the X chromosome and not on autosomes, although white is still partially dosage compensated on autosomes. This may result from the combination of two phenomena: On the X chromosome, excess of SU(VAR)3-7 induces preferential enrichment of heterochromatic silencing proteins and partial loss of MSLs. On autosomes, heterochromatic proteins recruitment is less visible and, in addition, the MSLs are massively present. Consequently the dosage compensation of a P(white) transgene linked to the X chromosome is more likely to be perturbed by excess of SU(VAR)3-7 than an autosomal insertion (Spierer, 2008).

In sum, in this study has revealed a role for SU(VAR)3-7 on global X chromosome morphology with an impact on the distribution of MSLs proteins, thus highlighting the contribution of SU(VAR)3-7 to the intriguing issue of X specific DCC targeting. It appears also that SU(VAR)3-7 is required for the viability of dosage compensated flies and the expression of a dosage compensated X-linked gene, suggesting a puzzling interplay between heterochromatin and the DCC. SU(VAR)3-7 plays a subtle role on dosage compensation: Flies need SU(VAR)3-7, especially the maternal protein, for correct dosage compensation but, at the same time, excess of SU(VAR)3-7 has a negative effect on dosage compensation. Future interest will focus on the fascinating issue of the molecular nature of heterochromatin/DCC intersection (Spierer, 2008).

Dual sex-specific functions of Drosophila Upstream of N-ras in the control of X chromosome dosage compensation

Dosage compensation in Drosophila involves the assembly of the MSL-2-containing dosage compensation complex (DCC) on the single X chromosome of male flies. Translational repression of msl-2 mRNA blocks this process in females. The ubiquitous protein Upstream of N-ras (Unr) is a necessary co-factor for msl-2 repression in vitro. In mammals Unr interacts with PABP (see Drosophila Pabp) within complexes that bind to distinct regions in the target transcripts. This study explored the function of Drosophila Unr in vivo. Hypomorphic Unr mutant flies show DCC assembly on high-affinity sites in the female X chromosomes, confirming that Unr inhibits dosage compensation in female flies. Unexpectedly, male mutant flies and Unr-depleted SL2 cells show decreased DCC binding to the X chromosome, suggesting a role for Unr in DCC assembly or targeting. Consistent with this possibility, Unr overexpression results in moderate loss of DCC from the male X chromosome and predominant male lethality. Immunoprecipitation experiments revealed that Unr binds to roX1 and roX2, the non-coding RNA components of the DCC, providing possible targets for Unr function in males. These results uncover dual sex-specific functions of Unr in dosage compensation: to repress DCC formation in female flies and to promote DCC assembly on the male X chromosome (Patalano, 2009).

Dosage compensation is the process that equalizes the level of X-linked gene expression between males (XY) and females (XX). In Drosophila, dosage compensation occurs by increasing transcription of the single male X chromosome by ~2-fold. Hyper-transcription requires the binding of the dosage compensation complex (DCC) to hundreds of sites along the male X chromosome. The DCC is composed of five proteins (MSL-1, MSL-2, MSL-3, MLE and MOF), the mutation of which causes male-specific lethality, and for this reason the DCC is also known as the male-specific lethal (MSL) complex. The DCC also contains two non-coding RNAs (roX1 and roX2) that appear to have redundant functions. MSL-2 is a limiting RING finger protein that, together with MSL-1, nucleates the assembly of the DCC. MLE (Maleless) is a helicase thought to be required for stable integration of roX RNAs into the DCC, whereas MSL-3 is a chromodomain protein, and MOF (Males absent on the first) is an acetyl-transferase that promotes the acetylation of histone H4 on lysine 16 (H4K16), a modification that specifically marks the compensated X chromosome. Other proteins, in addition to the DCC components, have been implicated in dosage compensation, including the H3S10 kinase JIL-1, the DNA supercoiling factor (SCF), the chromatin-binding protein SU(VAR)3-7, and the nuclear pore components Mtor and NUP153 (Patalano, 2009 and references therein).

In female flies, dosage compensation is inhibited because the expression of msl-2 is repressed by the female-specific RNA-binding protein Sex lethal (Sxl). Enforced expression of MSL-2 leads to the assembly of the DCC on both female X chromosomes and to lethality. Sxl binds to both untranslated regions (UTRs) of msl-2 pre-mRNA and inhibits first the splicing of a facultative intron in the 5' UTR of the transcript, and then its translation in the cytoplasm. Translational repression of msl-2 by Sxl occurs by a double-block mechanism whereby Sxl bound to the 3' UTR inhibits the recruitment of the small ribosomal subunit, and Sxl bound to the 5' UTR inhibits the scanning of those subunits that presumably have escaped the 3'-mediated control. Studies performed in cell-free translation extracts and cultured cells have shown that translational repression requires the recruitment of the co-repressor Upstream of N-ras (Unr) to sequences adjacent to the Sxl binding sites in the 3' UTR (Abaza, 2006; Duncan, 2006). Unr is an evolutionarily conserved RNA-binding protein that contains five cold-shock domains (CSDs) and two glutamine (Q)-rich regions. The first CSD (CSD1) mediates interactions with Sxl and msl-2 mRNA, whereas the N-terminal third of the protein carries most of the translational repression function in vitro (Abaza, 2008). Although Unr is a ubiquitous, primarily cytoplasmic protein that is present in both males and females, it binds to msl-2 only in females because its association depends on Sxl. Thus, Sxl provides a sex-specific function to Unr (Patalano, 2009).

To gain insight into the roles of Unr in development, hypomorphic mutant flies that lack the C-terminal half of Unr were analyzed, as well as flies that overexpress full-length Unr or a fragment containing CSDs 1 and 2. In Unr hypomorphic mutant females, the DCC was detected on a limited set of high-affinity sites on the X chromosomes, indicating that, as predicted from translation studies, Unr represses DCC formation in females. Unexpectedly, Unr mutant males showed decreased DCC recruitment to the X chromosome. Consistent with this, Unr knockdown in male Drosophila SL2 cells abrogated DCC binding without affecting the levels of DCC components or their nucleocytoplasmic distribution. In addition, flies overexpressing Unr showed preferential male lethality and DCC recruitment defects, and the X chromosome of both mutant and transgenic Unr males exhibited an altered morphology. Importantly, roX1 and roX2 RNAs co-immunoprecipitated with Unr in males, suggesting that Unr might function by targeting these non-coding RNAs. These results uncover new roles for Unr in the regulation of dosage compensation in males by a mechanism that is independent of msl-2 translation (Patalano, 2009).

Specific recruitment of Unr to the 3' UTR of msl-2 mRNA by Sxl is required for repression of msl-2 translation both in vitro and in cell culture (Abaza, 2006; Duncan, 2006). A prediction from these results is that Unr represses dosage compensation in female flies. Indeed, in hypomorphic mutant females lacking the C-terminal half of Unr, the DCC assembles on a set of X chromosomal sites. These sites map closely with positions previously described as being high-affinity sites, which are occupied by the DCC in conditions of low complex concentration. These observations suggest partial derepression of msl-2 translation in mutant females. Two of the high-affinity sites correspond to the loci for roX1 and roX2 RNAs (cytological positions 3F and 10C, respectively). Expression of these RNAs requires MSL-2 and their stability depends on their association to the DCC complex. The fact that roX levels were similarly low in mutant and wild-type females supports the notion that msl-2 translational derepression in the mutant is only partial. These results indicate that the N-terminal half of Unr exerts strong translational inhibition in vivo, and are consistent with in vitro data showing that amino acids 1-397 of Unr are sufficient for translational repression in functional tethering assays (Abaza, 2008). Appropriate Unr levels are essential for viability and development because moderate (~2-fold) overexpression of Unr results in complete lethality early in development for both males and females. Accordingly, keeping the correct stoichiometry between Unr and Sxl is important for translational control of msl-2, and might be necessary for the regulation of other substrates (Patalano, 2009).

