male-specific lethal 2
The male-specific lethal dosage compensation complex (MSL-DCC) selectively assembles on the X chromosome in Drosophila males and activates gene transcription by twofold through histone acetylation. An MSL recognition element (MRE) sequence motif nucleates the initial MSL association, but how it is recognized remains unknown. This study identified the CXC domain of MSL2 specifically recognizing the MRE motif and determined its crystal structure bound to specific and nonspecific DNAs. The CXC domain primarily contacts one strand of DNA duplex and employs a single arginine to directly read out dinucleotide sequences from the minor groove. The arginine is flexible when bound to nonspecific sequences. The core region of the MRE motif harbors two binding sites on opposite strands that can cooperatively recruit a CXC dimer. Specific DNA-binding mutants of MSL2 are impaired in MRE binding and X chromosome localization in vivo. These results reveal multiple dynamic DNA-binding modes of the CXC domain that target the MSL-DCC to X chromosomes (Zheng, 2014).
Dosage compensation in Drosophila is the mechanism by which X-linked gene expression is made
equal in males and females. Proper regulation of this process is critical to the survival of both sexes.
Males must turn the male-specific lethal (msl)-mediated pathway of dosage compensation on and
females must keep it off. The msl2 gene is the primary target of negative regulation in females.
Preventing production of Msl2 protein is sufficient to prevent dosage compensation; however, ectopic
expression of Msl2 protein in females is not sufficient to induce an insurmountable level of dosage
compensation, suggesting that an additional component is limiting in females. A candidate for this
limiting factor is Msl1, because the amount of Msl1 protein in females is reduced, as compared to
males. Two levels of negative regulation of msl1 have been identified in females. The predominant
regulation is at the level of protein stability, while a second regulatory mechanism functions at the level
of protein synthesis. It is thought that Msl2 stabilizes Msl1 by forming a complex with Msl1. The cluster of polyuridine tracts in the 3' UTR is necessary for a small reduction in Msl1 synthesis, presumably by direct binding of Sex-lethal. Overcoming these control mechanisms by overexpressing both Msl1 and Msl2 in females results in 100% female-specific lethality. Overexpressing Msl1 in females results in Msl1 association with sites on all female chromosome arms of salivary gland polytene chromosomes. Overexpressed Msl1 protein in males is associated with the X chromosome and also with multiple individual sites on the autosomes. The transient expression of Msl1 is not sufficient to recruit Msl2 to the autosomal sites in males or to any of the sites in females, but it indicates that Msl1 has affinity forchromatin. In contrast, overexpression of Msl2 caused by heat shock induction of a transgene results in exclusive association with MSl1 to sites only on the X chromosome (R. Kelly, unpublished data, communicated to Chang, 1998). Based on these results, it is possible that Msl1 privides the Msl complex with its affinity for chromatin, but that Msl2 is required to specifically target the complex to the X chromosome (Chang, 1998).
Drosophila Msl proteins are thought to act within a complex to elevate transcription
from the male X chromosome. Msl1, Msl2 and Msl3 proteins
are associated in immunoprecipitations, chromatographic steps, and in the yeast
two-hybrid system, but the Mle protein is not tightly complexed in these assays. Analysis focused on the Msl2-Msl1 interaction, which is postulated to play
a critical role in Msl complex association with the X chromosome. Using a modified
two-hybrid assay, missense mutations were isolated in Msl2 that disrupt its interaction with Msl1. In a
Drosophila virilis Msl2 homolog, 11 out of
12 mutated residues that cluster around the first zinc-binding site of the RING finger domain have been found to be conserved. All but one of
the residues (found to be important for Msl1 binding by reverse two-hybrid screening) are conserved in the D.virilis
protein; the single exception, S53, is adjacent to a conserved proline (P54) and was itself mutated to proline in the
screen. Four cysteine or histidine residues outside the RING domain are also conserved. Although these are not
positioned to form a canonical zinc finger structure, mutation of one of the residues (C107) to arginine disrupts the
interaction of Msl2 with Msl1. A second cysteine-rich region (amino acids 521-562), which is loosely related to the PHD motif and to
metallothioneins, is conserved at all cysteines and
histidines, including three positions not present in the published metallothionein alignment. The conservation of these
residues is compatible with a previous report that mutation of C540 and C542 to alanine diminishes, but does not
abolish msl2+ function in vivo. The acidic nature of an adjacent region (amino
acids 563-592) is also conserved, while a more distal proline-rich region (amino acids 681-701) is incompletely
conserved. A remarkable feature of the alignment is the presence of three gap regions having little or no homology. A short gap
(amino acids 116-128) separates the RING finger and N-terminus from the rest of the protein. The second gap (amino
acids 281-520) corresponds to the middle third of the Msl2 protein. This region contains most of the polymorphisms
and length variations present within published D.melanogaster msl2 sequences; in addition, neither the repeats nor the acidic character of this region of the D.melanogaster
protein are conserved in the D.virilis homolog. Finally, a gap following the second cysteine-rich domain (amino
acids 593-614) is conserved in length, but is not conserved in sequence. These data suggest that the functions of the
RING finger and other features of MSL2 have been conserved and that these domains may be positioned appropriately
in the protein by more or less randomly evolving spacers.
Two pre-existing D. melanogaster msl2 alleles, which fail to support male viability in vivo,
have lesions in the same region of the RING finger. These were tested in the two-hybrid system and are also defective in interaction with Msl1. Mutation of the second zinc-binding site has little effect on Msl1 binding,
suggesting that this portion of the RING finger may have a distinct function. These data support a model in which
MSL2-MSL1 interaction nucleates assembly of an MSL complex, with which Mle is weakly or transiently
associated (Copps, 1998).
Dosage compensation ensures that males with a single X chromosome have the same amount of most X-linked gene products as females with two X chromosomes. In Drosophila, this equalization is achieved by a twofold enhancement of the level of X chromosome transcription in males, relative to each X chromosome in females. The products of at least five genes, maleless (mle), male-specific lethal 1, 2, and 3 (msl-1, msl-2, msl-3) and males absent on the first (mof), are necessary for dosage compensation. MOF transcript is found in larvae and adults of both sexes. The proteins produced by these genes form a complex that is preferentially associated with numerous sites on the X chromosome in the somatic cells of males, but not females. Binding of the dosage compensation complex to the X chromosome correlates with a significant increase in the presence of a specific histone isoform (histone 4 acetylated at lysine 16) on the chromosome. Experimental results and sequence analysis suggest that the mof gene encodes an acetyl transferase that plays a direct role in the specific histone acetylation associated with dosage compensation. The predicted amino acid sequence of MOF exhibits a significant level of similarity to several other proteins, including the human HIV-1 Tat interactive protein Tip60 (see Drosophila Tip60), the human monocytic leukemia zinc finger protein MOZ, and the yeast silencing proteins SAS3 and SAS2. Also studied has been the role played by the various components of the complex in the targeting of MOF to the X chromosome. To this end, indirect cytoimmunofluorescence was used to monitor the binding of these components in males carrying either complete or partial loss-of-function mutations as well as in XX individuals in which formation of the dosage compensation complex has been induced by genetic means (Gu, 1998 and Hilfiker, 1997).
Dosage compensation works to heighten the activity of the single X chromosome in males. This heightened expression of the X chromosome in males is accomplished through the action of male-specific lethal (MSL) proteins. Immunostaining of chromosomes shows that the MSL proteins are associated
with all female chromosomes at a low level but are sequestered to the X chromosome in males.
Histone-4 Lys-16 acetylation follows a similar pattern in normal males and females, being higher on the
X and lower on the autosomes in males than in females. However, the staining pattern of acetylation
and the mof gene product, a putative histone acetylase, returns to a uniform
genome-wide distribution as found in females and in males that are mutant for the msl gene. Gene expression on the autosomes correlates with the
level of histone-4 acetylation. With minor exceptions, the expression levels of X-linked genes are
maintained with either an increase or decrease of acetylation, suggesting that the MSL complex
renders gene activity unresponsive to H4Lys16 acetylation. Evidence has also been found for the presence
of nucleation sites for the association of the MSL proteins with the X chromosome rather than individual
gene binding sequences (Bhadra, 1999).
