org Interactive Fly, Drosophila

RNA on the X-1


Transcriptional Regulation

The rox1 transcript is not observed in wild-type adult females. XO males and XXY females exhibit roX1 expression patterns identical to those of wild-type males and females, eliminating the possibility of a Y-linked regulator of roX1. The loss-of-function mutation tra1 transforms XX flies into pseudomales, but even though they are somatically male, thes flies do not express roX1. XX flies that are morphologically male, due to mutation in the Sex lethal gene, exhibit roX1 levels comparable to those observed in wild-type males. Therefore, Sxl+ function is responsible for the lack of roX1 transcripts in the female, but this suppression bypasses tra, a downstream effector of Sxl function (Meller, 1997).

Expression of roX1 and roX2 was analyzed in flies mutant for tra, tra2, doublesex, intersex and fruitless. Expression of roX1 and roX2 is not detected in any of the pseudomales generated by these mutations. These data suggest that roX1 and roX2 expression is independent of the cascade of genes essential for sex-specific differentiation. The whole complex of genes required for dosage compensation is required for activation of roX1 and roX2, as expression is not detected in females carrying the msl-2 transgene (to active the dosage compensation pathway) and carrying mutations for either msl-1, msl-3 or mle (Amrein, 1997).

At least one component of the dosage compensation system, mle, is required for roX1 expression in male larvae. Males mutant in mle show no staining for roX1 RNA in any tissues. The constitutive expression of an msl-2 transgene in females is sufficient to induce or stabilize all of the dosage compensation proteins and recruit them to the X chromosomes resulting in significant female mortality (Kelley, 1995). Salivary glands from females that carry the msl-2 transgene show strong roX1 staining, with subnuclear localization indistinguishable from that seen in males. Localization of MLE protein is not disrupted in roX1 mutants, indicating that roX1 is not essential for MLE binding to the X chromosome (Meller, 1997)

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

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

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

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

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

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

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

MLE functions as a transcriptional regulator of the roX2 gene

Dosage compensation is a process that equalizes transcription activity between the sexes. In Drosophila, two non-coding RNA, roX1 and roX2, and at least six protein regulators, MSL-1, MSL-2, MSL-3, MLE, MOF, and JIL-1, have been identified as essential for dosage compensation. Although there is accumulating evidence of the intricate functional and physical interactions between protein and RNA regulators, little is known about how roX RNA expression and function are modulated in coordination with protein regulators. A relatively short (about 350 bp) upstream genomic region of the roX2 gene, Prox2, harbors an activity that drives transcription of the downstream gene. This study has shown that MLE can stimulate the transcription activity of Prox2 and that MLE associates with Prox2 through direct interaction with a newly identified 54-bp repeat, Prox. These observations suggest a novel mechanism by which roX2 RNA is regulated at the transcriptional level (Lee, 2004).

When ectopically expressed, MSL2 induces dosage compensation even in female flies. Because roX RNAs are critical for dosage compensation, transcription of the corresponding genes, which are suppressed in female flies, must be activated in these transgenic female flies expressing MSL2. To understand the mechanism underlying transcription activation of the roX2 gene, attempts were made to identify an upstream genomic region potentially important for transcriptional regulation of the roX2 gene. Five genes, including roX2 and nod, are identified in the sense template upstream of rox3, and two genes, are in the antisense template. A 350-bp intergenic region, which has been titled Prox2, is present between CG11695 and the roX2 gene and contains a T-rich region at position -331 to -150 and a 54-bp repeated sequence at position -261 to -154 (Lee, 2004).

At present, the molecular mechanisms underlying roX gene transcription remain unknown. It has been shown that mutant embryos, lacking MLE, normally synthesize roX1 RNA, but roX1 RNA appears to be concentrated at its site of synthesis. Based on this observation, it has been proposed that MLE is required for the stability of roX1 RNA and its movement from the transcription site, but not for its synthesis. MLE has also been implicated in stable maintenance of the steady-state level of roX2 RNA. Expression of mleGET in place of the wild type MLE results in drastic reduction in the roX2 RNA level and formation of the MSL complex devoid of roX2 RNA. In addition to this post-transcriptional function, the present study provides two pieces of evidence of a direct involvement of MLE in transcription regulation of the roX2 gene: (1) MLE interacts with the upstream promoter region (i.e. Prox2) of the roX2 gene through association with a 54-bp repeat, Prox; (2) overexpression of MLE activates transcription driven by Prox2 either in a reporter construct or a chromosomal context (Lee, 2004).

This study has shown that ATP is not essential for the interaction of MLE with Prox. In addition, MLE ATPase activity is dispensable for transcriptional activation supported by Prox2. These results are consistent with the findings that MLE retains X chromosome binding ability despite various mutations introduced in the ATPase motifs and that ATPase activity is dispensable for transcriptional activation of the X-linked genes. Because mutations in the ATPase motifs of MLE affect the viability of male flies, the ATPase activity seems to be required for normal development of male flies. Then, by what mechanism does the ATPase activity of MLE influence dosage compensation? Based on poor binding of roX1 RNA to the X chromosome in flies expressing mleGET, it has been proposed that MLE ATPase activity plays an early role, perhaps in packaging roX2 RNA into growing MSL complexes. In support of this hypothesis, a recent study shows that in the absence of an ATP-dependent function of MLE, MSL complex can be assembled but is devoid of roX RNA. Thus, it is likely that in addition to transcriptional regulation by an ATP-independent function of MLE, roX2 RNA is post-transcriptionally regulated through association with MLS proteins, which require an ATP-dependent function of MLE (Lee, 2004).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Dosage compensation in Drosophila is mediated by a histone-modifying complex that upregulates transcription of genes on the single male X chromosome. The male-specific lethal (MSL) complex contains at least five proteins and two noncoding roX (RNA on X) RNAs. The mechanism by which the MSL complex targets the X chromosome is not understood. This study used a sensitized system to examine the function of roX genes on the X chromosome. In mutants that lack the NURF nucleosome remodeling complex, the male polytene X chromosome is severely distorted, appearing decondensed. This aberrant morphology is dependent on the MSL complex. Strikingly, roX mutations suppress the Nurf mutant phenotype regionally on the male X chromosome. Furthermore, a roX transgene induces disruption of local flanking autosomal chromatin in Nurf mutants. Taken together, these results demonstrate the potent capability of roX genes to organize large chromatin domains in cis, on the X chromosome. In addition to interacting functions at the level of chromosome morphology, it was found that NURF complex and MSL proteins have opposing effects on roX RNA transcription. Together, these results demonstrate the importance of a local balance between modifying activities that promote and antagonize chromatin compaction within defined chromatin domains in higher organisms (Bai, 2007).

The roX noncoding RNAs are critical components that regulate targeting of MSL complexes to the male X chromosome. roX RNAs are not stably expressed in wild-type females, and this study shows that NURF, an ATP-dependent chromatin-remodeling enzyme, is a repressor of roX transcription in females. Furthermore, regional decondensation of the male X chromosome found in Nurf mutants was dependent on the presence of a linked roX gene, roX1 for the distal X and roX2 for the proximal X. These results support a model in which the MSL complex assembles at roX genes and can act long distances along the X chromosome (Bai, 2007).

Previous analyses indicated that MSL proteins are required for transcription of roX genes. In the case of roX1, the MSL complex binds to the DHS and counteracts the activity of a constitutive repressor, establishing the male-specific pattern of roX1 transcription. This analysis of endogenous roX transcript levels and heterologous roX reporter constructs indicate that NURF mediates this repression and that, for roX1, NURF acts through the DNase hypersensitive sites (DHSs). This is confirmed by ChIP analysis that shows NURF is recruited to the roX1 DHS, demonstrating that regulation is direct (Bai, 2007).

The requirement for NURF in roX repression was detected in females that do not ordinarily express roX genes. In the absence of NURF, endogenous levels of both roX1 and roX2 are increased in females. In males, steady-state transcript levels are either unaffected (roX1) or increased approximately twofold (roX2). It is clear from the extreme decondensation of the NURF mutant male X chromosome and its suppression by roX and MSL complex mutants that the male X chromosome is very sensitive to the loss of the NURF complex. One model that may reconcile the apparent contradiction between lack of roX gene derepression in Nurf mutant males and the extreme male X chromosome phenotype is as follows: NURF may affect key initial levels of roX RNAs at embryonic stages when MSL complex binding is first initiated. The effect of improperly regulated complex assembly during initial stages could be progressively amplified during development resulting in a chromosome morphology defect at a time when roX gene expression is no longer regulated by NURF in males. Alternatively, antagonism at the level of roX transcription and at the level of chromatin morphology may be functionally independent events (Bai, 2007).