Unexpectedly, Unr mutant males showed decreased MSL-2 staining on the X chromosome, and Unr-depleted SL2 cells showed MSL-2 delocalization from the X chromosome and redistribution in the nucleoplasm. Reduced MSL-2 targeting to the X chromosome correlated with defective recruitment of other DCC components. These effects were independent of variations in MSL-2 levels, consistent with the observation that Unr does not bind to msl-2 mRNA in males (Abaza, 2006). Because DCC targeting defects have been observed under conditions of unbalanced concentrations of MSL proteins or disturbed MSL/roX ratios, it was reasoned that Unr might regulate the levels of other DCC constituents in males. Strikingly, however, the levels and nucleocytoplasmic distribution of all DCC protein components remained unaltered in Unr-depleted cells. Similarly, the levels of roX RNAs in Unr mutant flies or Unr-depleted cells were indistinguishable from those in the wild type. It is concluded that Unr does not interfere with the expression or localization of DCC components (Patalano, 2009).

In principle, Unr could affect DCC recruitment in males either directly or indirectly. A direct effect could be mediated by MLE and roX. Compared with other DCC proteins, binding of MLE to the X chromosome was more severely affected by Unr mutation or overexpression. MLE is loosely associated with the DCC: the presence of MLE in purified DCC complexes requires protection from RNA degradation and low salt conditions. In addition, RNase treatment of polytene chromosomes removes MLE from the DCC, suggesting that MLE recruitment to the X chromosome requires roX RNAs. Conversely, MLE is an RNA helicase necessary for roX incorporation into the DCC and its helicase activity is necessary for spreading of the DCC along the X chromosome. Thus, the binding of MLE and of roX RNAs to the X chromosome appear to be interdependent. A possible explanation for the role of Unr in males is that Unr affects the function of these DCC components. Unr is a CSD-containing protein and, in bacteria, CSD proteins associate with RNA helicases to modify the structure of RNA and regulate gene expression (reviewed by Horn, 2007). Indeed, mammalian Unr binds to the IRES of Apaf1 mRNA and modifies its conformation (Mitchell, 2003). Therefore, Unr might associate with MLE in order to promote the appropriate structure of the roX RNAs for incorporation into the DCC or for subsequent spreading along the X chromosome. In support of this hypothesis, Unr specifically binds to both roX1 and roX2 RNAs in males. In addition, as previously observed in blastoderm embryos, a fraction of Unr localizes to the nucleus of SL2 and salivary gland cells, where both MLE and roX concentrate (Patalano, 2009).

Unr could also function indirectly, via the regulation of chromatin structure, to promote DCC recruitment to the X chromosome. The Unr hypomorphic mutant and the transgenic Unr flies show abnormal packaging of the male X chromosome, consisting of bloated or knotted chromatin. The observation that staining of histone H3 appears normal suggests that the first level of chromatin compaction remains unaltered in Unr mutants. In order to regulate chromatin structure, Unr could interact with chromatin remodeling factors. For example, a member of the trithorax group, ALL-1 (MLL -- Human Gene Nomenclature Database), was found to interact with human Unr (CSDE1) in a yeast two-hybrid assay. Alternatively, Unr could control the expression of chromatin regulators that influence X chromosome morphology, such as ISWI, NURF, JIL-1 or SU(VAR)3-7. It is interesting to note that although mutations of most of these factors do not concur with loss of DCC binding, null mutations of Su(var)3-7 result in both a bloated X chromosome and depletion of the DCC from the X chromosome. Thus, Unr could regulate the expression of SU(VAR)3-7 -- or of other regulators with similar functions -- in order to modulate DCC recruitment. In summary, at this point the results do not allow conclusion of whether the chromatin-packaging and DCC-binding defects observed in males are dissociable events. Nevertheless, the fact that Unr binds to roX RNAs implicates a direct role of Unr in DCC recruitment. Further studies are necessary to clarify the relationship between the multiple nuclear functions of Unr (Patalano, 2009).

The results show that Unr performs opposing functions in the regulation of dosage compensation in males and females. Dosage compensation is evolutionarily linked to sex determination. In D. melanogaster, a single master protein regulates both processes: Sxl determines the female sexual fate and represses dosage compensation. However, Sxl is not sex-specifically expressed in other distant species of Diptera, raising the possibility that the use of Sxl for sex determination is a recent adaptation of the Drosophila genus (Pomiankowski, 2004). Perhaps, Sxl made use of an existing regulator of dosage compensation, namely Unr, and adapted its function to a new role in females. Further genetic studies and biochemical analyses will help to identify the interactors and substrates that mediate the diverse roles of Unr (Patalano, 2009).

Functional domains of Drosophila UNR in translational control

Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (Sxl) is an essential regulatory step of X chromosome dosage compensation in Drosophila. Translation inhibition requires that Sxl recruits the protein upstream of N-ras (Unr) to the 3' UTR of msl-2 mRNA. Unr is a conserved, ubiquitous protein that contains five cold-shock domains (CSDs). This study dissected the domains of Unr required for translational repression and complex formation with Sxl and msl-2 mRNA. Using gel-mobility shift assays, the domain involved in interactions with Sxl and msl-2 was mapped specifically to the first CSD (CSD1). Indeed, excess of a peptide containing this domain derepressed msl-2 translation in vitro. The CSD1 of human Unr can also form a complex with Sxl and msl-2. Comparative analyses of the CSDs of the Drosophila and human proteins together with site-directed mutagenesis experiments revealed that three exposed residues within CSD1 are required for complex formation. Tethering assays showed that CSD1 is not sufficient for translational repression, indicating that Unr binding to Sxl and msl-2 can be distinguished from translation inhibition. Repression by tethered Unr requires residues from both the amino-terminal Q-rich stretch and the two first CSDs, indicating that the translational effector domain of Unr resides within the first 397 amino acids of the protein. These results identify domains and residues required for Unr function in translational control (Abaza, 2008).

Translational control is widely used in development to regulate processes such as embryonic patterning, cell differentiation, synaptic plasticity, sex determination, and dosage compensation. Dosage compensation is the process that equalizes the expression of X-linked genes in those organisms in which sex determination relies on highly dimorphic sex chromosomes. In Drosophila, dosage compensation is achieved by increasing the transcriptional output of the single male X chromosome by approximately twofold, as a result of the activity of a ribonucleoprotein assembly known as the dosage compensation complex (DCC) or male-specific-lethal (MSL) complex. The DCC fails to assemble in females because the expression of one of its subunits, the protein MSL2, is blocked. The female-specific RNA-binding protein Sex-lethal (Sxl) prevents msl-2 expression via a dual mechanism that includes the inhibition of the splicing of a facultative intron in the 5' UTR of msl-2 pre-mRNA, and the subsequent translational repression of the unspliced message. Translational repression requires Sxl binding to specific U-rich sequences in both the 5' and 3' UTRs of msl-2 mRNA. Sxl binding to the 3' UTR is thought to inhibit the recruitment of the small ribosomal subunit to the mRNA, while Sxl binding to the 5' UTR blocks the scanning toward the AUG initiation codon of those subunits that presumably have escaped control through the 3' UTR. How Sxl inhibits these steps of translation initiation is unknown. Recently, a factor necessary for Sxl-mediated translational repression has been identified as the protein upstream of N-ras (Unr) (Abaza, 2006; Duncan, 2006). Unr is a conserved, ubiquitous protein that is recruited to the 3' UTR of msl-2 by Sxl, but its mechanism of action remains obscure (Abaza, 2008).