Three different approaches (chromosomal morphology, specific RNA quantitation, and binding of MSL proteins) were taken to investigate the role of the MSL complex on dosage compensation. Both chromosomal morphology and the measurement of specific transcripts reveal that the lack of MSL binding and histone H4 modification in the mle mutant males neither reduces X-chromosomal size specifically nor eliminates dosage compensation of most X-linked transcripts. Two X-derived transgenes, white and yellow, which are normally partially compensated on the autosomes, are elevated in expression by the homozygous mle mutation. Thus, transgenes do not lose dosage compensation in the mle::mle males; rather, they become fully compensated on the autosomes in the absence of the Mle protein. In addition, the presence of Msl-2 in H83M2 females (H83M2 is a stock in which Msl-2 is ectopically expressed in females) does not promote hypertranscription of the X-derived mini-white and yellow transgenes inserted into the autosomes. Rather the strong association of the MSL proteins and H4Ac16 enrichment with the two X chromosomes and concomitant reduction of H4Ac16 residues on the autosomes is correlated with reduced autosomal transgene expression. Sequestration of chromatin proteins from one location in the genome to another as a means to affect gene expression has also been described in the case of Sir silencing proteins in yeast (Marcand, 1996). In contrast, changes of the acetylation level on the X chromosome appear to have minimal consequences. MSL proteins do not associate with the X-derived transgenes or with a small X segment (>179 kb) in the autosomes. This result indicates that MSL proteins do not initiate binding to every gene on the X chromosome because the lack of binding within the >179-kb segment suggests the absence of potential nucleation sites in that region, while the same cytological bands are associated with MSL proteins when residing on a larger segment. Apparently, the MSL proteins associate with nucleation sites on the X that allow initial recognition followed by polymerization. It has been suggested that the presence of MSL proteins on the X is sufficient evidence to conclude they condition dosage compensation. However, it is now thought that measures of gene expression are necessary to understand the consequences of the msl mutations. Without such experiments, one can come to no conclusion about an involvement of male sex lethal proteins in any process (Bhadra, 1999 and references).
In this article, evidence is presented that chromosomal proteins are sequestered from the autosomes to the X in normal males. Therefore, when any of the msl loci are mutated, there is no sequestration. The X remains basically compensated and the autosomal expression is increased in general because MOF becomes uniformly distributed in the nucleus, resulting in a return of acetylation levels to those of females. The msl mutations have little effect in females because they are chromosomally balanced and therefore have no dosage effects operating. Also, there is a similar distribution of acetylation in mutants and normals. In contrast, in H83M2/+ females, the product of Msl-2 promotes sequestration of the acetylase to the X, which lowers the level of autosomal acetylation. As a result, the expression of transgenes inserted in the autosomes is reduced (Bhadra, 1999).
There is much to be learned before a full comprehension of the evolution of this situation can be formulated. However, one speculation might be that the degeneration of one member of a homologous pair of chromosomes that begins the formation of the Y would leave a small segment of the opposing homolog in the haplo state. A selective advantage would be afforded to individuals carrying any change, such as elevated acetylation, that would result in increased expression of haplo-insufficient genes in this region. Although a focus has been placed on the situation in which acetylation or lack thereof modifies the effect of sex-chromosomal dosage, it is also the case, as exemplified by the ectopic expression of Msl-2, that changes in acetylation will modify gene expression without involving a dosage effect. As the haplo region expands, the dosage in dosage-dependent regulatory genes would be altered. It is interesting to note that dosage-dependent regulatory genes are more likely to have an effect in the haplo condition than genes encoding metabolic functions. Their acetylation might be selected to eliminate their global effects on target genes throughout the genome, but some fraction must remain dosage dependent in normal males to produce compensation and the observed autosomal effects (Bhadra, 1999 and references).
Because an inverse effect is more common than a positive dosage response, there would be a tendency to increase gene expression throughout the genome, including rendering the haplo regions at least partially dosage compensated. To date there are over twenty genes identified that produce a trans-acting dosage effect on white. By analogy with modifiers of position-effect variegation, many of which have been identified as transcriptional regulators, there are scores of dosage-dependent modifiers of a single process. These genes appear to operate in a dosage-dependent cascade such that any one or several produce much the same effect on the target locus. Thus, statistically speaking, any region involving only a few percent of the genome will potentially carry a dosage-dependent modifier of any one target gene. Despite the large number of such modifiers, varying multiples does not in general generate cumulative effects beyond the inverse limits. This conclusion is derived from two different types of observations: (1) combinations of mutations do not exceed this limit and (2) large aneuploids produce trans-acting dosage effects that remain within this range. Thus, the combination of acetylation and the inverse dosage effect could provide for a synergistic expansion of the haplo regions. This combination would produce dosage compensation whether the affected target genes are haplo insufficient or not. The inverse dosage effect provides a numerical explanation of how the process of dosage compensation doubles the expression of many genes transcribed at widely differing rates, although the mechanism remains to be elucidated. The sequestration of acetylase to the X in increasingly larger quantities would lower the autosomal acetylation and thus mute the increased expression of the autosomes that would be expected to result from a lowered dosage of the evolving X. On the X, some component of the MSL complex or associated proteins would eventually render most genes unresponsive to the acetylation level. Indeed, there may be a selective advantage to maintaining transcriptional regulators as dosage dependent in order to maintain compensation (Bhadra, 1999 and references).
Such a situation might evolve if continued increases in acetylation caused a tendency for extensive overexpression of the X. Inactivation of a response to acetylation would hold the hyperactivation at the twofold level because of the effect of the X dosage. Moreover, there is a strong skewing in favor of TATA-less promoters on the X chromosome. Therefore, it is also possible that many individual X-linked genes have evolved features that render them more sensitive to the X dosage effects or less sensitive to the acetylation level. Consequently, with the available information, it is envisioned that as the heteromorphic sex chromosome situation evolved in Drosophila by degeneration of one member of a homologous pair of chromosomes to form the Y, dosage effects would come into play and produce compensation of most X-linked genes together with a tendency for the doubling of the expression of the autosomes in males. The sequestration of a histone acetylase to the X chromosome would mute the effect on the autosomes. The MSL complex on the X would inactivate the response to H4Lys16 acetylation for most genes, to maintain the hyperactivation at the twofold level. Thus it is suggested that sequestration of the MSL proteins occurs in males to nullify
on the autosomes and maintain on the X (an inverse effect produced by negatively acting
dosage-dependent regulatory genes) as a consequence of the evolution of the X/Y sex chromosomal
system. Thus, this model proposes a single mechanism that explains the five levels of transcription involved with the various cases of dosage compensation, the MSL binding on the X in males, and the gene expression pattern in the msl mutants (Bhadra, 1999).
While the hypothesis that the RNA on the X RNAs are involved in dosage compensation is attractive, some of the data with regard to the Rox RNAs are either lacking or
appear to be in conflict with previous knowledge about dosage compensation. The strongest evidence that rox1 might be involved in dosage compensation is the fact that it is associated with the X chromosome in male larval salivary gland nuclei. Using a similar technique, the same has been found to be true for rox2 RNA. However, this technique does not resolve the
distribution of these RNAs along the X chromosome because the extant methods for immunostaining of polytene chromosomes and RNA in situ hybridization are not
easily compatible. A protocol that gives an MSL banding pattern without removing the rox RNA molecules was developed by using less concentrated acid solutions
for relatively short fixation times. rox1 RNA and MSL-1 protein are colocalized to exactly the same
chromosomal bands. No signals were detected that show one of the two molecules alone. The same double staining was performed with rox2 RNA and MSL-1
protein and gave an identical result. These results show that MSL proteins and rox RNAs are distributed on the male X chromosome in exactly the
same pattern, providing strong evidence for a direct involvement of the rox RNAs in dosage compensation.
Simultaneous removal of rox1 and rox2 function leads to a loss of X Chromosome binding of the MSL proteins in male embryos (Franke, 1999).
In male Drosophila, histone H4 acetylated at Lys16 is enriched on the X chromosome, and most X-linked genes are transcribed at a
higher rate than in females (thus achieving dosage compensation). Five proteins, collectively called the MSLs, are required for dosage
compensation and male viability. Here it has been shown that one of these proteins, MSL1, interacts with three others, MSL2, MSL3 and MOF.
The latter is a putative histone acetyl transferase. Overexpression of either the N- or C-terminal domain of MSL1 has dominant-negative
effects, i.e. causes male-specific lethality. The lethality due to expression of the N-terminal domain is reduced if msl2 is
co-overexpressed. MSL2 co-purifies over a FLAG affinity column with the tagged region of MSL1, and both MSL3 and MOF co-purify with the FLAG-tagged
MSL1 C-terminal domain. Furthermore, the MSL1 C-terminal domain binds specifically to a GST-MOF fusion protein and co-immunoprecipitates with HA-tagged
MSL3. The MSL1 C-terminal domain shows similarity to a region of mouse CBP, a transcription co-activator. It is concluded that a main role of MSL1 is to serve as
the backbone for assembly of the MSL complex (Scott, 2000).