A principal activity of NURF is ATP-dependent nucleosome sliding in cis on DNA without displacement. It is not difficult to envisage how nucleosome sliding can be used to expose or block binding sites for transcription factors and thereby control transcription. Indeed there is much evidence from studies of the orthologous yeast Isw2 remodeling complex that nucleosome sliding can be used to repress transcription. Isw2 is needed to repress early meiotic genes and targets of the Tup1-Ssn6 complex. However, in these cases, repression is mediated through 5′ regulatory elements at the level of transcription initiation. As was seen in this study, NURF represses roX1 through a binding site present in the coding region, ~1 kb 3' of the transcription initiation site (Bai, 2007).

The location of NURF binding within the roX1 gene becomes more pertinent when the known distribution of the MSL complex is considered. Recent whole genome profiling of MSL complex distribution on X-chromosome targets indicates that the complex shows a strong preference for the 3′ ends of gene targets. This correlates with a previous, more restricted, analysis showing that acetylation of H4K16, the epigenetic mark established by the MSL complex, follows a similar distribution. One implication of this distribution is that the MSL complex may regulate transcription of targets (including roX genes) not at the level of transcription initiation, but by improving elongation. It is tempting to speculate that NURF may also control elongation, as suggested by studies of the yeast ISW1 complex (Bai, 2007).

This study found that the aberrant morphology of the male X chromosome in Nurf301 mutants is regionally suppressed by deletion of either roX1 or roX2, providing strong visual evidence that roX genes can function in cis over long distances (>1 Mb). These results are consistent with a model in which nascent roX RNAs normally assemble and nucleate “spreading” of MSL complexes along the X chromosome. The term “spreading” has been controversial as it is subject to a myriad of interpretations. What the authors of this paper mean by spreading is that following assembly, the MSL complex is much more likely to act regionally, in cis, than to be unconstrained. It has been proposed that in addition to roX genes, specific MSL interaction occurs at “high affinity sites” (also termed “chromatin entry sites”) whose identifying characteristics are yet to be defined. “Spreading” from roX genes and high affinity sites to the full MSL binding pattern could occur by scanning along the chromosome in a linear mode, but it could also occur solely by local release and recapture of the complex by regions in close physical proximity. Movement from one DNA molecule to another clearly can occur when roX genes or various segments of X are moved to autosomes (Bai, 2007).

High affinity sites and roX genes might normally function together to constrain the MSL complex largely to the X chromosome. An “affinities” model posits that there is a continuum of affinity sites for MSL complexes, ranging from high to low, and that only when high sites are locally concentrated can low affinity sites be recognized. This clearly falls under the general premise of the spreading model. In both cases, MSL targeting is a multistep process in which many binding sites are not recognized independently, in the absence of influence of neighboring sites in cis. While the image of X-chromosome morphology regionally controlled by the presence or absence of a roX gene is, to us, strong evidence for function of roX genes over long distances in cis, a more mechanistic view of MSL targeting clearly awaits additional data on the molecular nature of MSL–chromatin interactions (Bai, 2007).

Targeting of Painting of fourth to roX1 and roX2 proximal sites suggests evolutionary links between dosage compensation and the regulation of the fourth chromosome in Drosophila melanogaster

In Drosophila melanogaster, two chromosome-specific targeting and regulatory systems have been described. The male-specific lethal (MSL) complex supports dosage compensation by stimulating gene expression from the male X-chromosome, and the protein Painting of fourth (POF) specifically targets and stimulates expression from the heterochromatic 4th chromosome. The targeting sites of both systems are well characterized, but the principles underlying the targeting mechanisms have remained elusive. This study presents an original observation, namely that POF specifically targets two loci on the X-chromosome, PoX1 and PoX2 (POF-on-X). PoX1 and PoX2 are located close to the roX1 and roX2 genes, which encode noncoding RNAs important for the correct targeting and spreading of the MSL-complex. The targeting of POF to PoX1 and PoX2 is largely dependent on roX expression, and a high-affinity target region was identified that ectopically recruits POF. The results presented support a model linking the MSL-complex to POF and dosage compensation to regulation of heterochromatin (Lundberg, 2013).

High-affinity sites have been characterized for the MSL-complex and there are several published examples of short regions, including the roX1 and roX2 loci, that are capable of recruiting this complex when presented as transgenes. In contrast, until now no high-affinity sites for POF targeting have been identified. Translocated 4th chromosomes will not be targeted by POF, unless the proximal heterochromatic region is present and under conditions that favor heterochromatin formation (Johansson, 2007a). The characterization of POF targeting to PoX1 and PoX2 in females thus provides a unique opportunity to study the targeting of POF to a nonheterochromatic target and to further understanding of the evolution of these two targeting systems (Lundberg, 2013).

Considering the evolutionary relationship between POF and the MSL-complex (reviewed by Stenberg, 2011), it was intriguing to find POF targeting to two distinct regions on the X-chromosome, i.e., X:3E and X:10E-F. The apparent spreading of POF targeting in these two regions (resembling the spreading of the MSL-complex when it is targeted to roX transgenes) and the close location of these regions to roX1 and roX2 suggested a link with the MSL-complex and dosage compensation. It has been hypothesized that POF originated as a dosage compensation system, since POF targets the male X-chromosome in, for example, D. busckii and D. ananassae and in those species POF colocalizes with H4K16ac and the MSL-complex, respectively. However, the targeting of POF to endogenous PoX1 and PoX2 in D. melanogaster is restricted to females. This sex-specific targeting is not caused by sex-specific expression of the targeted genes, since comparable expression levels of >RE64691 as well as SelG and CG1840 are consistently found in male and female salivary glands (Lundberg, 2013).

Not only are the two targeted loci, PoX1 and PoX2, located in close proximity to roX1 and roX2, the targeting is also largely dependent on roX, because losses of roX1 alone or of roX1 and roX2 cause a clear decrease in the frequency of PoX targeting. Importantly, in all roX mutant conditions tested, complete loss of POF binding to the PoX sites was never found. Therefore, roX is not absolutely required for PoX targeting but rather it enhances or stabilizes the interaction. The dependence of targeting on roX is not caused by the close proximity of the PoX loci to the corresponding roX loci, because in the duplications tested the PoX1 and PoX2 are located on another chromosome, i.e., chromosome arm 3L, and the roX genes are not included in the duplicated region. Despite this, two of the duplications show targeting by POF, comparable to that to the endogenous loci. Furthermore, targeting to these transgenic regions was found to be largely dependent on roX, which indicates that roX can act in trans to enhance or stabilize POF targeting. The most parsimonious model to explain these observations is that it is the roX ncRNA species that enhance or stabilize targeting of POF to these non-chromosome 4 targets. This model is supported by the fact that roX2 overexpression seems to further increase the frequency of targeting. However, it should be stressed that endogenous roX expression in females is reported to be at very low levels or absent. In females, roX1 RNA has been reported in early embryos but it appears to be lost midway through embryogenesis, whereas in males expression is maintained through adulthood. roX2 RNA first appears a few hours after roX1, but only in male embryos (Lundberg, 2013).