Most of the current knowledge about Unr derives from mammalian systems. Human Unr (hUnr) is involved in c-fos mRNA destabilization and the translational repression of pabp mRNA. In both cases, Unr interacts with PABP within complexes that bind to distinct regions in the target transcripts. Mammalian Unr also regulates translation driven by the internal ribosome entry sites (IRESs) of a number of viral and cellular transcripts, including rhinovirus, poliovirus, c-myc, PITSLRE protein kinase, the pro-apoptotic factor Apaf-1, and Unr itself. At least in the case of Apaf-1, hUnr acts as an RNA chaperone, changing the conformation of the IRES to make it accessible to the activator PTB and, ultimately, the ribosome. RNA binding by hUnr is mediated by its five cold-shock domains (CSDs), an ancient β-barrel fold containing RNP1 and RNP2 motifs. Drosophila Unr (dUnr) contains an additional Q-rich amino terminus that is absent in its mammalian counterpart (Abaza, 2008 and references therein).

The CSD is a domain highly conserved in evolution used to bind single stranded nucleic acids. In addition, the CSD can support protein-protein interactions. Indeed, the CSD1 of Drosophila Unr sustains both binding to msl-2 mRNA and Sxl. Three specific residues within dCSD1 are responsible for these interactions: a tyrosine (Y) that is part of the RNP1 motif, and a lysine (K), and aspartic acid (D), which lay outside the RNP motifs. Although the assay used does not allow distinction between mRNA and protein binding, the location of these amino acids suggests that Y likely mediates msl-2 binding, while K and D may be involved in Sxl interaction. The data do not formally rule out that other domains of dUnr contribute separately to bind either Sxl or msl-2. However, this possibility is unlikely because the efficiency of binding of dCSD1 alone is identical to that of the full-length protein. The use of a dedicated CSD for RNA binding contrasts with the known properties of mammalian Unr. All five CSDs of hUnr are required to bind to the rhinovirus IRES (Brown, 2004). The fact that hUnr can bind to msl-2 mRNA in isolation while dUnr cannot, indeed suggests that the two proteins have different modes of RNA binding (Abaza, 2008).

In order to map the translational effector domain of Unr, tethering analysis was performed. Translational repression by tethered dUnr was less efficient than that observed for Sxl in its natural context, suggesting that Sxl function in 3' UTR-mediated repression is not limited to the recruitment of dUnr. Alternatively, the lesser efficiency of dUnr in repression could be due to aberrant conformation of the recombinant protein or to geometry constraints imposed on the tethered complex. In support for the latter, even though Sxl is critical for msl-2 translational repression, it does not function when tethered to the 3' UTR (Abaza, 2008).

Tethering assays show that dCSD1 is not sufficient for translational repression, indicating that elements in addition to Sxl and msl-2 binding are required for inhibition. These could include the interaction with other corepressors or with components of the translational apparatus. Similar to dCSD1, tethered hUnr could not support translational repression, implying that the translational effector domain is lacking from hUnr. An obvious domain absent in hUnr but present in its Drosophila counterpart is the N-terminal Q-rich domain. This domain contains 52 glutamines interrupted mainly by histidines, resulting in a highly polar stretch suitable for interactions. Certainly, Q-rich domains are present in proteins with diverse roles in gene expression and serve as protein-protein interaction and multimerisation modules. To test whether the Q-rich domain could confer translational repression, it was deleted from dUnr and fused to hUnr. dUnr lacking the Q-rich domain repressed translation less efficiently than the intact protein, indicating that the Q-rich domain was necessary for optimal repression. However, the Q-rich domain did not confer a significant translational repression activity to hUnr, suggesting that residues within the CSDs specific to the Drosophila protein were also relevant. Importantly, the fragment containing the Q-rich domain fused to dCSDs 1 and 2 showed a strong translational repression activity, indicating that the translational effector domain of dUnr is embedded within the first 397 amino acids of the protein. Consistent with these results, analysis of Unr mutant flies indicates that the N-terminal half of Unr exerts robust repression of dosage compensation in females (Abaza, 2008).

TIA-1, a splicing and translation regulator, contains a Q-rich C-terminal domain that interacts with the protein U1C facilitating the recruitment of the U1 snRNP to the 5' splice site. By analogy, the Q-rich domain of dUnr could facilitate the recruitment of corepressors, or components of the translation machinery that are so sequestered, to the 3' UTR of msl-2. One such component could be PABP. This translation factor has been shown to interact with hUnr in complexes binding to the coding region of c-fos mRNA and the 5' UTR of pabp mRNA, which are involved in destabilization and translational repression, respectively . However, it is not immediately obvious how PABP recruitment to the 3' UTR of msl-2 would result in repression, because PABP stimulates translation when tethered to the 3' as it does when it binds to the poly(A) tail. Furthermore, substantial translational repression by the Unr:Sxl complex occurs on nonadenylated msl-2 mRNA. Thus, even though PABP could play a role, additional factors are involved in translational repression by dUnr (Abaza, 2008).

In summary, these data delimit the functional domains of dUnr in msl-2 translational repression. Finding out which factors interact with the translational effector domain will help gain insight into the molecular mechanism of translation inhibition by this essential protein (Abaza, 2008).

Nuclear pore components are involved in the transcriptional regulation of dosage compensation in Drosophila

Dosage compensation in Drosophila is dependent on MSL proteins and involves hypertranscription of the male X chromosome, which ensures equal X-linked gene expression in both sexes. This paper reports the purification of enzymatically active MSL complexes from Drosophila embryos, Schneider cells, and human HeLa cells. A stable association of the histone H4 lysine 16-specific acetyltransferase MOF was found with the RNA/protein containing MSL complex as well as with an evolutionary conserved complex. The MSL complex interacts with several components of the nuclear pore, in particular Mtor/TPR and Nup153. Strikingly, knockdown of Mtor or Nup153 results in loss of the typical MSL X-chromosomal staining and dosage compensation in Drosophila male cells but not in female cells. These results reveal an unexpected physical and functional connection between nuclear pore components and chromatin regulation through MSL proteins, highlighting the role of nucleoporins in gene regulation in higher eukaryotes (Mendjan, 2006).

All Drosophila MSL proteins have mammalian orthologs. To address the evolutionary conservation, the human hMOF-containing complexes were purified from a stable HeLa cell line expressing hMOF tagged with one haemagglutinin (HA) and two FLAG epitopes (HA-2xFLAG-hMOF). The characterization of the interacting proteins revealed striking similarities in the complex composition between flies and humans (Mendjan, 2006).

Copurification of mammalian MSL orthologs showed that DCC is an evolutionary conserved protein complex. hMSL1, hMSL2, and hMSL3 were all present in the hMOF complex. Similar to Drosophila DCC, RNA helicase A (the ortholog of MLE) was not present in the complex, which is consistent with previous observations. Furthermore, two isoforms of hMSL3, hMSL3a and hMSL3c, were identified, copurifying with hMOF. The former represents the full-length protein, while the latter is an alternative splice isoform lacking the N-terminal chromobarrel domain (Mendjan, 2006).

In addition to the MSL proteins, most of the other proteins copurifying with TAP-MOF were also found in the hMOF complex. Z4 and Chriz/Chromator (Chr) lack clear mammalian orthologs, which could explain their absence. However, the Mtor ortholog TPR was identified in the HA-2xFLAG-hMOF purification. Human-specific proteins included the transcriptional coactivator HCF-1, O-linked N-acetylglucosaminetransferase OGT, and the forkhead and FHA domain containing transcription factor ILF-1/FOXK2. Interaction of hMSL3, hNSL1, hNSL2, hNSL3, and HCF-1 was further confirmed by Western blot analysis of eluted complex. Similar to the TAP-MOF and MSL-3FLAG complexes, the HA-2xFLAG-hMOF complex specifically acetylated histone H4 at lysine 16 on mononucleosomes (Mendjan, 2006).