In general, the amino acid sequences of the MSLs suggest regions or domains within the proteins that could be important for function in vivo. Indeed, this has been confirmed by mapping loss-of-function mutations to the domain, such as the helicase domain of MLE, the putative acetylase domain of MOF and the RING finger region of MSL2. The amino acid sequence of MSL1 is the least informative, containing no recognizable domains, although regions rich in acidic amino acids and possible PEST sequences have been identified. To identify regions within MSL1 that are important for function in vivo, it was determined which regions have dominant-negative effects when overexpressed. Two regions of MSL1, one near the N-terminus and the other at the C-terminus, are likely to be important for assembly of the MSL complex in vivo, because overexpression of either region causes male-specific lethality. Genetic evidence, decreased male viability of msl2 heterozygotes and increased male viability by co-overexpression of MSL2, suggests that the region of MSL1 at the N-terminus interacts with MSL2. This has been confirmed by co-purification of MSL2 with FLAG-tagged versions of MSL1 over FLAG affinity columns. Similarly, the C-terminal region of MSL1 interacts with both MOF and MSL3. Furthermore, expression of the C-terminal domain results in significant loss of MOF from the male X chromosome (Scott, 2000).
The N-terminal FN region of MSL1 that binds to MSL2 was chosen originally for expression in flies because it was predicted that almost half of FN (amino acids 96-172) would form a two-stranded, alpha-helical, coiled-coil structure. Coiled-coil structures are comprised of a heptad repeat (abcdefg)n where hydrophobic residues occupy positions a and d on the same side of the alpha-helix. The coiled-coil motif of GCN4 mediates dimerization. If a similar structure mediates the formation of the MSL1-MSL2 heterodimer, then part of the region of MSL2 that interacts with MSL1 should form a coiled-coil structure. The Ring finger domain region of MSL2 interacts with MSL1. It is predicted that the region immediately preceding the RING finger could form a coiled-coil structure. It is particularly significant that several of the mutations that disrupt the interaction with MSL1 in yeast introduce amino acid changes that either significantly disrupt the alpha-helix (leucine to proline) or introduce a charged amino acid into the predicted hydrophobic face of the alpha-helix. The RING domain is found in a number of proteins, including the V(D)J recombination-activating protein RAG1. The crystal structure of the RAG1 dimerization domain, which includes the RING finger, reveals that dimerization is stabilized by interaction between alpha-helices that form a hydrophobic core. The RING finger is thought to form the structural scaffold upon which the dimer interface is formed. It is tempting to speculate, by analogy with RAG1, that the association of MSL1 and MSL2 involves the interaction of amphipathic alpha-helices that depend on the RING finger domain. This could best be addressed by determining the crystal structure of the MSL1-MSL2 complex (Scott, 2000).
In vitro translated MSL1 C-terminal domain co-immunoprecipitates with in vitro translated HA·MSL3 but not HA·MOF. Thus, C interacts directly with MSL3 but the interaction with MOF requires either another factor present in fly extracts or post-translational modification of MSL1 or MOF. While the possibility of a nucleic acid component of the FC-MOF complex cannot be ruled out, the possiblity (post-translational modification of MSL1 or MOF) is favored since a silver stain of FLAG affinity-purified FC-MOF complex separated by SDS-PAGE shows only two main bands corresponding to the sizes expected for FC and MOF. The C-terminal domain of MSL1 is rich in serine and threonine residues, and contains several potential phosphorylation sites and a predicted PEST sequence. PEST sequences have been suggested to contribute to the instability of the MSL1 protein. However, the role of these sequences in MSL1 has not been determined. Indeed, an alternative function for the PEST sequences is suggested by the observations that the PEST domains of PU.1 and IB are required for their respective interactions with Pip and c-Rel. In both cases, phosphorylation of a serine residue within the PEST sequence is required for the respective protein-protein interactions. The recent finding that a serine/threonine kinase is associated preferentially with the male X chromosome raises the possibility that MSL1 or another MSL is phosphorylated by this enzyme (Scott, 2000).
In the sequential model for assembly of the MSL complex, the first step involves the binding of the MSL1-MSL2 complex to several 'high affinity' sites on the male X chromosome. Since the localization of both MOF and MSL3 to the X chromosome requires mle+ function, this suggests that the association of MOF and MSL3 with the MSL1-MSL2 complex is MLE dependent. MLE could either bind directly to MOF and/or MSL3, or somehow stabilize the MSL complex together with roX RNA. In support of the latter model, MOF and MSL3 bind directly to the C-terminal domain of MSL1. Furthermore, MLE did not co-purify with an FC-MOF-MSL3 complex over an affinity column. However, the affinity chromatography experiments were designed to maximize the likelihood of detecting protein-protein association and are not quantitative. It is possible that MOF and MSL3 may have a higher affinity for the C-terminal domain of MSL1 than full-length MSL1. Thus, one possible mechanism is that in vivo the C-terminal domain of MSL1 is not freely available to bind to MOF and/or MSL3, and that the binding of MLE to the MSL1-MSL2 complex causes a conformational change in MSL1, such that the C-terminal domain becomes more accessible (Scott, 2000).
Previous searches of the protein sequence database with the complete MSL1 sequence have failed to identify any significant similarities. However, when a search is carried out with just the C-terminal domain sequence, some similarity is found to a 254 amino acid region of mouse CBP. Although the similarity is not high, given that the similarity extends across almost the entire C-terminal domain of MSL1, and that both CBP and the MSL1 C-terminal domain bind to histone acetyl transferases (or putative histone acetyl transferases), it is thought that this homology may be significant. If this similarity reflects a conserved function, then it would be predicted that the MSL1-similar region of CBP, which has no known function, would associate with either an MOF-like histone acetyl transferase or an MSL3-like protein in mammalian cells (Scott, 2000).
It is not known how the MSL complex binds to the male X chromosome. None of the MSLs contain a recognizable DNA-binding motif. The F84 version of MSL1, lacking the first 84 amino acids, binds to MSL2, MSL3 and MOF but does not bind preferentially to the male X chromosome. This suggests that the male lethality that results from overexpression of F84 is due to this protein being able to bind to three MSLs, but not being able to bind to the X chromosome because the first 84 amino acids of MSL1 are required for recognition of the X chromosome. Alternatively, the lack of binding of F84 to the male X chromosome could be because the beginning of MSL1 is required for assembly of the MSL complex in vivo. However, if so, then it would be expected that F84 would have bound to the 'high affinity' sites since F84 does bind to MSL2. Assuming that MSL1 and MSL2 are the only components of the high affinity complex, it would then appear more likely that the first 84 amino acids of MSL1 are required for X chromosome binding rather than complex formation. However, there are several lines of evidence that suggest that the roX RNAs are part of the MSL complex, which raises the possibility that one or both of the roX RNAs could be part of the high affinity complex. Thus it will be of interest to determine if the MSL complex containing the F84 protein binds to roX RNA with a lower affinity than the complex containing full-length MSL1 (Scott, 2000).
In the male Drosophila, the X chromosome is transcriptionally upregulated to achieve dosage compensation, in a process that depends on association of the MSL proteins with the X chromosome. A role for non-coding RNAs has been suggested in recent studies. The roX1 and roX2 RNAs are male-specific, non-coding RNAs that are produced by, and also found associated with, the dosage-compensated male X chromosome. Whether roX RNAs are physically part of the MSL complex has not been resolved. roX RNAs are found to colocalize with the MSL proteins and are highly unstable unless the MSL complex is coexpressed, suggesting a physical interaction. roX2 RNA could be precipitated from male tissue-culture cells with antibodies to the proteins Msl1 and Mle, consistent with an integral association with MSL complexes. Localization of roX1 and roX2 RNAs in mutants indicates an order of MSL-complex assembly in which roX2 RNA is incorporated early in a process requiring the Mle helicase. It was also found that the roX2 gene, like roX1, is a nucleation site for MSL complex spreading into flanking chromatin in cis. These results support a model in which MSL proteins assemble at specific chromatin entry sites (including the roX1 and roX2 genes); the roX RNAs join the complex at their sites of synthesis; and complete complexes spread in cis to dosage compensate most genes on the X chromosome (Meller, 2000).