No high-affinity sites for POF targeting have previously been identified (Johansson, 2007a). It therefore came as a surprise that a short (6-kb) region from PoX2 functions as a strong ectopic target for POF in both males and females. The nonsex-specific targeting of POF to the P[w+ SelG CG1840] transgene, in contrast to Dp(1;3)DC246 and endogenous PoX, may be explained by a competition of targeting between POF and the MSL-complex in males. This competition will be more pronounced at the endogenous PoX sites and the duplications as these are also targets for the MSL-complex in males. This finding is supported by the fact that on polytene chromosomes, Dp(1;3)DC246 is targeted by MSL-complex in males while the P[w+ SelG CG1840] transgene is not targeted. A competition in targeting is also supported by the reduction frequency of PoX1 and PoX2 targeting by POF observed in females expressing a partial MSL-complex, i.e. w; P[w+ hsp83:msl2] msl3 females. It is important to note that the targeting of POF to the P[w+ SelG CG1840] transgene was not caused by genomic location of this transgene since the same attP docking site (3L:65B2) was used as for the duplications of the PoX1 and PoX2 loci. The lack of targeting of POF to translocated parts of the 4th chromosome and the strong targeting to the PoX2 transgene suggest that the PoX regions may be POF targets that are functionally separable from the 4th chromosome genes. Since both Setdb1 and HP1a are detected on the transgene, it appears likely that POF recruitment leads to, or is connected with, the formation of GREEN (HP1a and H3K9me enriched) chromatin structure (Filion, 2010) (Lundberg, 2013).

The targeting of POF to the 4th chromosome depends on its well-characterized heterochromatic nature and on the presence of HP1a and Setdb1. It is therefore important to note that links between the MSL-complex, roX1 and roX2 and heterochromatic regions have been reported previously, though they remain to be understood. It is known that in roX1 roX2 mutant males, the MSL-complex is still detected on the X-chromosome, albeit at a reduced number of sites, but binding is also found in the chromocenter and at a few reproducible sites on the 4th chromosome. In contrast to the X-chromosome, where the MSL-complex is believed to stimulate gene expression, loss of roX RNA reduces expression from genes located in the chromocenter and on the 4th chromosome. It has been suggested that roX RNAs participate in two distinct regulatory systems, the dosage compensation system and a system for the modulation of heterochromatin. Although the mechanism by which roX RNAs enhance binding of POF to PoX loci remains elusive, the observation supports a model linking dosage compensation to modulation of heterochromatin. Additional factors supporting a model linking heterochromatin to dosage compensation are the proposed binding of HP1a to the male X-chromosome and the fact that a reduction in the histone H3S10 kinase JIL-1 results in the spreading of heterochromatic markers (such as H3K9me2 and HP1a) along the chromosome arms, with the most marked increase taking place on the X-chromosomes. The JIL1 kinase, which is believed to counteract heterochromatin formation, is highly enriched on the male X-chromosome and is reported to be loosely attached to the MSL-complex). It is noteworthy that POF, which targets genes in a heterochromatic environment, i.e., on the 4th chromosome, has an intrinsic ability to target the male X-chromosome, as seen in, e.g., D. ananassae, and the targeting to X-chromosome sites reported in this study is dependent on roX RNAs. At the same time the MSL-complex, which binds to and stimulates expression of genes on the male X-chromosome, has an intrinsic ability to target heterochromatin as seen in the roX1 roX2 mutant background. The link between these two systems is intriguing and promises to increase understanding of balanced gene expression (Lundberg, 2013).

High-affinity targeting to the PoX1 and PoX2 loci therefore provides a novel system for further studies on targeting mechanisms involved in chromosome-wide gene regulation, the evolutionary relationship between POF and dosage compensation and the evolution of balanced gene expression, and the results favor a model involving not only the X-chromosome but also balance to heterochromatin (Lundberg, 2013).

High-affinity sites form an interaction network to facilitate spreading of the MSL complex across the X chromosome in Drosophila

Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation toward targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses, this study shows that high-affinity sites (HAS), landing platforms of the male-specific lethal (MSL) complex, are enriched around topologically associating domain (TAD) boundaries on the X chromosome and harbor more long-range contacts in a sex-independent manner. Ectopically expressed roX1 and roX2 RNAs target HAS on the X chromosome in trans and, via spatial proximity, induce spreading of the MSL complex in cis, leading to increased expression of neighboring autosomal genes. It was shown that the MSL complex regulates nucleosome positioning at HAS, therefore acting locally rather than influencing the overall chromosomal architecture. The study proposes that the sex-independent, three-dimensional conformation of the X chromosome poises it for exploitation by the MSL complex, thereby facilitating spreading in males (Ramírez, 2015).

This study provides a first step toward understanding the role of chromosome conformation in dosage compensation in D. melanogaster. HAS, the landing regions of the MSL complex on the X chromosome, frequently reside in proximity to TAD boundaries. HAS are enriched in Hi-C contacts to each other and to other X chromosomal regions and that this organization remains comparable between male and female cells (Ramírez, 2015).

This analysis revealed that HAS are characterized by a combination of DNA sequence (MREs), chromatin state (active), and gene architecture, which drives the specificity of the MSL complex toward the X chromosome. The data suggest that when the MSL complex binds to HAS, it then spreads (either via an active mechanism or via diffusion) to spatially close regions to place the histone H4 lysine 16 acetylation (H4K16ac) mark on active genes. A 'conformation-based affinity' model is proposed based on the strategic location of HAS at highly interconnected regions of the D. melanogaster X chromosome that efficiently distribute the MSL complex over the X chromosome by attracting the MSL complex to cis-interacting HAS on the X chromosome. This system ensures that only this chromosome is specifically and globally targeted. By spreading from those HAS over short (3D) distances, all active genes on the X chromosome are then reached and acetylated without influencing the autosomes. It is suggested that this system is resilient to major perturbations, exemplified by the large autosomal insertion from chromosome 3L and the ectopic expression of the roX genes that produce viable cells and flies, respectively (Ramírez, 2015).

MNase-seq analysis shows a direct effect of the MSL complex on nucleosome organization specifically on HAS and not on the TSS, despite prominent binding of MSL1/2 to promoter regions. The MSL complex may act similar to a pioneer DNA binding protein to establish nucleosome patterns at HAS and may act on neighboring active regions rather than modifying TAD boundaries. This system may be unique to flies because the Drosophila dosage compensation evolved a fine-tuning transcription activation mechanism rather than a complete shutdown of gene transcription as seen in mammalian X chromosome inactivation. It would be very interesting to see how nucleosome positioning is affected upon Xist binding in mammals (Ramírez, 2015).

Although many factors, including the CCCTC-binding factor (CTCF) as well as tRNA and housekeeping genes, have been shown to be enriched at boundaries, by dissecting the targeting and spreading activity of the MSL complex for the X chromosome, this study offers a plausible explanation behind the advantages of HAS localization. HAS are enriched at the X chromosomal boundaries and not at autosomal boundaries, where all other boundary factors will bind indiscriminately. Furthermore, it was found that the few HAS that are not near a boundary also occupy locations of an elevated number of long-range contacts, indicating that HAS form interaction hubs for the spreading of the MSL complex (Ramírez, 2015).

Hi-C as well as in vivo immunofluorescence show that active roX genes have more contacts and are closer to each other than inactive regions. These observations are in line with previous reports showing that active chromatin compartments interact more often with each other and that active chromatin localizes to the interface of the chromosomal territory. The results imply that different transcriptional programs in each cell line or tissue are likely to be associated with a particular arrangement of long-range contacts, suggesting that the dosage compensation must be flexible to act over such diverse conformations without disturbing them. This idea is consistent with the observation that the chromosome conformation remains unchanged after knockdown of the MSL complex, and stays in contrast to mammalian X inactivation, which involves chromatin condensation, gene inactivation, and alterations in chromosome conformation (Ramírez, 2015).

Dosage compensation mechanisms in flies and mammals lead to opposite outcomes; namely, gene activation versus gene repression. However, both systems use lncRNAs transcribed from the dosage-compensated X chromosome. roX1 and roX2 RNA are expressed from the male hyperactivated X chromosome in D. melanogaster, whereas Xist is expressed from the inactivated X chromosome in mammalian females. Recent work has shown that Xist spreads to distal sites on the X chromosome. Interestingly, this spreading is dependent on the spatial proximity of sites distal to the Xist gene. This is further exemplified by ectopic expression of Xist from chromosome 21, where Xist spread only in cis on this chromosome. In this study, ectopic insertion of roX transgenes on autosomes demonstrated that the roX/MSL complex can reach the X chromosome and rescue male lethality. Therefore, acting in trans is a special feature of roX RNAs (in conjunction with the MSL complex) not observed for Xist, indicating that the two systems utilize the respective lncRNAs differently. In both systems, however, the lncRNAs need to be functional because the stem loop structures of the roX RNAs are required for dosage compensation in D. melanogaster, whereas Xist needs the "A repeat domain" to induce mammalian X chromosome inactivation. The distinct mechanisms utilized by the Xist and roX RNAs exemplify the great versatility by which lncRNAs can be involved in the global regulation of single chromosomes and might reflect important differences between the two systems. In mammals, only one of the two X chromosomes needs to be inactivated. Therefore, a trans action of Xist RNA on the sister X chromosome would be detrimental to the organism. In contrast, the dosage-compensated X chromosome is present singularly in males in Drosophila. However, because the roX RNAs can act in trans, it may be disadvantageous to target the activating MSL complex to active genes on autosomes, hence the need for specific target regions (the HAS) unique to the X chromosome (Ramírez, 2015).