Taken together, the data demonstrate that MOF interactions are evolutionary conserved and that the DCC is an evolutionary ancient complex that acetylates histone H4 at lysine 16 (Mendjan, 2006).

The purification of the MSL complex revealed quite an unusual complex composition. One would expect that a complex thought to modulate transcription and/or chromatin structure would contain a significant number of classical transcription factors, some of the numerous components associated with RNA polymerase II, or at least subunits of the ubiquitous chromatin remodeling and modifier complexes. However, none of these components was found. Instead, there seems to be a core MSL complex that interacts substoichiometrically with nucleoporins (Mtor, Nup153, Nup160, Nup98, and Nup154), interband binding proteins (Z4, Chromator/Chriz), and exosome components (Rrp6, Dis3) (Mendjan, 2006).

The results suggest that MOF is a subunit of two independent complexes in mammals and fruit flies. Several lines of evidence support this notion. This includes coimmunoprecipitation experiments and glycerol gradient centrifugation. Furthermore, hMOF was recently found in the MLL1 methyltransferase complex together with HCF-1, MCRS2, WDR5, NSL1, and PHF20, but this complex did not contain hMSL1. Finally, purification of the hMSL3 complex provides further evidence that hMSL3 does not associate with many of the MOF-interacting proteins. Therefore, it is suggested that the NSL complex contains at least MOF, NSL1, NSL2, NSL3, MCRS2, MBD-R2, and WDS, and in humans also HCF-1 and OGT (Mendjan, 2006).

The results presented here also suggest a molecular mechanism as to how the MOF complexes bifurcate. Both MSL-1 and NSL1 contain a PEHE domain in their C terminus. The NSL1 PEHE domain interacts directly with hMOF in vitro, and Drosophila MSL-1 has been shown to interact directly with MOF through the same domain. Furthermore, MSL-1 is required for full activity of MOF in vitro and for the assembly of the DCC on the male X chromosome. MSL-1 and NSL1 are the only two genes with a PEHE domain in the Drosophila genome, suggesting that it is an evolutionary conserved MOF-interacting domain. It is postulated that MSL1 and NSL1 serve as mutually exclusive bridging factors that assemble two different complexes around MOF, a histone H4 lysine 16-specific acetyltransferase (Mendjan, 2006).

In the current study, focus was placed on the mechanism of DCC function in Drosophila. All three purifications resulted in enzymatically active complexes with consistent copurification of MSL-1, MSL-2, MSL-3, MOF, roX1, and roX2 but not of MLE or JIL-1. The absence of MLE was expected, since its interaction with MSLs has reported to be salt and detergent sensitive. It is likely that JIL-1, like MLE, is sensitive to the purification conditions used in this study (Mendjan, 2006).

To examine the function of the new interacting proteins in dosage compensation, mutant flies were studied and RNAi was used in cell culture. In Z4 mutants or in MBD-R2-depleted SL-2 cells, MSL localization on the X chromosome was not affected. Consequently, these proteins are not required for MSL recruitment, or they have an alternative function with MOF that is independent of its role in dosage compensation (Mendjan, 2006).

However, an unexpected link was discovered between dosage compensation and the nuclear pore. Depletion of either Mtor or Nup153 but not of other nucleoporins or NXF1 delocalized MSL proteins from the X chromosome. The effects observed were not due to a general transport defect, since all the five MSL proteins and roX2 RNA remained nuclear in Mtor- and Nup153-depleted cells, and no accumulation was observe of bulk mRNA in these cells. Consistent with these observations, Mtor and Nup153 are required for proper dosage compensation of several classical MSL-dependent dosage-compensated genes in SL-2 cells. The expression of these genes was not affected in female Kc cells (Mendjan, 2006).

An important question raised from this study is whether the observed effects are due to a soluble fraction of Mtor and Nup153 in the nucleus or due to their function as components of the NPC. The latter is favored: (1) Nup153 staining is exclusively peripheral; (2) depletion of Nup153 delocalizes Mtor from the nuclear periphery and increases the soluble pool of Mtor in the nucleoplasm, but MSL proteins still remained delocalized in Nup153-depleted cells; (3) the fact that several nucleoporins, which exist together only at the nuclear pore, were copurified with the MSL complexes strongly favors the idea that there is an interaction between the DCC and the intact NPC. This interaction is substoichiometric but with clear functional importance for DCC assembly or maintenance on the X chromosome (Mendjan, 2006).

A wealth of information has been generated in budding yeast regarding nuclear organization and gene regulation. For instance, yeast telomeres associate with the nuclear periphery and form a transcriptionally silenced chromatin domain. However, a number of recent studies have shown that nuclear periphery is not just a domain of gene inactivation but also of activation. Consistent with these observations, yeast MLP1 and MLP2 (Mtor orthologs in yeast) associate with transcriptionally active genes and are involved in relocalization of active genes to the nuclear periphery. Furthermore, MLPs are involved in chromatin domain formation and pre-mRNA quality control (Mendjan, 2006 and references therein).

Interestingly, in Schneider cells, male embryos, salivary glands, and imaginal discs, the Drosophila male X chromosome appears localized at or near the nuclear periphery and in most cases even follows the nuclear rim curvature. The inactive X in mammals also localizes close to the nuclear periphery as the Barr body. Like the Drosophila male X chromosome, the inactive X has to be globally controlled (inactivated) and is characterized by a special histone modification (trimethylation of lysine 27 of histone H3). Another common feature between mammals and Drosophila is that noncoding RNAs play an essential role. A possible model that can account for these intriguing similarities is that the nuclear periphery is used to generate transcriptional domains that can be transcriptionally active or inactive in order to achieve coregulation of gene expression for a subset of genes. In the case of the Drosophila male X chromosome, hundreds of genes with different basal transcriptional properties need to be coactivated by a factor of two. This kind of a subtle transcriptional coregulation of a whole chromosome may be achieved by partial compartmentalization of the X chromosome mediated by the nucleoporin-MSL interaction, allowing the formation of hyperacetylated chromatin domains with unique transcriptional and/or posttranscriptionalproperties (Mendjan, 2006).

It is important to emphasize that Mtor and Nup153 may be required for general chromatin organization (not just individual chromosomes) through their interaction with chromatin-associated proteins. The DCC might mediate X-chromosomal tethering to the nuclear pore as a mechanism to coregulate a large set of genes by creating chromosomal loops or domains. This could happen by direct or indirect interactions of MSLs with Mtor/Nup153 located at or near high-affinity sites along the X chromosome, which are the binding sites of the DCC. Interactions with nuclear pore components may also be used to 'economize resources' and/or for efficient coupling of transcription to processing of the newly transcribed coregulated messages (Mendjan, 2006).

In summary, the purification of the MSL complex has revealed an unexpected link between dosage compensation and the NPC. In the context of data from other systems, this allows formulation of new hypotheses about the mechanism of dosage compensation that will be exciting to test in the future (Mendjan, 2006).

The dosage compensation complex shapes the conformation of the X chromosome in Drosophila

The dosage compensation complex (DCC) in Drosophila globally increases transcription from the X chromosome in males to compensate for its monosomy. A male-specific conformation of the X chromosome was discovered that depends on the associations of high-affinity binding sites (HAS) of the DCC. The core DCC subunits MSL1-MSL2 are responsible for this male-specific organization. Contrary to emerging concepts, it was found that neither DCC assembly nor the conformation of the male X chromosome are influenced by nuclear pore components. It is proposed that nuclear organization of HAS is central to the faithful distribution of the DCC along the X chromosome (Grimaud, 2009).