Wild-type males have complete MSL complexes bound to hundreds of bands along the X chromosome in a highly reproducible pattern where they mediate hypertranscription. Genetic analysis of debilitated MSL complexes lacking either Mle, Msl3 or Mof subunits, or containing enzymatically dead Mof acetyltransferase or Mle helicase, has shown that partial MSL complexes can bind to only ~35 bands that have been termed chromatin entry sites (formerly called high-affinity sites). It has been proposed that these sites direct dosage compensation to the X chromosome by serving as sites of assembly and subsequent spreading of the MSL complex into flanking chromatin in cis. roX2 has been identified as a chromatin entry site based on the ability of roX2 transgenes to attract the MSL complex to autosomal insertion sites, even in the absence of Msl3. To test whether roX2 could also support spreading of MSL complexes into flanking autosomes, multiple lines of transgenic larvae were created carrying genomic roX2 transgenes. It was found that the roX2 chromatin entry site also supports variable spreading of MSL complexes into flanking chromatin. The spreading displays the same characteristics as has been reported for a roX1 transgene: bidirectionality, dependence on insertion site, and substantial variability even from nucleus to nucleus in the same animal. These results strongly support the idea that most or all chromatin entry sites will act as nucleation points for MSL spreading in cis (Meller, 2000).
To determine whether the roX RNAs and MSL proteins are associated in the same binding pattern on the X chromosome, the immunostaining protocol was adapted to allow detection of roX RNAs by in situ hybridization to spread polytene chromosomes. It was found that the MSL proteins, roX1 and roX2 RNAs are precisely colocalized along the male polytene X chromosome. In addition, a subtle enrichment for roX1 RNA has been consistently detected in several bands surrounding its site of synthesis (Meller, 2000).
MSL proteins are found at decreased levels when they are unable to assemble into a wild-type complex. Therefore, it was asked whether roX RNAs might exhibit the same behavior, indicative of a requirement for complex assembly for their integrity. roX1 RNA was expressed constitutively from an H83roX1 transgene and it was determined whether roX1 RNA could accumulate and perhaps even associate with the X chromosomes in females, which lack MSL complexes. It was found that ectopic roX1 RNA fails to bind the X chromosome in females as assayed by in situ hybridization. To determine whether the roX1 RNA produced from the H83roX1 transgene could be stabilized by the presence of the MSL complex, its behavior was examined in males lacking endogenous roX1, or females that form MSL complexes as a result of ectopic expression of Msl2. It was found that transgenic roX1 RNA associates with the X chromosome in males and in Msl2-expressing females. Thus, roX1 RNA is dependent on the MSL complex for its stability (Meller, 2000).
The idea that the MSL complex assembles at chromatin entry sites rests on the observation that these are the only places partial MSL complexes bind when one subunit is removed by mutation. Therefore, it was determined whether roX RNAs might also be associated with these partial complexes in the absence of spreading into flanking chromatin. To visualize chromatin entry sites, females were used that ectopically express Msl2 but are mutant for msl3 or mle. They assemble partial MSL complexes at the same sites as dying mutant males, but are viable and produce excellent polytene chromosomes. Surprisingly, a difference was found in the localization of roX1 and roX2 RNAs in msl3 mutants. Although roX1 RNA is detected only at cytological position 3F, the site of its synthesis, roX2 is consistently seen at many chromatin entry sites including 3F. This result suggests that roX2 may assemble first into partial MSL complexes and then be exported to other chromatin entry sites. These roX2-containing complexes may subsequently acquire a second roX RNA species (such as roX1) or be present in distinct complexes from roX1 (Meller, 2000).
mle mutants were examined for localization of roX RNAs. It was found that roX2 RNA is no longer able to move to other chromatin entry sites, but is only detected at its site of synthesis, like roX1. This demonstrates that the Mle helicase plays an early role, perhaps in packaging roX2 RNA into growing MSL complexes. Blocking assembly at this point prevents roX2 RNA from reaching other chromatin entry sites (Meller, 2000).
To further test the idea that roX2 assembly may occur at an earlier step than roX1, it was determined whether roX2 RNA assembles correctly in the absence of roX1. It was found that roX2 RNA is localized to the male X chromosome in the absence of roX1, with no apparent perturbation of its staining pattern. It is concluded that roX2 RNA, like the MSL protein complex, is independent of roX1 for localization on the X chromosome (Meller, 2000).
MSL complexes can be immunoprecipitated from Schneider SL2 tissue-culture cells, which exhibit male-like X chromosomal MSL complex and histone H4Ac16 by immunostaining. Therefore, reverse transcription (RT)-PCR analyses was performed to determine whether roX RNA could be coprecipitated with MSL complexes from SL2 cell extracts. Focus was placed on roX2 because the genetic experiments had predicted that it may be included at an early step in MSL complex assembly. RT-PCR products were first confirmed from male larval total RNA, SL2 cell total RNA, and SL2 cell protein lysates, using primers flanking an intron of 141 bp. Surprisingly, a smaller RNA with a 270 bp intron removed is the predominant product detected in both male larvae and SL2 cell total RNA. The intron encompasses the 141 bp intron and utilizes the same 3' splice site. The spliced roX2 RNA is detected in immunoprecipitates from SL2 cells using anti-Msl1 and anti-Mle antibodies. It is concluded that the MSL proteins are physically associated with the spliced form of roX2 RNA (Meller, 2000).
Until recently, it had been assumed that X-chromosome-specific cis-acting sequences would be associated with most genes bound by the MSL proteins. However, studies of roX1 have suggested that, if provided with a chromatin entry site (a roX1 transgene), the MSL complex could be attracted in cis to genes on autosomes that were never before dosage-compensated. The complex could not normally gain access to the autosomes in wild-type males because the MSL chromatin entry sites are found only on the X chromosome. Here, it has been shown that roX2 transgene can also function as a chromatin entry site for spreading of MSL complexes into autosomal chromatin (Meller, 2000).
In addition to roX genes functioning as chromatin entry sites, it is thought that the RNAs themselves may play roles in MSL complex assembly or function on the X chromosome. Although large ribonucleoprotein machines are well known in the form of ribosomes, spliceosomes and telomerases, RNAs have not been considered important factors in transcriptional regulation. There have been recent reports, however, of transcriptional control by non-coding RNAs in several diverse systems. A prominent example is the Xist RNA, which has been shown to play a central role in silencing most of the genes along one of the two X chromosomes in mammals. Another example is SRA, a novel non-coding RNA discovered as a coactivator of steroid hormone receptors. Individually, roX RNAs do not appear essential for MSL complex assembly on the X chromosome, but the phenotype of mutant embryos that lack roX1, roX2 and an unknown number of neighboring genes suggest that assembly of the MSL complex is delayed or abolished when both RNAs are removed (Meller, 2000 and references therein).
These data are consistent with assembly of roX RNAs into MSL complexes: (1) roX2 RNA can be immunoprecipitated from male tissue-culture cells, using anti-Msl1 or anti-Mle antibodies; (2) the roX RNAs are highly unstable in the absence of MSL proteins even if their synthesis is driven by a strong constitutive or inducible promoter; (3) movement of roX2 RNA from its site of synthesis to other chromatin entry sites requires Mle helicase. These observations are most easily understood if the MSL proteins physically contact the roX RNAs, protecting them from cytoplasmic export or degradation and targeting them instead to the X chromosome (Meller, 2000).
One clear difference between roX1 and roX2 RNAs is that roX2 RNA can travel to ~35 other chromatin entry sites along the X chromosome in the absence of Msl3. Maturation of the complex is thought to take place at these sites and then to initiate spreading into flanking chromatin. In the same nuclei lacking Msl3 protein, roX1 RNA is found only at its site of transcription. This suggests that the protein and RNA components of the MSL complex may assemble in a stepwise manner at several sites, and that roX2 may play a role in early events (Meller, 2000).
These results support three interrelated roles for roX genes in dosage compensation: (1) to produce RNA components of the MSL complex; (2) to provide sites for complex assembly and, (3) to mark the X chromosome for dosage compensation by acting as nucleation sites for spreading the MSL complex in cis to surrounding genes. A model has been presented for an ordered pathway of MSL-roX assembly, in which roX2 RNA joins partial MSL complexes at an early step and is exported to other chromatin entry sites in a process dependent on the Mle helicase. The roX2-containing complexes may complete assembly at other chromatin entry sites, perhaps by acquiring a second roX RNA species (such as roX1). Only mature complexes are functional to spread and dosage compensate flanking genes on the X chromosome (Meller, 2000).