To fully understand the occurrence of HAS at sites with extensive long-range interactions on the X chromosomes, it could be helpful to consider evolutionary models proposing that X chromosomes tend to evolve faster than autosomes (faster X effect). Under the faster X effect, traits only beneficial for males can introduce significant changes specific to the X chromosome on a short evolutionary timescale. Based on these and other observations suggesting that the X chromosome in flies is different from autosomes, it is assumed that selective pressures on males favored the occurrence of HAS at regions of increased interactions, like TAD boundaries. Future analyses of different Drosophila species will open exciting opportunities to study the evolutionary changes of HAS in the context of X chromosomal architecture. Moreover, conformation-based affinity could be a generic mechanism for other regulatory elements to exert their functions. It remains to be seen in which contexts the in cis versus in trans action of different lncRNAs is essential for their function and how chromosome conformation, long-range contacts, HAS, and regulation of transcription have co-evolved for dosage compensation (Ramírez, 2015).

Modulation of heterochromatin by male specific lethal proteins and roX RNA in Drosophila melanogaster males

The ribonucleoprotein Male Specific Lethal (MSL) complex is required for X chromosome dosage compensation in Drosophila males. Beginning at 3 h of development the MSL complex binds transcribed X-linked genes and modifies chromatin. A subset of MSL complex proteins, including MSL1 and MSL3, is also necessary for full expression of autosomal heterochromatic genes in males, but not females. Loss of the non-coding roX RNAs, essential components of the MSL complex, lowers the expression of heterochromatic genes and suppresses position effect variegation (PEV) only in males, revealing a sex-limited disruption of heterochromatin. MLE, but not Jil-1 kinase, was found to contribute to heterochromatic gene expression. To determine if identical regions of roX RNA are required for dosage compensation and heterochromatic silencing, a panel of roX1 transgenes and deletions was tested; the X chromosome and heterochromatin functions were found to be separable by some mutations. Widespread autosomal binding of MSL3 occurs before and after localization of the MSL complex to the X chromosome at 3 h AEL. Autosomal MSL3 binding was dependent on MSL1, supporting the idea that a subset of MSL proteins associates with chromatin throughout the genome during early development. It is postulated that this binding may contribute to the sex-specific differences in heterochromatin that have been noted (Koya, 2015).

A central question raised by this study is how factors known for their role in X chromosome dosage compensation also modulate autosomal heterochromatin. Although the MSL proteins were first identified by their role in X chromosome compensation, homologues of these proteins participate in chromatin organization, DNA repair, gene expression, cell metabolism and neural function throughout the eukaryotes. Furthermore, flies contain a distinct complex, the Non-Sex specific Lethal (NSL) complex, containing MOF and the MSL orthologs NSL1, NSL2 and NSL3. The essential NSL complex is broadly associated with promoters throughout the fly genome, where it acetylates multiple H4 residues. In light of the discovery that the MSL proteins represent an ancient lineage of chromatin regulators, it is unsurprising that members of this complex fulfill additional functions (Koya, 2015).

An alternative hypothesis for the dosage compensation of male X-linked genes proposes that the MSL proteins are general transcription regulators, and recruitment of these factors to the male X chromosome reduces autosomal gene expression, thus equalizing the X:A expression ratio. Arguing against this idea are ChIP studies finding that the MSL complex, and engaged RNA polymerase II, are increased within the bodies of compensated X-linked genes. In agreement with this, a study that normalized expression to genomic DNA concluded that compensation increases the expression of male X-linked genes. The current study now reveals that autosomal heterochromatic genes are indeed dependent on a subset of MSL proteins for full expression. However, native heterochromatic genes make up only 4% of autosomal genes, and their misregulation is not expected to compromise genome-wide expression studies normalized to autosomal expression (Koya, 2015).

Expression of heterochromatic genes is thought to involve mechanisms to overcome the repressive chromatin environment. It is possible that a complex composed of roX RNA and a subset of MSL proteins participates in this process. This would explain why heterochromatic genes are particularly sensitive to the loss of these factors. Alternatively, it is possible that roX and MSL proteins participate in heterochromatin assembly. This would explain the simultaneous disruption of heterochromatic gene expression and suppression of PEV at transgene insertions (Koya, 2015).

Heterochromatin assembly is first detected at 3-4 h AEL, a time when MSL3 is bound throughout the genome. Intriguingly, studies from yeast identify a role for H3K4 and H4K16 acetylation in formation of heterochromatin. Active deacetylation of H4K16ac is necessary for spreading of chromatin-based silencing in yeast, demonstrating the need for a sequential and ordered series of histone modifications (Koya, 2015).

As MOF is responsible for the majority of H4K16ac in the fly, a MOF-containing complex could fulfill a similar role during heterochromatin formation. While this study found a significant effect of MOF in expression only on the X and 4th chromosomes, it is possible that examination of a larger number of genes would reveal a more widespread autosomal effect (Koya, 2015).

In roX1 roX2 males the 4th chromosome displays stronger suppression of PEV and more profound gene misregulation than do other heterochromatic regions. This is consistent with the observation that heterochromatin on the 4th chromosome is genetically and biochemically different from that on other chromosomes. Loss of roX RNA leads to misregulation of genes in distinct genomic regions, the dosage compensated X chromosome and autosomal heterochromatin. This study found that the regulation of these two groups is, to some extent, genetically separable. MSL2, which binds roX1 RNA and is an essential member of the dosage compensation complex, is not required for full expression of heterochromatic genes in males. Ectopic expression of MSL2 in females induces formation of MSL complexes that localize to both X chromosomes, inducing inappropriate dosage compensation. As would be expected from the lack of a role for MSL2 in autosomal heterochromatin in males, ectopic expression of this protein in females has no effect on PEV (Koya, 2015).

Elegant, high-resolution studies reveal that MLE and MSL2 bind essentially indistinguishable regions of roX1. Three prominent regions of MLE/MSL2 binding have been identified, one overlapping the 3' stem loop. This stem loop incorporates a short 'roX box' consensus sequence that is present in D. melanogaster roX1 and roX2, and conserved in roX RNAs in related species (Koya, 2015).

An experimentally supported explanation for the concurrence of MLE and MSL2 binding at the 3' stem loop is that MLE, an ATP-dependent RNA/DNA helicase, remodels this structure to permit MSL2 binding. The finding that disruption of this stem blocks dosage compensation but does not influence heterochromatic integrity is consistent with participation of roX1 in two processes that differ in MSL2 involvement. However, a region surrounding the stem loop is required for the heterochromatic function of roX1, as roX1Δ10, removing the stem loop and upstream regions, is deficient in both dosage compensation and heterochromatic silencing. Further differentiating these processes is the finding that low levels of roX RNA from a repressed transgene fully rescue heterochromatic silencing, but not dosage compensation. An intriguing question raised by this study is why the sexes display differences in autosomal heterochromatin (Koya, 2015).

The chromatin content of males and females are substantially different as XY males have a single X and a large, heterochromatic Y chromosome. It is speculated that this has driven changes in how heterochromatin is established or maintained in one sex. A search for the genetic regulators of the sex difference in autosomal heterochromatin eliminated the Y chromosome and the conventional sex determination pathway, suggesting that the number of X chromosomes determines the sensitivity of autosomal heterochromatin to loss of roX activity. Interestingly, the amount of pericentromeric X heterochromatin, rather than the euchromatic 'numerator' elements, appears to be the critical factor. The recognition that heterochromatin displays differences in the sexes, and that a specific set of proteins are required for normal function of autosomal heterochromatin in males suggests a useful paradigm for the evolution of chromatin in response to genomic content (Koya, 2015).