This study assessed the conformation of the X chromosome by measuring the 3D distances between selected HAS for the DCC. FISH probes were developed specific for the ultraspiracle (usp) locus located at cytological position 2C, the roX2 locus located at 10C, and the Nup153 locus located at 14F, containing HAS. The specificity of each probe for the selected locus was verified by FISH on polytene chromosomes of third instar larvae. They were then used to determine the 3D distances between pairs of loci in various tissues and genetic conditions. Each two-color FISH experiment was followed by MSL2 immunostaining, which reveals the X-chromosomal territory and distinguishes male and female tissues. Interphase nuclei in the epidermal layer of wild-type (yw) embryos were selected at stages 9-12, in which dosage compensation is effective. A calculation of the average nuclear radius, together with a comparison of the relative 3D distances of two autosomal loci, demonstrated the absence of general differences between male and female nuclei. Then the 3D distances between the different HAS located on the X chromosome were compared. Due to the pairing between homologous chromosomes in Drosophila, only one FISH signal for each probe was observed in the majority of female nuclei. Analysis of male and female nuclei using the roX2 and usp probes revealed a continuum of distances with a striking difference between male and female nuclei. The significance of distance differences between the loci in the two sexes was evaluated by a two-tailed Mann-Whitney U-test. The two loci were significantly closer in males than in females. In 27.5% of the male nuclei, usp and roX2 were within 0.5 microm, whereas this was only the case in 4% of the female nuclei. Visualization of the male X chromosome by MSL2 immunostaining showed that usp and roX2 resided within the MSL2 domain in 75% of the nuclei, and that the long-distance association between these two loci occurred within this domain (Grimaud, 2009).

The usp and Nup153 loci are physically separated by 14.5 Mb on the X chromosome. Strikingly, this increased linear distance was not reflected in space. The loci were found within 0.5 microm in 29.4% of the male nuclei and in only 9.9% of the female nuclei. Accordingly, the roX2 and Nup153 loci were also significantly closer in males. In this case, the physical proximity of these sites, separated by only 5 Mb, is reflected by a smaller average distance between them in female nuclei also (24.2% of the signals within 0.5 microm). Taken together, the data document long-range associations between three strong DCC-binding sites. The fact that this proximity was sex-specific shows that the X chromosome in male nuclei differs in conformation from the female X. This conformation may be brought about by the clustering of the HAS in space. Alternatively, a male-specific chromosome conformation may promote close contacts between HAS (Grimaud, 2009).

In order to determine whether the observed long-range associations were specific for DCC-binding sites, two further probes were developed that label X chromosomal sequences that do not bind DCC in male nuclei. The probe located around the gene dpr8 at cytological position 12F resides in an area of ~300 kb that is devoid of DCC. Two-color FISH on whole-mount embryos showed no significant distance difference between dpr8 and usp in female or male nuclei. In contrast to the three HAS, the dpr8 locus was found outside of the territory in ~70% of the nuclei. Remarkably, the 3D distances between dpr8 and the roX2 or Nup153 loci, which are relatively close on the physical map of the chromosome (3 Mb and 2 Mb, respectively), were significantly shorter in female nuclei than in males, as if X-chromosomal sequences not bound by DCC were driven out of the area of clustering into the periphery of the DCC territory. At later stages of development, the observations made for dpr8 were reproduced with even more striking values. The use of a fifth probe located around the CG9650 gene at cytological position 7A, which is 60 kb away from a HAS, confirmed that DCC-free sequences present on the X do not reside in the DCC territory and do not preferentially associate in male nuclei. The CG9650 locus does not particularly reside in the vicinity of the roX2, the Nup153, or the dpr8 loci in female or in male nuclei. This locus is relatively close to the usp locus in female nuclei, which mainly reflects the linear placement of the loci on the X chromosome. In male nuclei, the loci significantly separate. In summary, the data show that HAS tend to cluster in male nuclei, but sites unbound by the DCC are more likely to be excluded from the core of the dosage-compensated chromosomal territory (Grimaud, 2009).

In order to analyze whether DCC subunits affect the conformation of the male X chromosome, MSL1, MSL2, MSL3, or MOF were depleted by inducible RNAi. The efficiency of the specific ablation of those proteins using the SGS3-GAL4 driver was verified on polytene chromosomes. Knockdown of MSL1 prevented the binding of MSL2 and vice versa, in line with earlier genetic analyses. Crossing homozygous flies of the 69B-GAL4 driver line with flies homozygous for the RNAi constructs directed against each DCC member, male third instar larvae were obtained where each of MSL1, MSL2, MSL3, or MOF were strongly depleted in the wing discs. The effectiveness of the knockdown was confirmed by strongly reduced numbers of adult male flies obtained from these crosses. The 3D distances between the usp and roX2 loci or between the usp and Nup153 loci were monitored under those conditions and it was found that the absence of MSL1 or MSL2 strongly affected the association between these loci. Remarkably, the distance distribution in male MSL1RNAi or MSL2RNAi nuclei resembles that obtained in female nuclei for the same probes. The sex-specific chromosome conformation therefore, depends on these core DCC subunits, in line with earlier observations that MSL1-MSL2 alone are capable of directing the DCC to HAS. Conversely, the efficient knockdown of MSL3 or MOF did not affect the male-specific proximity between the roX2 or the Nup153 and the usp loci. MSL3 and MOF are rather implicated in the spreading of the DCC from HAS to transcribed sequences, which constitute low-affinity binding sites for DCC (Larschan, 2007). The preferential interaction of MSL3 with the transcription-dependent H3K36 methylation mark promotes recruitment of the other DCC components to these low binding affinity sites. Acetylation of H4K16 mediated by MOF would facilitate the acquisition of opened chromatin structure, increasing the probability of a new event of transcription onto these novel DCC-binding sites (Grimaud, 2009).

Recognition of HAS by MSL1-2 is an early event in the multistep process of recruitment of DCC to the X chromosome. The sex-specific organization of HAS, which is mediated by MSL1-2, may thus contribute to assuring the efficient distribution of the complex. The X chromosome is envisioned to be structured in zones of decreasing DCC concentrations. DCC may be assembled at the site of roX RNA transcription within the X chromosome territory, containing HAS for MSL1-2. Sites of highest affinity for DCC may be clustered more internally, surrounded by layers of lower affinity, including active genes marked by H3K36 methylation. About 25% of X-chromosome sequences are bound by DCC, and these constitute the nuclear domain stained by MSL2 antibody. X-chromosomal sequences that do not attract DCC—like intergenic regions, inactive genes, and genes that escape transcriptional dosage compensation are interspersed with DCC-bound domains on the chromosome map. The fact that such a sequence (like dpr8 or CG9650) consistently resided outside of the MSL2 domain suggests that sequences not bound by the DCC may be arranged as an outer layer around the DCC-bound territory. Such an arrangement would be inverse to the one suggested for the inactive X chromosome on mammals (Grimaud, 2009).

The mechanism responsible for the clustering of DCC HAS are as yet unknown. A 'self-assembly' mechanism is favored, based purely on concentrations of factors and interaction affinities. Accordingly, roX genes would initially establish a local focus of DCC enrichment within the X territory due to the assembly of the complex around the nascent roX transcript. HAS that encounter this focus during random chromosome movements will tend to dwell longer in its vicinity. Their presence will in turn lead to further concentration of DCC, establishing a positive feedback loop between DCC enrichment and the clustering of binding sites that will lead to a concentration gradient of DCC centered around the roX genes. Sites of lower affinity due to either the presence of degenerate sequence elements or the H3K36 methyl mark-associated with active transcription will be bound by DCC and incorporated into the domain, whereas loci without DCC-binding sites will tend to be excluded. Because the recognition of HAS relies mainly on MSL1-2, knockdown of MSL3 or MOF does not affect their clustering. However, loss of MSL3-MOF is also lethal for males, which illustrates the importance of distributing the DCC on to the bulk of binding sites at transcribed genes. Further 3D structuring of the chromosome may then depend on transcription; for example, by cohabitation of dosage-compensated transcription units in transcription factories. However, unlike the situation in mammals where the female X chromosomes show extreme differences in expression, the male and female X chromosomes of Drosophila differ in their activity state by up to only twofold. The example of the dosage-compensated X chromosome thus serves as a prominent example for the intimate relationship between chromosome conformation and coordinate regulation of a large number of genes (Grimaud, 2009).