In this report, an examination was carried out of how mutations in the principal sex determination gene, Sex lethal (Sxl), impact the H4 acetylation and gene expression on both the X and autosomes. When
Sxl expression is missing in females, the sequestration occurs concordantly with reductions in autosomal H4Lys16 acetylation and gene expression on the
whole. When Sxl is ectopically expressed in SxlM mutant males, the sequestration is disrupted, leading to an increase in autosomal H4Lys16 acetylation and overall
gene expression. In both cases relatively little effect is found on X chromosomal gene expression (Bhadra, 2000).
In SxlM males, in which Sxl is ectopically expressed, the slow accumulation of the Sxl protein during development eventually prevents significant Msl-2 expression and hence reduces the Msl complex association with the X chromosome. This results in X and autosomal gene expression quite similar to that found in the mle mutant, i.e., little response of the X-linked genes, but an overall increase in autosomal expression. Similarly, the association of the Msl proteins in ~50% of the cells of heteroallelic Sxl females causes sequestration of MOF to the two X chromosomes. This sequestration reduces the H4Ac16 on the autosomal loci, resulting in a lowered expression. There is a concomitant increase of acetylation on the X chromosome, but little overall response of the X-linked genes (Bhadra, 2000).
In the SxlM and Sxlf;mle genotypes a low level of Msl-1/Msl-2 shows chromosomal binding to some degree, although binding takes present in distinct patterns in the two cases. This low level of binding, however, is insufficient to sequester all the available Males absent on the first (Mof) present in the cell. Previous and present data suggest that in the absence of a functional Msl complex, Mof still associates with the chromosomes and is active in modifying H4. The reduced amount of Msl-1/Msl-2 appears saturated with Mof, allowing the remainder to be uniformly distributed across the genome, which modulates gene expression (Bhadra, 2000).
The general trends of X and autosomal gene expression in SxlM and Sxlf mutants match the published autoradiographic data when the latter is considered as absolute levels rather than relative X to autosomal ratios. Autoradiographic grain counts over the X chromosome were changed very little in SxlM larvae compared to normal, but the counts over the autosomes were increased. Conversely, in Sxlf females, the autosomal counts were lower than in normal females with little change over the X. Because the data reported here are anchored to rRNA levels, which in turn do not vary per unit of DNA, the 'per cell' expression trends can be determined on an absolute rather than a relative comparison basis; they indicate greater changes of the autosomes compared to the X chromosome (Bhadra, 2000 and references therein).
The loss of individual components of the Msl complex in the msl mutant males releases the Mof acetylase from the X and a uniform H4 acetylation distribution results. Accordingly, autosomal gene expression is generally increased, reflecting an inverse effect of the X on the autosomes, because the normally sequestered acetylase is now dispersed: this results in higher acetylation levels on the autosomes. An increase or decrease of acetylation level on the X is not reflected in major changes in gene expression, suggesting that some member of the Msl complex insulates genes on the X from responding to the much increased acetylation level (Bhadra, 2000 and references therein).
The collective data indicate that models that posit an association of the Msl complex with a gene for dosage compensation to occur are not supported. First of all, genetic destruction of the complex does not eliminate dosage compensation of most X-linked genes. However, one could perhaps argue that this action eliminates compensation of the regulatory genes on the X, which, because they are now dosage dependent, will compensate most of the 'housekeeping' genes that were assayed. This alternative is not favored for three reasons. (1) Ectopic expression of MSL2 in females as a transgene or in Sxlf mutants (present study) have the Msl complex present on their Xs but gene expression in general is not increased as predicted if the MSL complex alone conditions hyperactivation. (2) Dosage compensation also occurs in metafemales (3X chromosomes with diploid autosomes), where there is no complete complex and this compensation is related to that occurring in males. (3) Autosomal insertions of many X-derived genes still exhibit some degree of compensation despite the fact that these genes have no association with the Msl complex. Thus, there are several circumstances known in which compensation occurs without the Msl complex. The function of the Msl complex on the X chromosome appears to be to inhibit the response of most X-linked genes to high levels of histone acetylation (Bhadra, 2000 and references therein).
When these data are taken together along with previous studies on mle, a consistent model is supported, indicating that the effect of the Sex lethal gene is mediated through its control of the presence or absence of Msl-2. When Msl-2 protein is expressed, the sequestration of the MSL complex occurs with a resultant increase of H4Lys16 acetylation on the X at the expense of acetylation on the autosomes. In the absence of MSL-2, there is a uniform genomewide distribution of Mof and H4Lys16 acetylation. In general, gene expression on the autosomes responds positively to the level of acetylation, but the X is refractory to it in the presence of the Msl complex. In this way, the twofold inverse-dosage effect of the X is used to achieve a proper level of dosage compensation, but the effect on the autosomes is diminished. Thus, as the heteromorphic sex chromosomes have evolved, both the X and the autosomes have maintained nearly equal expression between the sexes (Bhadra, 2000).
In order to understand the mechanisms of dosage compensation occurring in Drosophila it is necessary to identify all the components of the MSL complex
and to determine how they may interact to mediate upregulation of transcription on the male X. JIL-1 colocalizes with the
MSL complex proteins on the male X; JIL-1 can molecularly interact with MSL complex proteins; ectopic expression of MSL2 in females
causes a concomitant upregulation of JIL-1 to the female X; this upregulation is abolished in msl mutants that are unable to assemble the complex.
Thus, these results strongly indicate that JIL-1 can associate with the MSL dosage compensation complex. The ability of JIL-1 to phosphorylate histone H3 in
vitro (Jin, 1999) further suggests a model where JIL-1's role in the MSL complex is to assist in regulating transcription possibly through modification of
chromatin (Jin, 2000).
The MSL complex is believed to promote dosage compensation in males by targeting MOF, the histone acetylase responsible for the increased H4Ac16
modification found on the male X chromosome. This modification is thought to lead to a more diffuse chromatin structure and enhanced
accessibility of the DNA for transcription. However, it is becoming increasingly evident that in addition to acetylation, phosphorylation may also play a key role
not only at the level of modulation of transcription factor activity, but also more generally at the level of chromatin structure. For example, histone H3
phosphorylation has been found to occur in a small subset of nucleosomes in mitogenically stimulated cells. This phosphorylation appears to be regulated by mitogen-activated signal transduction cascades indicating a direct link between signal transduction pathways
and chromatin structure. JIL-1 has been shown to phosphorylate histone H3 in vitro (Jin, 1999) suggesting that the MSL complex may affect chromatin
structure not only by histone acetylation, but also by histone phosphorylation. However, the finding that JIL-1 interacts with the MSL complex through its kinase
domains also raises the possibility that one or more of the proteins in the MSL complex itself could be a substrate for JIL-1. Regardless of whether the
substrate(s) for JIL-1 is histones or components of the MSL complex (or both) it is likely that the JIL-1 kinase serves as a regulator of MSL complex function
through site-specific phosphorylation (Jin, 2000).
The finding that JIL-1 associates with the MSL complex is based on physical interaction assays such as coimmunoprecipitation and GST pull-down experiments.
These approaches require a fairly stable interaction between JIL-1 and the MSL complex making it unlikely that this association depends on the presence of the
roX RNAs. This is in contrast to the MLE product that is not found as part of the MSL complex during chromatographic separation, a
finding consistent with it being the only one of the known MSL complex proteins to be lost from the male X chromosomes upon treatment with RNase. Moreover, the finding that addition of GST-JIL-1 fusion protein to S2 cell extracts can be used to pull down the MSL complex indicates
that this association can occur in vitro and suggests that the JIL-1 kinase can interact with the preassembled MSL complex. However, since this preassembled
complex may already contain endogenous JIL-1 it does not address whether the formation of the MSL complex requires the presence of JIL-1 protein or
whether JIL-1 is present in the MSL complex in stoichiometric amounts (Jin, 2000).
In Drosophila, dosage compensation is controlled by the male-specific lethal (MSL) complex consisting of MSL proteins and roX RNAs. The MSL complex is specifically localized on the male X chromosome where it acts to increase its expression ~2-fold. A model for the targeted assembly of the MSL complex has been proposed in which initial binding occurs at ~35 dispersed chromatin entry sites, followed by spreading in cis into flanking regions. Here, one of the chromatin entry sites, the roX1 gene, was analyzed to determine which sequences are sufficient to recruit the MSL complex. Association and spreading of the MSL complex from roX1 transgenes was found in the absence of detectable roX1 RNA synthesis from the transgene. The recruitment activity was mapped to a 217 bp roX1 fragment that shows male-specific DNase hypersensitivity and can be preferentially cross-linked in vivo to the MSL complex. When inserted on autosomes, this small roX1 segment is sufficient to produce an ectopic chromatin entry site that can nucleate binding and spreading of the MSL complex hundreds of kilobases into neighboring regions (Kageymama, 2001).