Protein Interactions

A cDNA fragment containing the putative Males absent on the first (Mof) catalytic domain (aa 518 to 827) was expressed and it was determined that the recombinant peptide can acetylate Drosophila histones with a preference for histone H4. This pattern is similar to that for a related yeast protein, Esa1p. Active full-length Mof could not be expressed, Mof was isolated as a component of a partially purified MSL complex. Tissue culture cells were used for the initial characterization of the MSL complex. S2 cells are male, based on the following criteria: they do not express the Sxl (Sex-lethal) gene product, which is necessary for female differentiation, and they express Msl2, a limiting component of the dosage compensation machinery whose synthesis is prevented by Sxl. S2 cells can be stably transfected, allowing the use of commercially available antibodies recognizing epitope tags. Transient transfection of S2 cells with Msl2 tagged at its carboxy terminus with the HA epitope reveals that the localization of the HA epitope is coincident with the location of endogenous Mof. After selection with hygromycin, most cells exhibit HA staining on the male X chromosome, the location of which is revealed by antibodies to H4Ac16 (Smith, 2000).

Immunoprecipitation of nuclear extracts from Msl2-HA cells with the 12CA5 (anti-HA) antiserum results in the same proteins as those obtained from S2 cells with an Msl1 antiserum. In salivary gland nuclei, Mle is released from the male X chromosome with RNase treatment. Furthermore, the roX1 and roX2 RNAs are found along the X chromosome with a distribution that mimics that of the MSL complex. Therefore, attempts were made to obtain a partially purified complex containing Mle and a roX RNA and to see whether the presence of either of these components depended on the other. 'RNA-friendly' conditions were developed to increase the chances of purifying Mle and roX RNA-containing complex. The method involved a cell line expressing Flag-tagged MSL3 and sonication under low-salt conditions, immunoprecipitation with Flag antibodies followed by peptide elution, and a second immunoprecipitation with either an MSL antibody or with the corresponding preimmune serum. By using this two-step procedure, a faint band was detected by silver staining that corresponds to Mle protein. Clear enrichment of Mle was seen in the Msl1 immunoprecipitate relative to the preimmune serum. However, following a brief treatment with 0.4 M NaCl, the Mle levels were significantly reduced (Smith, 2000).

To determine if roX RNAs are expressed in S2 cells, Northern blot analysis was performed and it was observed that roX2, but not roX1, is expressed in these cells, consistent with the observation that roX1 is dispensable in flies. The size of the major roX2 transcript observed by Northern analysis was ~ 600 nucleotides. To test if roX2 RNA is present in the Mle-containing immunoprecipitates, RNA was extracted from the immunoprecipitation pellets and a RT-PCR was performed with roX2-specific primers in the linear range. The results show a clear enrichment of roX2 RNA in the immune over the preimmune serum precipitates (Smith, 2000).

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

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).

Targeting the chromatin-remodeling MSL complex of Drosophila to its sites of action on the X chromosome requires both acetyl transferase and ATPase activities

Dosage compensation in Drosophila is mediated by a multiprotein, RNA-containing complex that associates with the X chromosome at multiple sites. Investigated were the roles that the enzymatic activities of two complex components, the histone acetyltransferase activity of Mof and the ATPase activity of Mle, may have in the targeting and association of the complex with the X chromosome. Mle and Mof activities are necessary for complexes to access the various X chromosome sites. The role that histone H4 acetylation plays in this process is supported by the observations that Mof overexpression leads to the ectopic association of the complex with autosomal sites (Gu, 2000).

The normal association of the MSL complex at hundreds of sites along the X chromosome appears to be a process with at least three major steps. The first is the formation of functional complexes at the two entry sites where the roX RNAs are transcribed. It should be noted that although the Msl1 and Msl2 proteins are able to access the X chromosome at the entry sites and to recruit Mle, further complex assembly can only occur in the presence of the roX RNAs. This contention is supported by the observation that, in the absence of the two roX genes, no complex is seen to form in embryonic stages where it is normally evident. A caveat is that removal of roX2 was accomplished by using a deletion of such size that other roX-like genes or other unidentified components of the complex or genes whose product is required for complex stability, closely linked to roX2, may have been deleted as well. In any event, since the roX RNAs are unstable unless they are associated with the complex, the process of assembly can proceed only at the entry sites containing the roX genes. Once complexes are formed, they access the X chromosome through all of the entry sites, presumably via the affinity of their Msl1/Msl2 components for these sites. Finally, the complexes spread from the entry sites to the many other sites along the X chromosome where they are normally found. This last step requires the histone acetyltransferase activity of Mof. It is suggested that the spreading process involves the acetylation of neighboring nucleosomes, thereby altering the conformation of adjacent chromatin and rendering it more accessible to the entry of additional MSL complexes. The latter may require the presence of acetylated histone H4 tails in order to stabilize their chromatin association. This conclusion is consistent with the observation that, in S2 cells overexpressing Mof, the resulting abnormal ectopic acetylation of histone H4 at Lys16 leads to the association of the MSL complex along autosomal chromatin. This may mirror the normal situation in vivo where complexes, initially attracted to the entry sites, acetylate histone H4 at Lys16 and thereby make adjacent chromatin regions accessible to more complexes. The affinity of the MSL complex for histone H4 tails implied in this model is reminiscent of a similar role played by histone tails in the spreading of complexes containing SIR2, 3 and 4 during the silencing of mating type loci and telomeric heterochromatin formation in yeast. Although critical to the spreading process, the role played by the ATPase function of Mle, either directly or in conjunction with roX RNA, is not sufficiently understood to be incorporated in the model (Gu, 2000).

It is thought that the process just described can provide the following explanations for the gaps in Msl binding that occur along the X chromosome, or at ectopic autosomal sites where the complex has been caused to form at the site of a roX transgene. It is possible that the spread of H4 acetylation and complex association may be stopped by some insulator or some as yet uncharacterized boundary elements. This would not necessarily require that the entry sites be entirely responsible for the pattern seen along the X chromosome. The interphase chromosome is believed to consist of a series of rosettes formed by loops of the chromatid fiber anchored to a central core by dispersed regions that have affinity for one another. In such an arrangement, a cluster of complexes that have been stopped by some boundary element could acetylate the nucleosomes on a neighboring loop, initiating a spreading process on the other side of a gap (Gu, 2000).

The above considerations raise a number of questions that remain to be resolved. Is the pattern of complex association on the X chromosome tissue specific? Is it dependent on a tissue-specific distribution of the entry sites (other than those containing the roX loci, which must remain invariant in all tissues)? Is the tissue-specific distribution established when the complex first forms in early embryogenesis and is the pattern perpetuated through the mitotic divisions that give rise to a particular tissue? To answer these questions will require a thorough melding of cytological and biochemical approaches (Gu, 2000).

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

In Drosophila, dosage compensation is controlled by the male-specific lethal (MSL) complex consisting of MSL proteins and roX RNAs. The MSL complex is specifically localized on the male X chromosome 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. The roX1c3.4 transgene recruits the MSL complex in the absence of functional mof as well as in cells lacking msl3, as expected for an authentic chromatin entry site. However, in mle mutant animals, surprisingly, the transgene does not bind detectable partial complexes. By careful re-examination, it was found that the endogenous roX1 locus at 3F shows two strong MSL binding sites as a tight doublet in the absence of msl3, but shows a faint, single MSL signal in mle mutants. This suggests that there are two distinct MSL binding sites located at 3F, one site within the roX1 gene that requires MLE, and an additional site, similar to the majority of entry sites, that can attract partial MSL complexes in the absence of MLE. It should be noted that even at entry sites that attract partial complexes independent of MLE, the strength or stability of binding to those sites is weak. Since the requirement for MLE is also a characteristic of the endogenous roX2 locus (at 10C), it is possible that this is a common feature of entry sites that encode roX RNAs (Kageymama, 2001).