The RING finger protein MSL2 in the MOF complex is an E3 ubiquitin ligase for H2B K34 and is involved in crosstalk with H3 K4 and K79 methylation

This study demonstrates that RING finger protein MSL2 in the MOF-MSL complex is a histone ubiquitin E3 ligase. MSL2, together with MSL1, has robust histone ubiquitylation activity that mainly targets nucleosomal H2B on lysine 34 (H2B K34ub), a site within a conserved basic patch on H2B tail. H2B K34ub by MSL1/2 directly regulates H3 K4 and K79 methylation through trans-tail crosstalk both in vitro and in cells. The significance of MSL1/2-mediated histone H2B ubiquitylation is underscored by the facts that MSL1/2 activity is important for transcription activation at HOXA9 and MEIS1 loci and that this activity is evolutionarily conserved in the Drosophila dosage compensation complex. Altogether, these results indicate that the MOF-MSL complex possesses two distinct chromatin-modifying activities (i.e., H4 K16 acetylation and H2B K34 ubiquitylation) through MOF and MSL2 subunits. They also shed light on how an intricate network of chromatin-modifying enzymes functions coordinately in gene activation (Wu, 2011).

This study describes a histone E3 ligase activity for the MOF-MSL complex. This activity mainly targets nucleosomal H2B K34, a site distinct from well-characterized H2B K120. Both MSL2 and MSL1, but not MOF and MSL3, are indispensable for the optimal activity. Importantly, H2B K34ub by MOF-MSL directly stimulates H3 K4 and K79 methylation through trans-tail regulation. It also affects H2B K120ub by regulating RNF20/40 chromatin association. The significance of MSL1/2-mediated H2B K34ub is underscored by data indicating that MSL1/2 activity is evolutionarily conserved in Drosophila DCC. Altogether, these results shed lights on the intricate network of chromatin-modifying enzymes that often function coordinately in gene activation (Wu, 2011).

Several early studies suggest that histone H2B can be ubiquitylated at sites other than K120 in vivo. For example, a proteomic study using a mouse brain sample identifies five ubiquitylation sites on histone H2B: K34, K46, K108, K116, and K120. Ubiquitylation of endogenous yeast H2B at multiple lysine residues in addition to K123 (K120 equivalent) has also been reported. Despite these observations, functions of these additional H2B ubiquitylation events as well as their respective Ub-conjugating enzymes and E3 ligases are largely unknown. Therefore, this study represents a step toward understanding the potential complexity of H2B ubiquitylation and their functions in transcription regulation beyond H2B K120ub. Comparing the activity of MSL1/2 to that of RNF20/40, several conclusions can be drawn: (1) MSL1/2 activity is lower than that of RNF20/40, contributing to low abundance of H2B K34ub in cells; (2) MSL1/2 has less strict substrate specificity, capable of weakly ubiquitylating H3 and H4 in vitro; and (3) MSL1/2 is mainly a monoubiquitin ligase, but it is able to add polyubiquitin chain to histones at lower efficiency. Given the distinct substrate specificity of MSL1/2 and RNF20/ 40, it is likely that similar to other histone modifications (e.g., methylation, acetylation), H2B ubiquitylation is catalyzed by a multitude of E3 ubiquitin ligases with distinct substrate preferences. Future studies on other RING finger-containing proteins will reveal yet-uncharacterized E3 enzymes and histone targets (Wu, 2011).

Like H2B K120ub, H2B K34ub plays important roles in regulating H3 K4/K79 methylation. It directly stimulates H3 K4 methylation by MLL and H3 K79 methylation by DOT1L in vitro. This result implies that trans-tail interactions between H2Bub and H3 methylation have certain plasticity in terms of H2B ubiquitylation site requirement. This is consistent with a previous report that disulfide mimics of ubH2B (uH2Bss) at K125 and K116 sites are able to stimulate DOT1L activity in vitro (Chatterjee, 2010). Given the demonstrated trans-tail regulation in this study, especially that H2B K34 resides between DNA gyres, it is important to further examine the basis for H2Bub-mediated stimulation of methyltransferase activity. It would be particularly interesting to test whether H2B K34ub stimulates H3 methylation by altering intrinsic nucleosome stability and/or nucleosome breathing mode (Böhm, 2011). In addition to a direct effect on H3 methylation, MSL1/2 also affects H3 methylation indirectly through regulating recruitment of RNF20/40 to chromatin and thus activity of RNF20/40. It was possible to differentiate these two effects by overexpression of RNF20/40 in MSL2 knockdown cells. Both immunoblot and ChIP assays support that MSL1/2 regulates H3 methylation through both direct and indirect mechanisms, further strengthening the case that MSL1/2 regulates an intricate network of histone modifications for transcription activation in cells (Wu, 2011).

Since it was not possible to detect any MOF-independent MSL1/2 heterodimer fraction on a size-exclusion column, it is likely that MSL1/2 always functions as an integral part of the MOF-MSL complex. Taking advantage of the fact that different histone modifying activities require different MOF-MSL components, it was possible to distinguish contributions of H2B K34ub and H4K16ac to transcription activation. MSL1 knockdown, which affects both H2B K34ub and H4 K16ac, has much more profound effects on HOX gene expression than MSL3 knockdown, which only affects H4 K16ac. It indicates functions of MOF-MSL in transcription regulation beyond HAT (Wu, 2011).

The finding that MOF-MSL regulates H2B K120ub and H3 K79me2, two important marks for transcription elongation, supports a role of MOF-MSL in later transcription events. Interestingly, the dMSL complex is implicated in the regulation of transcription elongation in Drosophila: (1) it is recruited to the bodies of X-linked genes, and (2) recruitment of the dMSL complex to ectopic loci with X chromosome enhancer sequences depends on active transcription, and (3) using global run-on sequencing (GRO-Seq), a recent study shows that the dMSL complex functions to facilitate progression of RNA Pol II across the bodies of active X-linked genes (Larschan, 2011). It would be interesting to test whether MOF-MSL in mammals regulates gene expression in the same manner and whether H2B K34ub (relative to H4 K16ac) contributes to the cascade of events associated with elongating Pol II. Future genome-wide analyses of H2B K34ub and MSL1/2 in mammalian cells will be very informative to further dissect the functions of different histone-modifying activities of MOF-MSL in transcription regulation (Wu, 2011).