MSL1 and MSL2 are thought to form a core complex that binds to chromatin entry sites, while MLE and other components including roX RNAs may be added sequentially. To assess whether all the MSL components are required to bind to the roX1 exons, MSL1 localization on the roX1c3.4 transgene was analyzed in msl mutant animals. Since msl mutant males show poor morphology of polytene chromosomes, females carrying a Hsp83-MSL2 transgene were analyzed. Females normally lack MSL2 and can not form MSL complexes, but when MSL2 is constitutively expressed in females, the complex forms and is localized to both X chromosomes (Kageymama, 2001).
The roX1 and roX2 genes of Drosophila produce male-specific non-coding RNAs that co-localize with the Male-Specific Lethal (MSL) protein complex. This complex mediates up-regulation of the male X chromosome by increasing histone H4 acetylation, thus contributing to the equalization of X-linked gene expression between the sexes. Both roX genes overlap two of ~35 chromatin entry sites, DNA sequences proposed to act in cis to direct the MSL complex to the X chromosome. Although dosage compensation is essential in males, an intact roX1 gene is not required by either sex. Flies have been generated lacking roX2 and this gene is also found to be non-essential. However, simultaneous removal of both roX RNAs causes a striking male-specific reduction in viability accompanied by relocation of the MSL proteins and acetylated histone H4 from the X chromosome to autosomal sites and heterochromatin. Males can be rescued by roX cDNAs from autosomal transgenes, demonstrating the genetic separation of the chromatin entry and RNA-encoding functions. Therefore, the roX1 and roX2 genes produce redundant, male-specific lethal transcripts required for targeting the MSL complex (Meller, 2002).
The X-localization of roX transcripts, their male-specificity and regulation of roX RNA accumulation by the MSL proteins all have suggested a role for these RNAs in dosage compensation. However, failure to detect a phenotype in males mutated for roX1 and the absence of precise mutations in roX2 has previously precluded formal tests of this possibility. Furthermore, slight differences between the roX RNAs, such as the ability of transcripts from roX2, but not roX1, to move to all entry sites on the X chromosome in a fly mutated for msl3, and the identification of flies and cell lines lacking roX1, but none lacking roX2, have supported the notion that roX2 might be an essential gene in males. However, the mutation strategy used in this study, generation of a lethal deletion and restoration of essential gene functions with a genomic cosmid, has produced healthy males lacking roX2 RNA. When the roX2 deletion was combined with a roX1 mutation, male-specific lethality was revealed. Heat shock-driven expression of either roX RNA restores the viability of doubly mutated males, indicating that the male-specific phenotype is due to lack of roX RNA only. These experiments show that roX2 is not essential in males, and the synthetic lethality detected when both roX genes are mutated eliminates the possibility of undetected roX transcripts with similar function. The roX2 deletion removes the roX2-associated chromatin entry site at 10C, and all but 40 bp of the roX1 site is removed in roX1 ex6. Because male viability is rescued by roX RNA in trans, male lethality is consequential to loss of the roX transcripts and not to mutation of the chromatin entry sites. It is concluded that the RNA products of the roX genes are redundant male-specific lethals. The roX2 deletant that was generated is still complex, since two genes flanking roX2 are disrupted by Df(1)52 and not restored by cosmid [w +4Delta4.3]. The rescue of male viability by roX cDNAs also demonstrates that male lethality is not an unintended consequence of other mutations associated with the Df(1)52 chromosome or the rescuing [w + 4Delta4.3] transgene (Meller, 2002).
The involvement of the roX RNAs in dosage compensation can be functionally demonstrated by the rescue of females forced to express msl2. Production of the MSL2 protein triggers the formation of intact MSL complexes that bind to the X chromosome and enhance the transcription of X-linked genes. Female expression of msl2 leads to an inappropriate up-regulation of both X chromosomes resulting in a high level of lethality that can be blocked by mutations in another of the protein-coding msl genes. Removal of both roX RNAs similarly rescues msl2-expressing females. Partial rescue of female lethality by elimination of only one of the roX RNAs is consistent with previous results indicating that females expressing msl2 from the [w + H83M2-6I] transgene are a sensitized genetic background in which changes in the level of components of the MSL complex may be detected. These studies specifically demonstrate a block of dosage compensation when both roX RNAs are eliminated (Meller, 2002).
In contrast to the absence of escapers from mutations in the protein-coding msl genes, the doubly mutant X chromosomes used in this study did allow a low number of escaper males. This is a surprising finding in light of the disruption in MSL localization and loss of H4Ac16 enrichment on the X chromosome. It suggests that in spite of the clear importance of the roX RNAs for histone acetylation, compensation of the X chromosome is not completely compromised in the roX - males that were generated. It is possible that the roX RNAs are peripheral to formation of the MSL complex. Residual MSL2 binding on the X chromosome of roX - males would be consistent with this. Partial complexes with reduced activity could also form in the absence of the roX RNAs, and the simultaneous enrichment at the chromocenter of MSL1, MSL2 and H4Ac16 in roX - males supports the idea that some or all of the MSL proteins may still assemble into a complex capable of acetylating histone H4. It is also possible that the available roX1 mutations are not complete loss-of-function alleles (Meller, 2002).
Two lines of evidence support the idea that the MSL proteins, in the absence of the roX RNAs, can act to regulate gene expression. (1) MSL1, MSL2 and H4Ac16 are similarly redistributed in roX - males, suggesting that a protein complex able to direct histone acetylation still forms in the absence of roX RNA, although its localization is disrupted. (2) The sites of autosomal accumulation of the MSL proteins in roX - males are often puffed, suggestive of locally high rates of transcription. Autosomal sites of MSL3 binding have been reported in wild-type males, and MSL2 has been detected at several sites, including three specifically noted as binding MSL3. The one focused upon, 21B, displays fairly weak MSL2 staining in wild-type males but is often strongly stained and puffed in preparations from roX - males. It is suggested that an MSL complex lacking roX transcripts normally functions at a few autosomal sites in wild-type males. It is plausible that these represent a handful of genes with sex-specific expression. Removal of roX RNA releases the bulk of the MSL proteins from the X chromosome, and these are consequently available at high titers for association with the autosomal sites. Puffing of these sites suggests that elevated levels of the MSL proteins can hyper-activate transcription (Meller, 2002).
Alternatively, it is possible that all targeting of the MSL proteins to chromatin requires an RNA cofactor, but only roX1 and roX2 serve to direct the MSL proteins to the X chromosome. This would evoke the presence of other roX-like RNAs that specify a more restricted set of targets, perhaps a few autosomal genes. The potential utility of a system of gene activation that can be redirected by deploying an assortment of transcripts is quite attractive; however, in this instance the restriction to a single sex would limit the range of target genes considerably (Meller, 2002).
Mislocalization of MSL2 is strikingly different in roX - males than in those lacking mle, msl3 or mof, where residual MSL2 is observed binding only to ~35 chromatin entry sites. This points to a role for roX RNA in correct targeting of the intact complex to the X chromosome. The mechanism driving relocation of the MSL proteins to heterochromatin is at this point speculative. Two of the components of the MSL complex, MSL3 and MOF, contain variant chromodomain motifs. Chromodomains are involved in targeting HP1 to heterochromatin, and localization depends on the interaction of this domain with histone H3 methylated on lysine 9, a modification found in heterochromatic regions. The variant chromodomains of MSL3 and MOF have been shown to bind RNA in vitro, and it is likely that the roX RNAs are their normal ligands. Removal of roX transcripts could allow these domains to engage in inappropriate protein-protein interactions that target the remaining members of the MSL complex to heterochromatin (Meller, 2002).
Dosage compensation ensures equal expression of X-linked genes in males and females. In Drosophila, equalization is achieved by hypertranscription of the male X chromosome. This process requires an RNA/protein containing dosage compensation complex (DCC). RNA interference of individual DCC components has been used to define the order of complex assembly in Schneider cells. Interaction of MOF with MSL-3 leads to specific acetylation of MSL-3 at a single lysine residue adjacent to one of its chromodomains. Localization of MSL-3 to the X chromosome is RNA dependent and acetylation sensitive. The acetylation status of MSL-3 determines its interaction with roX2 RNA. Furthermore, RPD3 interacts with MSL-3 and MSL-3 can be deacetylated by the RPD3 complex. It is proposed that regulated acetylation of MSL-3 may provide a mechanistic explanation for spreading of the dosage compensation complex along the male X chromosome (Buscaino, 2003).