Functional msl3 and mof are not required for MSL binding to chromatin entry sites including roX1, but in their absence the complex fails to associate with the additional hundreds of sites seen in its wild-type pattern on the X chromosome. This has been interpreted as an inability of the complex to spread between chromatin entry sites in the absence of either MSL3 or MOF. To test this hypothesis at the level of an individual entry site, a strain was examined in which MSL1 showed frequent spreading from a roX1 transgene (roX1c3.4-51A). It was found that lack of functional msl3 or mof prevents spreading, as it does on the endogenous X chromosome. Taken together, the MLE helicase protein is essential for the MSL complex to bind to the roX1 transgene, while the MSL3 chromodomain protein and MOF histone acetyltransferase are required only for spreading (Kageymama, 2001).

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

MSL complexes bind the single male X chromosome in Drosophila to increase transcription approximately 2-fold. Complexes contain at least five proteins and two noncoding RNAs, roX1 and roX2. The mechanism of X chromosome binding is not known. A 110 bp sequence in roX2 has been identified, characterized by high-affinity MSL binding, male-specific DNase I hypersensitivity, a shared consensus with the otherwise dissimilar roX1 gene, and conservation across species. Mutagenesis of evolutionarily conserved sequences diminish MSL binding in vivo. MSL binding to these sites is roX RNA dependent, suggesting that complexes become competent for binding only after incorporation of roX RNAs. However, the roX RNA segments homologous to the DNA binding sites are not required, ruling out simple RNA-DNA complementarity as the primary targeting mechanism. These results are consistent with a model in which nascent roX RNA assembly with MSL proteins is an early step in the initiation of dosage compensation (Park, 2003).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The MRG domain mediates the functional integration of MSL3 into the dosage compensation complex: Interaction of MSL3 with RNA

The male-specific-lethal (MSL) proteins in Drosophila melanogaster serve to adjust gene expression levels in male flies containing a single X chromosome to equal those in females with a double dose of X-linked genes. Together with noncoding roX RNA, MSL proteins form the 'dosage compensation complex' (DCC), which interacts selectively with the X chromosome to restrict the transcription-activating histone H4 acetyltransferase MOF (Males-absent-on-the-first) to that chromosome. MSL3 is essential for the activation of MOF's nucleosomal histone acetyltransferase activity within an MSL1-MOF complex. By characterizing the MSL3 domain structure and its associated functions, it has been found that the nucleic acid binding determinants reside in the N terminus of MSL3, well separable from the C-terminal MRG signatures that form an integrated domain required for MSL1 interaction. Interaction with MSL1 mediates the activation of MOF in vitro and the targeting of MSL3 to the X-chromosomal territory in vivo. An N-terminal truncation that lacks the chromo-related domain and all nucleic acid binding activity is able to trigger de novo assembly of the DCC and establish an acetylated X-chromosome territory (Morales, 2005).

The MSL1 interaction surface maps to the C-terminal half of MSL3. This part of MSL3 is characterized by similarities to the MRG domain that subsumes MRG15, MSL3, and related proteins in multiple species into the so-called MRG family. The msl3 gene is related to the Drosophila mrg15 gene, suggesting an early gene duplication event. Accordingly, MRG sequences in MSL3 are highly conserved between D. melanogaster and Drosophila virilis. The MRG domain consists of three blocks of strong sequence similarity separated by short amino acid stretches of lesser conservation. Interestingly, these 'linker' regions harbor rather long insertions in MSL3 of flies and humans. The C terminus of MSL3 may thus be organized by folding of MRG signature sequences, which are disconnected in the primary sequence, into a compact unit from which the MSL3-specific structures 'loop out.' Consistent with this idea, it was found that every deletion in the C terminus of MSL3 compromises interaction with MSL1. Most of these deletions affect at least one of the blocks of MRG sequence similarity, most likely leading to global misfolding. However, one deletion that abolishes MSL1 binding (Delta328-433) selectively removed MSL3-specific sequences between two MRG blocks. There is considerable conservation of these sequences in the Drosophila species for which sequence information has recently become available, suggesting a conserved function, but whether this sequence contains a dedicated MSL1 interface remains to be explored. In any case, this analysis suggests that the MRG sequence similarity reflects a functional domain. The MRG-MSL1 contact is essential for targeting MSL3 to the X-chromosomal territory, confirming the functional importance of the interactions defined in vitro. It is suggested that MRG modules in other MRG family members may also constitute protein-protein interaction units (Morales, 2005).

In vitro analysis showed that MSL3 interacts better with single-stranded nucleic acids than with dsDNA. The significance of ssDNA interaction, if any, is unclear at the moment. In contrast, there is evidence that MSL3 interacts with roX RNA in vivo and in vitro, but the domain involved in RNA binding had not been defined. Biochemical analysis demonstrates that the nucleic acid binding structures reside in the N-terminal half of MSL3, which also contains the CRD. Previously, it has been suggested that RNA interaction of MSL3 is affected by its acetylation at lysine 116, close to the CRD. In the current studies, a fragment comprising the first 140 amino acids (and hence the CRD as well as K116) was not sufficient for nucleic acid binding, but sequences up to amino acid 259 contributed significantly. To what extent the CRD of MSL3 contributes to RNA binding needs to be established. The CRDs of MSL3 and MOF appear more related to each other than to canonical chromodomains. They lack the alpha-helix supporting the ß-sheet bundle and aromatic residues that may be involved in recognition of methylated histone N termini. The CRD of MOF also appears not to be sufficient for RNA binding. A further interesting similarity between MOF and MSL3 is that nucleic acid interactions are not the primary targeting determinant for either MOF (Morales, 2004) or MSL3. Although impairment of the CRDs leads to somewhat increased binding of the corresponding GFP fusion protein to autosomes, their concentration on the X-chromosomal territory is still obvious. However, the CRDs and noncoding RNA may have functions that are not assayed for in simple recruitment experiments. It is also possible that the CRDs of MOF and MSL3 provide partially redundant functions for DCC assembly. In contrast, mutations in MOF or MSL3 that abrogate their interaction with the C terminus of MSL1 prevent faithful recruitment to the X chromosome. Obviously, the recruitment assay employed may just reveal the strongest binary interaction that MSL3 or MOF are involved in. However, the fact that overexpression of an MSL3 lacking all nucleic acid binding capacity was able to complement an MSL3 deficiency and to trigger the accumulation of MOF and H4K16 acetylation on the X-chromosomal territory emphasizes the importance of the MSL protein interactions for the assembly of a functional DCC (Morales, 2005).

MSL complexes can be formed in vitro in the absence of RNA. A deficiency of roX RNA in vivo can be partially overcome by overexpression of the 'platform' proteins MSL1 and MSL2. It is possible that transient overexpression of MSL3 overcomes the RNA requirement and that under normal conditions of limiting MSL protein concentrations RNA is required for faithful DCC assembly (Morales, 2005).

The remarkable stimulation of MOF's HAT activity upon association of MSL3 with an MSL1-MOF complex was not due to enhanced binding of MSL3 to nucleic acids but rather required contact of MSL3 with the MSL1 scaffold. MOF and MSL3 are brought into proximity by interaction with adjacent structures in the C terminus of MSL1 (Morales, 2004). It is possible that the MSL1 scaffold stabilizes an otherwise transient and therefore nonproductive direct contact between MSL3 and MOF (Morales, 2004). The existence of such a contact has been inferred from the fact that MSL3 can be acetylated by MOF. However, when it comes to acetylation, MSL1 is a much better substrate for MOF than MSL3 (Morales, 2004). The new data reinforce a previous model of an acetylation 'checkpoint' built into DCC assembly. Accordingly, the regulatory potential of H4K16 acetylation would only be fully realized upon binding of MOF with MSL1 and the completion of the complex by association of MSL3 (Morales, 2004). Such a checkpoint would render full activation of MOF dependent on proper DCC assembly and hence 'maleness' and serve to restrict the critical epigenetic mark to the X chromosome (Morales, 2005).

Targeting determinants of dosage compensation in Drosophila

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

MSL complex is attracted to genes marked by H3K36 trimethylation using a sequence-independent mechanism

In Drosophila, X chromosome dosage compensation requires the male-specific lethal (MSL) complex, which associates with actively transcribed genes on the single male X chromosome to upregulate transcription 2-fold. On the male X chromosome, or when MSL complex is ectopically localized to an autosome, histone H3K36 trimethylation (H3K36me3) is a strong predictor of MSL binding. Mutants lacking Set2, the H3K36me3 methyltransferase, were isolated, and it was found that Set2 is an essential gene in both sexes of Drosophila. In set2 mutant males, MSL complex maintains X specificity but exhibits reduced binding to target genes. Furthermore, recombinant MSL3 protein preferentially binds nucleosomes marked by H3K36me3 in vitro. These results support a model in which MSL complex uses high-affinity sites to initially recognize the X chromosome and then associates with many of its targets through sequence-independent features of transcribed genes (Larschan, 2007).

