male-specific lethal 2
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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 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).