Several remaining issues for H2B K34ub await future studies. First, how is H2B K34ub regulated in vivo? A series of elegant yeast genetic studies and in vitro biochemical studies show that H2B K120ub is extensively regulated by factors associated with Pol II transcription. They include the PAF complex, Pol II CTD, and Kin28 kinas. In addition, H2B K120ub is dynamically controlled by ubiquitin proteases such as Ubp8 and Ubp10. Given the robust activity of MSL1/2 in vitro and low H2B K34ub level in cells, it is likely that H2B K34ub is under extensive regulation. It is important to examine whether regulation of H2B K34ub and H2B K120ub is the same, or whether it involves different mechanisms despite their shared downstream effects on H3 methylation (Wu, 2011)

Second, what is the function of H2B K34ub in regulating chromatin structures? K34 resides in a highly conserved region in H2B from yeast to mammal. This region consists of a stretch of eight basic residues. Like the basic patch on the H4 N-terminal tail (14-20 aa), this H2B region is also proposed to play important roles in the formation of chromatin fibers by facilitating interactions with neighboring nucleosomes. This H2B basic patch overlaps with a conserved 'HBR' motif (30-37 aa) identified in S. cerevisiae, whose deletion leads to derepression of 8.6% of yeast genes. Given that both H2B K34 and H4 K16 are substrates of MOF-MSL and both reside within a basic patch, it is important to determine whether these two distinct histone modifications play any synergistic roles in regulating higher-order chromatin structures (Wu, 2011).

Finally, does dH2B K31ub play a role in Drosophila dosage compensation? Given the E3 ligase activity of the dMSL complex, it is important to test whether dH2B K31ub marks Drosophila male X chromosome and whether inactive dMSL2 leads to defects in expression of X-linked genes in male fly. It has been reported that dMSL1/2, but not other dMOF complex components, binds to large chromosome domains defined as high-affinity sites (HAS) or chromosome entry sites (CES) and initiates the spreading of H4 K16 acetylation mark along male X chromosome. It is important to examine whether the dMSL1/2 activity plays any role in setting up these HAS/CES sites in vivo. One common feature of HAS/CES is their relatively low nucleosome density compared to other chromatin regions. In light of the E3 activity of Drosophila dMSL1/2, it is important to test whether dMSL1/2 prefers to bind the nucleosome-free region or whether this is a result of nucleo- some destabilization caused by dH2B K31ub (Wu, 2011).

Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context

The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at 'entry sites' that contain a consensus sequence motif ('MSL recognition element' or MRE). However, this motif is only ~2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. This study asked whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), it was found that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. These sites were tested for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. Ectopic MSL expression in Kc cells was shown to lead to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, these results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. This analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome (Alekseyenko, 2012).

This study considered the roles of chromatin environment and flanking sequence composition in selection of functional binding sites by a sequence-specific protein complex. It is generally not clear whether the chromatin features that are often observed at the binding sites of proteins contribute directly to binding selectivity or are simply a consequence of binding. In the dosage compensation system of the X chromosome in Drosophila, a unique opportunity is presented to address this question because it is possible to compare the chromatin environment of MSL binding sites in female cells, in the absence of the complex, to male cells, where the functional sites are bound. Binding data from an RNAi experiment were used in which a component of the sex determination pathway was knocked down in females to induce dosage compensation. Bioinformatic analysis of a large number of profiles from the modENCODE project suggests that a pre-existing active chromatin context plays a critical role in establishing the initial binding of the MSL complex on the X. The surprising discovery was made that GC content in the DNA surrounding functional binding sites has a characteristic profile (Alekseyenko, 2012).

In summary, the results strongly support a model in which an active chromatin composition helps define the initial entry sites selected by the MSL complex. Functional MSL binding results in increased lability of local nucleosomal composition, and H4K16 acetylation and JIL-1 binding along the bodies of virtually all active X-linked genes. This work provides key insights into the order of events leading to dosage compensation in Drosophila, and can also serve as a model for using genome-wide data sets to understand how sequence-specific factors find their ultimate targets (Alekseyenko, 2012).

The results support roles for local chromatin environment and flanking GC content in discrimination of true target sites of the MSL dosage compensation complex. The model (see Model for binding site selection by a chromatin associated factor) depicts the GC content and active chromatin marks surrounding MREs in female Kc cells that predict binding by MSL complex in male S2 or BG3 cells (or after MSL induction in female Kc cells). MREs that do not pre-exist in a favorable environment are not bound by MSL complex and thus are non-functional. Definition of the favorable chromatin features that pre-exist factor binding may be a general tool, in addition to DNA motif analysis, for prediction of functional binding sites (Alekseyenko, 2012).

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).

Previous work suggested that MSL2 may tether the MSL-DCC to DNA and that an intact CXC domain is required for X-chromosome discrimination. To assess the DNA-binding specificity intrinsic to MSL2 comprehensively, the Drosophila genome was surveyed for MSL2-binding sites in vitro by DNA immunoprecipitation (DIP). Recombinant MSL2 was incubated with sheared genomic DNA (gDNA) purified from male Drosophila S2 cells. MSL2-bound DNA was recovered and sequenced (Villa, 2016).

Considering the lack of X-chromosome binding selectivity seen in previous in vitro studies, it was not expected to find that MSL2 preferentially retrieved DNA from distinct genomic loci, with a notable enrichment of sequences from the X chromosome. On the X chromosome, the MSL2 binding pattern was remarkably similar to the in vivo pattern that marks the positions of high-affinity binding sites (HAS; or chromatin entry sites) of the MSL-DCC. A total of 57 DIP sites coincided with in vivo HAS, although they show different signal intensities. The results were similar if DIP followed by sequencing (DIP-seq) was performed with gDNA extracted from female cells or synthesized in vitro by whole-genome amplification (excluding the contribution of male-specific RNA contaminants or DNA modifications). It therefore appears that recombinant MSL2 has an intrinsic ability to enrich X-chromosomal sequences from complex genomic DNA (Villa, 2016).

Next, the contribution was assessed of the three known MSL2 domains to DNA binding. Deletion of the RING finger domain that mediates MSL2 interaction with MSL1 and contains E3 ligase activity had no obvious effect. Unexpectedly, however, deletion of a region rich in proline and basic amino acid residues (the Pro/Bas domain) that may bind RNA resulted in the complete loss of DNA binding (Villa, 2016).

Upon deletion or mutation of the CXC domain, binding to a subset of sites was much reduced. Statistical analyses revealed 56 regions that specifically required a functional CXC domain for binding. Notably, these 'CXC-dependent' sites displayed a higher enrichment on the X chromosome. A total of 37 sites mapped to MSL2 in vivo peaks (HAS) on the X chromosome, and 2 sites corresponded to rare cases of autosomal sites that show MSL2 enrichment in vivo. The data suggest that MSL2 interacts with DNA via two domains, CXC and Pro/Bas, and that the CXC domain is the major determinant of the selectivity for the X chromosome. While binding-site specificity can be achieved by cooperation between different transcription factors, the finding suggests that cooperation between two different DNA-binding surfaces within this one protein may also refine its overall binding specificity (Villa, 2016).

Sequence analyses within CXC-dependent and CXC-independent binding sites for MSL2 yielded two distinct motifs. Whereas the CXC-independent binding sites shared low-complexity GA repeats, the CXC-dependent peaks centre around a more complex variation of the MSL response element (MRE), with a notable 5' extension. Remarkably, this novel motif can predict in vivo MSL2 binding (HAS) better than the MRE, as its position weight matrix (PWM) is superior in classifying whether MRE hit regions overlap HAS. Applying low thresholds, 2,667 instances of this motif were found throughout the genome, with an approximately twofold enrichment on the X chromosome. Higher-scoring matches to the consensus sequence tend to be more strongly enriched on the X chromosome. For example, the 34 best matches are 9.8-fold enriched on the X chromosome. However, 18 of those instances were not bound in vitro by MSL2 in a CXC-dependent manner, indicating that the recognition sequence represented by a PWM cannot fully explain this binding mode (Villa, 2016).