As in male flies, MSL-2 is central to the assembly of the dosage compensation complex in SL-2 cells, since its depletion by RNAi leads to disassembly of the complex. MOF protein requires prior assembly of MSL-1, MSL-2, and MSL-3, while the MSL-3 protein requires prior assembly of at least MSL-1 and MSL-2 proteins. Since in MOF dsRNA-treated cells approximately 10% of MOF protein can still be detected by Western blot analysis, it remains possible that small amount of MOF enzyme may be sufficient for MSL-3 localization to the X chromosome. In MOF mutant flies, MSL-3 protein localization on polytene chromosomes is restricted only to chromatin entry sites, as detected by immunostaining of the polytene chromosomes, suggesting that incomplete knockdown of MOF may be a plausible explanation for the apparent unaffected MSL-3 localization in these cells. However, it is also important to note that due to the limited size of SL-2 cells, it is difficult to resolve entry sites in SL-2 cells in comparison to the polytene chromosomes. Alternatively, MSL-3 localization to the X chromosome in MOF dsRNA-treated cells could also be a feature specific to SL-2 cells. Interestingly, depletion of MSL-2, MSL-3, or MOF led to dissociation of MLE from the X chromosome. It is therefore suggested that MLE localization is sensitive to the assembly of the rest of the complex in SL-2 cells (Buscaino, 2003).
Genetic equality between males and females is established by chromosome-wide dosage-compensation mechanisms. In the fruitfly Drosophila melanogaster, the dosage-compensation complex promotes twofold hypertranscription of the single male X-chromosome and is silenced in females by inhibition of the translation of msl2, which codes for the limiting component of the dosage-compensation complex. The female-specific protein Sex-lethal (Sxl) recruits Upstream-of-N-ras (Unr) to the 3' untranslated region of msl2 messenger RNA, preventing the engagement of the small ribosomal subunit3. This study reports the 2.8 Å crystal structure, NMR and small-angle X-ray and neutron scattering data of the ternary Sxl-Unr-msl2 ribonucleoprotein complex featuring unprecedented intertwined interactions of two Sxl RNA recognition motifs, a Unr cold-shock domain and RNA. Cooperative complex formation is associated with a 1,000-fold increase of RNA binding affinity for the Unr cold-shock domain and involves novel ternary interactions, as well as non-canonical RNA contacts by the α1 helix of Sxl RNA recognition motif 1. These results suggest that repression of dosage compensation, necessary for female viability, is triggered by specific, cooperative molecular interactions that lock a ribonucleoprotein switch to regulate translation. The structure serves as a paradigm for how a combination of general and widespread RNA binding domains expands the code for specific single-stranded RNA recognition in the regulation of gene expression (Henning, 2014).
Translational repression of msl2 mRNA is coordinated by Sxl binding to uridine-rich stretches in both untranslated regions (UTRs): binding to the 3' UTR inhibits the recruitment of the small ribosomal subunit whereas binding to the 5' UTR inhibits the scanning of those subunits that presumably have escaped the 3' UTR-mediated control. At the 3' UTR, the recruitment of Unr by Sxl to bind in close spatial proximity is critical for translational repression. The region of Sxl containing residues 122-301 (Drosophila RNA binding domain 4, dRBD4) shows full translational repression activity, while the RNA recognition motifs (RRMs) alone (residues 123-294, dRBD3) are necessary and sufficient for RNA binding. Only the first cold-shock domain (CSD1) of Unr is required for complex formation with Sxl and msl2 mRNA. Notably, CSD1 and Sxl do not interact in the absence of RNA, suggesting a cooperative binding mechanism. A 46-nucleotide region in the msl2 3' UTR containing two uridine-rich Sxl-binding sites is sufficient for complex formation and translational repression. To identify the minimal region required for Unr and Sxl binding, ternary complex formation was analysed by electrophoretic mobility shift assays (EMSA) using wild-type and variant RNAs. Binding of dRBD4 and Unr to the wild-type RNA indicated the presence of two complexes. The number of complexes was reduced to one by mutation of either Sxl-binding site, and site F supported complex formation with a higher affinity than site E. Mutation of the sequences surrounding site F affected Unr binding, while more distal mutations did not impair complex formation. These data indicate the formation of 2:2:1 and 1:1:1 dRBD4-Unr-RNA complexes representing the two bands of slower mobility, which was further confirmed by static light scattering measurements (Henning, 2014).
Taken together these data demonstrate that the triple zipper and the non-canonical RNA contacts mediated by Sxl RRM1 are critical for translational regulation by Sxl and Unr. It is important to note that these interactions are essential for msl2 translational repression, but are dispensable for the regulation of transformer pre-mRNA splicing19, as recognition of the uridine-rich 5' region of msl2 RNA by Sxl dRBD3 in the ternary complex is virtually identical to that previously observed for transformer pre-mRNA11. Therefore, recognition of a uridine-rich RNA sequence by Sxl can play distinct roles in regulating splicing and translation depending on the binding of Unr in close proximity (Henning, 2014).
The data also explain why human Unr can form a complex with Drosophila Sxl and msl2 RNA as all residues involved are conserved (His 213, Asp 237 and Arg 239). In contrast, CSD1 alone can bind a variety of distinct RNA sequences with similar affinity in the absence of Sxl. This indicates that strong and specific RNA recognition for the GCACG motif in msl2 RNA depends on the presence of Sxl in the ternary complex. Interestingly, C11 does not conform to the previously reported consensus sequence for human Unr CSD1, but is nevertheless strictly conserved in the msl2 mRNA of organisms that may employ D. melanogaster-like dosage compensatio. Consistent with this, CSD1 Asp 237 and Arg 239, which recognize C11, are conserved in CSD1 but not in CSD2-5 of Unr proteins or in cold shock domains of other proteins (Henning, 2014).
Although the Drosophila dosage-compensation mechanism is not conserved in mammals, it is expected that ternary interactions involving RRM and CSD domains with RNA may be important for other biological functions. For example, human orthologues of the proteins examined in this study, such as the Sxl orthologue HuR or the RNA binding protein RBM6, share triple-zipper and α1-helix residues, which could mediate similar interactions (Henning, 2014).
Sandwiching of single-stranded RNA by multiple proteins has been observed previously, for example in small nuclear ribonucleic particles or the exon junction complex, but the intertwined recognition observed in this study is particularly intriguing. Moreover, the combination of these two general and abundant RNA binding domains (RRM and CSD), which are also involved in other RNA binding events, generates a new and unique binding specificity for single-stranded RNA. The intertwined cooperative binding of Sxl and Unr establishes a functionally active arrangement of multiple RNA binding domains from two distinct proteins, thus extending principles recently observed for multi-domain RNA binding proteins (Henning, 2014).
These results show that repression of a biological process with dramatic consequences for viability depends on the establishment of a specific set of novel molecular interactions. This is of particular significance considering that a limited set of RNA binding modules has been identified in the mRNA interactome. The Unr-Sxl-msl2 complex illustrates how the combinatorial activity of general RNA binding domains expands the code for RNA recognition by establishing unique and distinct ribonucleoprotein architectures and thus greatly amplifying the opportunities for regulation of gene expression (Henning, 2014).
Dosage compensation in Drosophila involves a global activation of genes on the male X chromosome. The activating complex (MSL-DCC) consists of male-specific-lethal (MSL) proteins and two long, noncoding roX RNAs. The roX RNAs are essential for X-chromosomal targeting, but their contributions to MSL-DCC structure and function are enigmatic. Conceivably, the RNA helicase MLE, itself an MSL subunit, is actively involved in incorporating roX into functional DCC. This study determined the secondary structure of roX2 and mapped specific interaction sites for MLE in vitro. Upon addition of ATP, MLE disrupted a functionally important stem loop in roX2. This RNA remodeling enhanced specific ATP-dependent association of MSL2, the core subunit of the MSL-DCC, providing a link between roX and MSL subunits. Probing the conformation of roX in vivo revealed a remodeled stem loop in chromatin-bound roX2. The active remodeling of a stable secondary structure by MLE may constitute a rate-limiting step for MSL-DCC assembly (Maenner, 2013).