MSL complex colocalizes with H3K36 trimethylation on X-linked genes: To investigate the relationship between MSL complex recruitment and histone methylation, ChIP-on-chip analysis of SL2 cells was performed with antibodies that recognize H3 trimethylated at K36 (H3K36me3) or dimethylated at K4 (H3K4me2). The SL2 cell line exhibits a male phenotype with respect to dosage compensation. NimbleGen tiling arrays were used; these contain the entire X chromosome and left arm of chromosome 2, tiled at 100 bp resolution. A general histone H3 antibody was used as a control for histone occupancy, and three biological replicates for tiling arrays indicated a high degree of reproducibility. As expected, the H3K36me3 and H3K4me2 modifications were associated with the 3' and 5' ends of transcribed genes, respectively, as previously reported for S. cerevisiae, mammals, and chicken. Close to 100% of transcribed genes on the X and 2L chromosomes were methylated at H3K36 and H3K4, largely independent of transcript level as previously reported for other organisms. Similar results were observed for MSL3-TAP, specifically on the X chromosome, but a lower fraction of transcribed genes on the X was bound (approximately 80%). With improved computational analysis, 1014 genes on the X chromosome scored positive for MSL binding in SL2 cells (up from previous estimate of 675 genes). 67% of the newly scored MSL-bound genes in SL2 cells were identified previouslyw was clearly bound in at least one cell type (Larschan, 2007).

To determine whether MSL binding colocalizes with H3K36me3 or H3K4me2, the correlation was examined between the data sets at the gene level. Of the 1014 MSL-bound genes in SL2 cells, 93% were positive for H3K36me3, and 83% were positive for H3K4me2. Interestingly, it was previously reported that a small percentage of untranscribed genes were bound by MSL3-TAP (7%), and the current study found that these genes also carried the H3K36me3 histone modification. In addition, untranscribed genes bound by MSL have significantly higher levels of H3K36me3 than untranscribed genes that are unbound by MSL complex. A likely explanation is that some nontranscribed genes are located near transcribed genes with very extensive H3K36me3 and MSL signals or within domains that have continuous strong signal over many kilobases. Specifically, 82% of MSL3-TAP-bound genes are transcribed, while 93% percent of MSL3-TAP-bound genes carry the H3K36me3 modification. Therefore, H3K36me3 is an even better predictor of MSL binding on the X than transcription state as defined by Affymetrix expression arrays. Similar results were observed for clone 8 cells, a Drosophila cell line derived from the wing disc (Larschan, 2007).

Colocalization in terms of whole genes could occur without coincident binding along the gene. It was previously reported that MSL3-TAP binds over the body of transcribed genes specifically on the X chromosome with a bias toward the 3' end. To determine whether H3K36me3 on the X chromosome and MSL complex colocalize spatially within transcription units, average gene profiles were compared for H3 methylation modifications and MSL3-TAP. It was found that H3K36me3 and MSL3-TAP exhibit a similar 3' biased profile, whereas H3 lysine 4 dimethylation is associated with the 5' end of transcription units, as reported in other organisms. Furthermore, at the probe level, a strong positive correlation is observed between MSL binding and H3K36me3 association. In contrast, a weaker correlation is observed with H3K4me2 that associates with the 5' ends of genes. These results demonstrate that H3K36 trimethylation is a 3' biased mark associated generally with active transcription units and that it is a very strong predictor of MSL binding on the X chromosome (Larschan, 2007).

MSL complex attracted to chromosome 2L by a roX2 transgene binds neighboring 2L genes marked by transcription and H3K36me3: When either a roX1 or a roX2 genomic transgene is inserted on an autosome, it attracts MSL complex to its site of insertion, with occasional signs of additional binding to neighboring regions along the autosome. Ectopic binding along the autosome is greatly increased when the X chromosome in the same nucleus is deleted for both roX1 and roX2. Such binding generally extends >1 Mb bidirectionally from the site of the roX transgene insertion, as measured by immunofluorescence for the MSL proteins. One interpretation is that nascent roX RNAs compete for attraction of the MSL proteins for assembly at their site of synthesis and that, after local assembly, MSL complex becomes competent to search for targets in its new chromosome environment. To determine whether ectopic binding on a normally untargeted chromosome would provide clues to the specificity of MSL binding, ChIP-on-chip analysis was performed on MSL3-TAP male larvae mutant for both roX1 and roX2 on the X chromosome and containing a roX2 transgene inserted at position 26D8-9 (near the CG9537 gene) on chromosome 2L. When assayed by immunostaining of polytene chromosomes, such males consistently show MSL binding in interbands along chromosome 2L, surrounding the site of the transgene insertion. At the level of genomic tiling arrays, ChIP results map this binding at high resolution. As a control, an additional array was used that contains the 3R chromosome and the entire X. It was found that the domain of MSL binding extends greater than 2 Mb in each direction from the insertion site on 2L, while binding to 3R was undetected. Importantly, the targets of binding are transcribed 2L genes, with the averaged binding profile showing enrichment over the bodies of genes, with a bias toward 3'ends. Each of these characteristics is typical of target genes on the X chromosome in wild-type larvae, cells, and embryos. Furthermore, when the 2L pattern of ectopic MSL binding in larvae was compared to the wild-type distribution of H3K36 trimethylation in tissue culture cells, a strong correlation was found between MSL binding and K36me3 within 1 Mb of the site of the roX transgenic insertion. Interestingly, although MSL-bound genes are consistently marked with H3K36me3, at greater than 1 Mb distances from the transgene insertion site, MSL complex increasingly skips some H3K36me3-bound genes while binding others. Overall, it was found that MSL targets selected on 2L were transcribed genes enriched for H3K36 trimethylation and that MSL binding showed a 3′ bias analogous to that normally found on X chromosome targets. These results raise the strong possibility that, once targeted to a chromosomal domain by a high-affinity site, MSL complex recognizes general marks for transcription such as H3K36me3 or other 3′-associated features rather than an X-specific sequence element at each individual target (Larschan, 2007).

Set2 is required for H3K36 trimethylation and for viability in both males and females in Drosophila : To investigate whether H3K36me3 plays a functional role in MSL complex targeting, a genetic approach was taken to inactivate the methyltransferase responsible for H3K36me3 in Drosophila. In S. cerevisiae, the Set2 histone methyltransferase is responsible for di- and trimethylation of H3K36. The CG1716-encoded protein has been identified as the likely functional homolog of ySet2 in Drosophila based on the presence of SRI and SET domains. Two initial tests were pursued to examine CG1716 function, the first in yeast and the second in Drosophila tissue culture cells. To test the function of CG1716 in yeast, an inducible CG1716 expression vector was transformed into set2Δ mutant S. cerevisiae that lack detectable H3K36me3. When CG1716 was induced by growth in media containing galactose, H3K36me3 (and some H3K36me2) was restored, demonstrating that a CG1716 cDNA functionally complements the yeast set2Δ. Also, the CG1716-encoded protein can interact with the RNA Pol II CTD as observed for S. cerevisiae Set2, further confirming the identity of CG1716 as the functional homolog of the S. cerevisiae SET2 gene. To test the function of CG1716 in Drosophila tissue culture cells, RNAi was used to target CG1716. A strong reduction of CG1716 mRNA was found to correlate with a significant loss of H3K36me3 by Western blot, immunostaining, and ChIP analysis. H3K4me2, a distinct chromatin mark for transcribed genes, was largely unaffected. ChIP analysis allowed quantification of a 3- to 5-fold reduction in H3K36me3 and only very small changes in H3K4me2. Based on these results, a Drosophila mutant was isolated that disrupts the CG1716 gene, henceforth referred to as the Set2 gene (Larschan, 2007).