PWMs model the base readout of DNA sequences with the implicit assumption that each nucleotide at a given position contributes to binding independently of other positions. Physical interactions of neighbouring base pairs, however, alter the structural conformation of the DNA double helix (often referred to as the DNA shape), which may manifest as variations in the minor groove width, roll, helix twist, and propeller twist. Many proteins depend on both base identity and localized helix shape to recognize their binding site. Using a pentamer-based model built from all-atom Monte Carlo simulations of DNA structures, DNA shape parameters were calculated at each base position of the low-stringency motif hits, with 20-base-pair (bp) extensions on either side. To complement these position-centred features regional mono- and dinucleodide frequencies (k-mers) were also calculated in 4-bp windows along the hit sequences. Principal component analysis (PCA) revealed that a combination of DNA shape and k-mer features was able to separate the two classes of sequences: those that were bound in a CXC-dependent manner (CXC-bound) and those that were not bound in a CXC-dependent manner (non-CXC-bound). Sequences in the latter group were either not bound at all (2,502) or were bound independently of the CXC domain. This suggested that at least some of the DNA features might improve binding prediction. Indeed, classification models constructed with the additional feature sets performed much better than a PWM-score model in predicting CXC-dependent binding sites on all motif hits (Villa, 2016).

Guided by the good performance of the classification model using both the PWM-hit-score and k-mer features, mutations were predicted that would convert robust CXC-bound sites to non-CXC-bound sites. The model suggested that the best discriminating residues would localize to the 5' part of the motif and not to the GA-rich region. To test these predictions, the DIP experiment was modified by mixing appropriately diluted DNA oligonucleotides, representing either a native site or its mutated version, into the genomic DNA. The efficiency of DNA retrieval of experimental oligonucleotides and control genomic loci was quantified by quantitative PCR (qPCR). The results confirmed the predictions, leading to the conclusion that the main determinants for CXC-dependent binding reside within the first eight bases at the 5' end of the consensus motif. Notably, this is the part of the motif that diverges most from the MRE (Villa, 2016).

To investigate further the role of MSL2-binding sites in X chromosome dosage compensation, attempts were made to monitor the interactions of MSL2 with HAS in vivo, with minimal contributions from other DCC subunits. Genetic studies had shown that the assembly of a mature MSL-DCC bound to the non-coding roX RNA in male flies is compromised by inactivating the RNA helicase maleless (MLE). Under those circumstances, the remaining MSL2-MSL1 sub-complex is bound to a small subset of HAS. This scenario was recreated in S2 cells by using RNA interference (RNAi) against mle expression, and MSL2 binding was found to be preferentially retained at HAS, corresponding to CXC-dependent binding sites. The 25 HAS that were most resistant to MLE depletion revealed a shared sequence, bearing a strong resemblance to the CXC-dependent motif. By contrast, the 25 sites most sensitive to MLE depletion (bound only by the complete DCC) shared a GA motif similar to the one found in CXC-independent in vitro binding sites. This suggests that under physiological conditions, the MSL2-MSL1 sub-complex directly contacts a subset of HAS in a CXC-dependent manner in the absence of associated protein and RNA subunits (Villa, 2016).

It is possible that these chromosomal interactions represent an intermediate of MSL-DCC assembly. To test this hypothesis, de novo MSL-DCC assembly was initiated in female Kc cells by reducing the expression of the sex-lethal gene Sxl. The SXL protein prevents MSL2 expression and thus the dosage compensation program in female cells. Upon depletion of SXL, binding of newly expressed MSL2 to CXC-dependent HAS was stronger and occurred earlier when compared to CXC-independent ones. Consistent with this finding, hierarchical clustering of MSL2 signals from the common set of Kc and S2 peak regions revealed 30 sites that acquire strong MSL2 binding ability 3 days after SXL depletion. De novo motif discovery on these sites revealed a consensus sequence that resembles the one in the CXC-dependent sites. The data strongly suggest that those sites identified in vitro as CXC-dependent are pioneering binding sites for MSL2 in vivo. These are therefore refered to as PionX sites, and to their defining motif as the PionX motif (Villa, 2016).

The notion that PionX sites are important for dosage compensation is further supported by evolutionary considerations. Drosophila miranda represents a unique system to study how newly evolving X chromosomes acquired dosage compensation. The D. miranda neo-X chromosome is a sex chromosome that began to evolve just 1 million-2 million years ago. Owing to the relatively short evolutionary time span, the neo-X chromosome still retains many autosomal features, but has already acquired partial dosage compensation. Recent work has identified the MSL-DCC-binding sites on all D. miranda X chromosomes19. De novo motif analysis yielded the typical GA-rich MREs for the older, fully compensated X-chromosomal arms XL and XR. Notably, though, the consensus sequence derived from the neo-X chromosome clearly resembled the PionX signature (Villa, 2016).

The neo-Y chromosome originated from the fusion of one Müller-C chromosome to the Y chromosome, resulting in evolutionary pressure on the second Müller-C chromosome to become the neo-X chromosome. The PionX motif (but not the MRE) is particularly enriched on the D. miranda neo-X chromosome but not on the related Drosophlia pseodoobscura Müller C autosome, supporting the idea that this motif represents a new X-chromosome-specific feature. Careful comparison of neo-X-chromosome sequences with the homologous regions in D. pseudoobscura revealed that the novel MSL-DCC-binding sites were acquired by diverse molecular mechanisms, including point mutations and short insertions/deletions of precursor sequences. About half of them originated from precursor sequences contained in a D. miranda-specific helitron transposon. The homologous neo-Y helitron does not contain PionX motifs -- only precursor sequences in which the 5' CAC motif and the 3' GA-rich element are separated. On the neo-X chromosome these two parts are fused by a 10-bp deletion to form PionX consensus motifs. The insertion of a PionX consensus motif derived from the D. miranda neo-X chromosome into an autosome of D. melanogaster led to strong, ectopic binding of the MSL-DCC. By contrast, the corresponding homologous neo-Y-chromosome sequence, in which the 5' and GA sequences are split by a 10-bp insertion, did not recruit the complex. A similar experiment used the strongest DCC-binding site from the neo-X chromosome and the corresponding neo-Y-chromosomal fragment, showing MSL-DCC recruitment to the former, but not to the latter. While the neo-Y-chromosomal fragment does not contain a PionX motif, the evolved neo-X chromosome contains nine of them. Collectively, these observations suggest that PionX motifs play an important role in de novo acquisition of dosage compensation (Villa, 2016).

In summary, this study provides three lines of argument suggesting that PionX sites are X-chromosome-specific determinants that function early in the series of events that lead to exclusive targeting of the X chromosome and correct dosage compensation. First, PionX sites are bound by an MSL2-MSL1 sub-complex in the absence of all other subunits, a state that may reflect an early intermediate of MSL-DCC assembly at HAS. Second, PionX sites are the first to be occupied during de novo establishment of dosage compensation. Finally, PionX motifs arose during the early phase of neo-X-chromosome evolution in D. miranda (Villa, 2016).

A pertinent conceptual advance from this study is the understanding that not all HAS contain the same amount of information. The subset of PionX sites are not necessarily sites of highest MSL2 occupancy in vivo, but contribute an important qualitative element of X-chromosomal discrimination. This discrimination is not wholly apparent from the consensus motif as it also relies on the shape of the DNA at the MSL2-binding site (Villa, 2016).

The initial recruitment of MSL2 to PionX sites on the X chromosome may trigger the distribution of the complex to nearby non-PionX HAS within the chromosomal territory, thereby further amplifying the difference in MSL2 occupancy between the X chromosome and the autosomes. It is likely that other factors contribute to the stability of the targeting system in vivo, such as the cooperativity of MSL2 domains within what is presumed to be a dimeric complex; the assembly of functional complexes within the X-chromosomal territory owing to transcription of roX RNA from the X chromosome; synergistic interactions between different MSL-DCC complexes and with the CLAMP protein at clustered MREs24; and a supportive organization of the conformation of the X chromosome (Villa, 2016).

male-specific lethal 2: Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation | References

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