Since the discovery of Xist RNA as a crucial epigenetic regulator involved in mammalian X inactivation about 20 years ago, an increasing number of long, noncoding (lnc) RNAs have been found associated with chromatin-modifying complexes. Their hypothetical functions include roles as versatile scaffolds for protein complexes and in targeting associated regulators to genomic loci, either as nascent RNA or through R-loop or triple helix formation. Considering the conformational flexibility and structural diversity of RNA, its regulatory potential is enormous. However, a detailed mechanism of action has only been resolved in a few cases (Maenner, 2013 and references therein).
The highly elaborate dosage compensation systems that evolved to compensate for sex chromosome monosomy provide instructive examples for regulatory functions of lnc RNAs. The inactivation of one X chromosome in female mammals is orchestrated by the interplay of at least three lnc RNAs. In Drosophila melanogaster, dosage compensation involves doubling the transcription of many X-linked genes. This activation is achieved by a ribonucleoprotein dosage compensation complex (DCC) consisting of five male-specific-lethal (MSL) proteins and two lnc RNAs, roX1 and roX2 (for 'RNA-on-the-X'). These RNAs are considered scaffolds for the proper assembly of the MSL proteins. The MSL-DCC only forms in male flies due to the male-specific expression of two key components: the core subunit MSL2 and the roX RNAs. The remaining protein subunits-MSL1, MSL3, the acetyltransferase MOF (Males-absent-on-the-first), and the RNA helicase MLE (Maleless)-are also expressed in female cells and have additional functions outside of the MSL-DCC (Maenner, 2013).
Genome-wide mapping of the chromosomal interactions revealed that most of the MSL-DCC interacts with the bodies of actively transcribed genes on the X chromosome. According to a popular model, the X-specific targeting of the MSL-DCC involves an initial recognition of a limited number of 'chromosomal entry' or 'high-affinity' sites ('CES' or 'HAS,' respectively), which are distributed along the X. Accordingly, the complex transfers from these sites to active genes in the nuclear vicinity. Once tethered to the active chromatin, the MSL-DCC activates transcription through acetylation of lysine 16 of histone H4 (H4K16ac) by MOF, which is thought to unfold the chromatin fiber and facilitate the production of mature mRNAs (Maenner, 2013).
RoX1 and roX2 RNAs differ greatly in size (3.7 kb versus 600 nt) and show very little sequence similarity. Despite these differences, each alone can support the assembly of a functional MSL-DCC. Inactivation of both roX genes abolishes dosage compensation and is lethal for males. Exploring the common denominator between the two RNAs by phylogenetic comparison and functional analyses, Kuroda, Park, and colleagues discovered a series of conserved sequence motifs (GUUNUNCG), the 'roX-boxes,' which reside in the 3' ends of the roX RNAs. In silico analysis of structural motifs predicts that prominent roX-boxes participate in the formation of stable stem-loop structures (SLroX). The integrity of SLroX structure is essential for roX function. Furthermore, expressing an RNA consisting solely of six tandem repeats of SLroX2 suffices to recruit the MSL proteins to the X chromosome and acetylate H4K16 on the X in the absence of endogenous roX RNA. These data highlight the importance of the SL for roX RNA function, but the underlying molecular mechanism remains unclear. Although four of the five MSL proteins are able to bind RNA in vitro, so far no specific interaction of any protein with SLroX has been demonstrated (Maenner, 2013).
Several lines of evidence suggest a primary role for the RNA helicase MLE in incorporating roX into the MSL-DCC. RoX1 is initially expressed in preblastoderm embryos of both sexes, where it is stabilized by maternal MLE until dosage compensation is established. At later developmental stages, MLE is required for steady-state assembly and targeting the MSL-DCC to the X chromosome. Inactivating the ATPase activity of MLE by point mutation (MLEGET) impairs X-chromosomal targeting of the DCC. A mutation that selectively inactivates the helicase function greatly limits the spreading of the MSL-DCC onto the active gene bodies (Maenner, 2013).
It is unclear where in the nucleus roX RNAs and MSL proteins assemble to form a functional DCC. This may happen at the site of roX gene transcription. Another possibility is that HASs are sites of MSL-DCC formation. It was recently found in a genome-wide study of chromosomal interactions that MLE strongly enriched at some 240 HAS sites on the X, in close proximity to MSL2. Since the specific chromatin immunoprecipitation (ChIP) methodology applied in this study emphasizes direct chromatin interactions, it was consider that MLE may affect the assembly or reorganization of the MSL-DCC at HAS. In line with this argument, the recent ChIRP (chromatin isolation by RNA purification) profiling of chromatin interactions also found roX2 RNA enriched at HAS (Maenner, 2013).
The current study suggests that the assembly of the ribonucleoprotein MSL-DCC is initiated by an obligatory, energy-consuming RNA remodeling step, during which MLE recognizes the SLroX structure and converts it into a more extended form. Accordingly, the stable, low-energy SL conformation that roX2 adopts in vitro represents an inactive, closed conformation, which needs to be actively opened to expose binding sites for MSL proteins (Maenner, 2013).
This study has found that the specific interaction of MSL2, the key identifier of the MSL dosage compensation complex, with roX RNA depends on the prior remodeling of a prominent stem-loop structure in the 3' end of roX2. This hairpin, together with a related structure in roX1, is indispensable for roX function in flies. Previous failures to detect specific MSL interactions with SLroX2 are now at least in part explained by the observation that SLroX2 itself does not serve as a binding site but has to be converted into an alternative, open conformation through the ATP-dependent remodeling action of MLE. The data describe a function for a nuclear helicase in lncRNA conformation. The occurrence and functional importance of the proposed conformational switch during organismal development remains to be tested in future studies (Maenner, 2013).
A role for MLE in resolving long dsRNA stems was already suggested more than a decade ago. A mutation in MLE, the mlenapts allele, causes the failure to express the para-encoded Na+ channel. Para transcripts are subject to consecutive RNA editing and splicing. Adenosine-to-inosine editing is guided by the formation of a stable dsRNA secondary structure that needs to be resolved for subsequent splicing. The mutated helicase binds to and stabilizes the stem structure rather than unwinding it, as it would normally do to allow access of the splicing machinery. Interestingly, the ds editing structure of the para transcript also contains two A-U motifs in the vicinity of the edited sequence, which resemble the roX-box motif. It is suggested that MLE functions more generally to unwind long base-paired RNA structures to expose ssRNA sequences for downstream interactions (Maenner, 2013).
The data show that MLE preferentially binds to RNA containing an SLroX even in the absence of ATP and that this interaction is improved by increasing the stability of the SL. In the presence of ATP, the specificity of interaction with this particular binding site is reinforced. Because the MLEGET mutant that binds nucleotides with reduced affinity does not show the effect, it is assumed that the ATP-dependent disruption of the SL strengthens the MLE interaction with roX. The SLroX structures share a conserved sequence, the roX-box, which contributes to MLE affinity. The four consecutive A-U pairs may facilitate the unwinding of the stem and/or provide a binding site for MLE and/or other MSL subunits in their single-stranded conformation (Maenner, 2013).
It is hypothesized that RNA remodeling by MLE physically connects the two male-specific principles that are key to the formation of the MSL-DCC: the male-specific MSL2 protein and the roX RNAs. Numerous observations already suggested a close interdependency of their functions. In the absence of MSL2, roX RNAs are unstable and, conversely, any MSL2 that is not in a complex with roX will be degraded. The enrichment of MLE on the X chromosome depends on MSL2 and MSL1. MSL2 may physically connect the MLE-roX module to the MSL1-MSL3-MOF module. If MSL1 is mutated to abolish the MSL2 interaction, roX RNAs are no longer incorporated into the MSL-DCC. It is currently unclear whether MLE would remain part of a stable MSL-DCC complex once it has remodeled the roX RNA. Earlier findings that MLE readily dissociates during purification of MSL complexes indicate that MLE was rather loosely bound. The helicase may only fulfill a transient function during the biogenesis of the complex. The striking enrichment of MLE at 240 HAS on the X chromosome in direct neighborhood of MSL2 identifies these loci as primary sites of MLE action (Maenner, 2013).
In summary, it is proposed that the active remodeling of roX RNA by the ubiquitous RNA helicase MLE constitutes an obligatory, regulated step in the biogenesis of the MSL-dosage compensation complex. This study paves the way for a mechanistic dissection of this fundamental conformational switch. Lessons learned from the dosage compensation system will undoubtedly enhance the understanding of other lncRNA-containing chromatin regulators (Maenner, 2013).
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