Imprecise excision of a P element upstream of the Set2 gene was induced to create a series of Set2 deletion strains, and Set21 was selected for further analysis. dSet21 eliminates most of the coding region including the catalytic SET domain without extending bidirectionally into the neighboring CG1998 gene. Since the Set2 gene is located on the X chromosome, hemizygous males were initially isolated, and they were found to die as late third-instar larvae. To demonstrate that this lethality was due to loss of Set2, and not to any additional defects that might have been induced during P element excision, a transgene was constructed encompassing only the genomic region of Set2; it was able to fully rescue the Set21 mutants. Using the rescued males as fathers, homozygous mutant females were subsequently examined, and the Set21 mutation was found to cause late larval lethality in both sexes. To further analyze the viability of Set2 mutants at the cellular level, homozygous mutant Set2 eyes were created in the context of heterozygous mutant adult females, using the GMR-hid system. set2 mutant eyes were diminished in size and rough compared to wild-type eyes, which is a qualitative assay suggesting that Set2 is important for normal cell proliferation (Larschan, 2007).

To determine whether or not H3K36me3 was affected in the set2 mutant, polytene chromosome squashes of mutant larvae were were immunostained. H3K36me3 was significantly depleted in the Set21 mutant when compared to wild-type. As a control for the specificity of this defect, the same nuclei were immunostained for the interband protein Z4, which showed similar staining in wild-type and mutant. Set21 mutant larvae were further analyzed by ChIP to quantify the H3K36me3 levels in wild-type and Set21 mutants. H3K36me3 in the Set21 mutant was found to be dramatically decreased at the transcribed genes tested, to levels comparable to an untranscribed gene (CG15570). Changes in H3K4me2 varied from slight to none. Thus, Set2 is required for viability and methylation of H3K36 in Drosophila (Larschan, 2007).

Set2 contributes to optimal MSL complex targeting at transcribed genes, but not at high-affinity sites: To examine whether MSL complex targeting requires H3K36me3, polytene chromosomes of Set21 mutant larvae were immunostained with antibodies directed against MSL complex, but no difference in MSL pattern or intensity was detected at this level of resolution. Upon initial consideration, this result would appear to rule out a requirement for H3K36me3 in MSL targeting. However, when attempts were made to validate this observation with ChIP assays conducted with two independent fly stocks and ChIP protocols (both anti-MSL2 and MSL3-TAP IPs), it was found that wild-type and Set21 mutant larvae showed significant differences at many specific gene targets. Nine genes with high, medium, or low levels of MSL complex binding were assayed for recruitment of MSL2 and MSL3-TAP in wild-type and Set21 mutant third-instar larvae by ChIP analysis. Highly reproducible 2- to 10-fold decreases were observed in MSL2 and MSL3-TAP association at all nine genes assayed. In contrast, MSL complex association with previously reported 'high-affinity sites', such as roX1, roX2, and 18D11, was largely unaffected in the Set21 mutant (Larschan, 2007).

Such a result might be attributed to indirect effects in Set21 mutant larvae as opposed to specific defects in MSL targeting. To address this, roX RNA and msl2 mRNA levels were measured, and it was found that they were not affected significantly in the Set21 mutant, suggesting that H3K36me3 does not affect MSL complex recruitment indirectly by affecting expression of MSL components. Western and polytene staining analysis of Msl1 and Msl2 also indicate that protein levels are largely unchanged. It was also found that ChIP for H3K4me2 and RNA polymerase II were not significantly affected in set2 mutants, further supporting a direct role for H3K36me3 in stabilization of MSL complex at target genes (Larschan, 2007).

To address the functional role of H3K36me3 in transcription of genes bound by MSL complex, the transcript levels of MSL complex target genes were compared in wild-type and Set21 mutant larvae. Transcription of MSL target genes is not strongly affected in Set21 mutant larvae, although genes that exhibit the strongest loss of MSL complex binding (CG13316, CG12690, CG32555, and CG32575) exhibit decreases in transcript level. Dosage compensation involves a 2-fold upregulation of transcription, limiting the expected transcriptional changes to a 50% decrease in transcript. Furthermore, when H4K16 acetylation at these genes was examined, significant residual levels were found (10-fold over autosomal controls or untranscribed genes), even when very small amounts of MSL complex remain. Thus, residual MSL complex function may be largely sufficient for transcriptional upregulation in the Set21 mutant, yet MSL complex targeting is significantly reduced (Larschan, 2007).

Together, these results suggest that a subset of MSL binding sites is particularly sensitive to H3K36me3 levels, while others, including three previously defined high-affinity sites are not. Since MSL binding is diminished significantly but not ablated in the Set21 mutant, these results support a model in which recognition of H3K36me3 is one contributing factor to MSL complex targeting that functions with additional features of transcribed genes (Larschan, 2007).

An important caveat to the conclusion that H3K36me3 functions together with other recognition features is that the heterozygous mothers of hemizygous Set21 mutants carry a functional Set2 gene and thus could provide a maternal supply of wild-type Set2 mRNA or protein to the mutant embryos. This maternal contribution of H3K36me3 could be sufficient to initially establish MSL binding, which might be maintained through development, independent of the initial recognition mark. Thus, if the maternal contribution of H3K36me3 could be eliminated, it was hypothesized that an even more significant defect would be observed in MSL complex recruitment. To address this possibility genetically, a stock designed to create homozygous set2 mutant germline clones was constructed using FLP-FRT-mediated recombination in an ovoD dominant female sterile mutant. After recombination, the set2 mutant germ cells would no longer carry ovoD and thus should produce oocytes that would lack any maternal Set2 mRNA or protein. Despite recombination to remove ovoD from germ cells, no functional oocytes were produced, demonstrating that Set2 is essential for oogenesis. Therefore, the maternal contribution of Set2 remains in these studies; its elimination might reveal an even more significant role or H3K36me3 in MSL recruitment than has been reported (Larschan, 2007).

Recombinant MSL3 binds preferentially to nucleosomes trimethylated at H3K36: Eaf3, the yeast member of the conserved MSL3/MRG family of proteins, has been implicated in a physical and functional interaction of Rpd3(S) complexes with H3K36me3, raising the attractive hypothesis that MSL3 plays an analogous function in MSL complex. Furthermore, the distinction between high-affinity MSL binding sites such as roX1, roX2, and 18D11 and the majority of MSL targets is that high-affinity sites are MSL3 independent. Therefore, sensitivity to loss of H3K36me3 might be a specific characteristic of MSL3-dependent targets. To test the idea that MSL3 contributes to specific recognition of H3K36me3-modified nucleosomes, gel shift analyses was performed with recombinant MSL3 protein produced in baculovirus using nucleosomes assembled in vitro. Using an EMSA assay system where specifically modified recombinant nucleosomes were assembled, it was found that purified MSL3 protein showed increased affinity to nucleosomes pretreated with active Set2, and thus marked with H3K36 methylation, as opposed to nucleosomes that were unmodified at H3K36. This preferential binding was only detected in nucleosomes bearing linker DNA, suggesting that affinity for free DNA may be contributing to the binding of MSL3 to the nucleosomes methylated at H3K36. Titrations were performed to measure the relative affinity of MSL3 association with methylated compared to unmethylated nucleosomes. The increased affinity of MSL3 for methylated nucleosomes is best observed at the 4.4 nM concentration. These results provide additional evidence supporting a model in which H3K36me3 is a 3' chromatin mark required for the robust, wild-type MSL binding pattern on the X chromosome (Larschan, 2007).

This study has found that ectopic spreading of MSL complex to the 3' ends of transcribed genes on autosomes indicates that a sequence-independent mechanism can define MSL complex target genes. Furthermore, trimethylation of H3K36 is required for optimal MSL complex targeting to transcribed genes on the male X chromosome subsequent to initial recognition of the X. In the absence of H3K36me3, MSL complex can associate with high-affinity sites on the X chromosome but exhibits reduced binding to target genes. Since MSL binding is reduced but is not eliminated, a model if favored in which association with H3K36me3 is a contributing factor that functions with recognition of one or more additional 3' features of transcribed genes such as nascent mRNAs or RNA Pol II CTD phosphorylation (Larschan, 2007).

In addition to a function for Set2 in MSL complex targeting, this study demonstrates that Set2 is essential for viability of both sexes in Drosophila. Conservation of the Set2 H3K36 methyltransferase function from S. cerevisiae to Drosophila was observed, as predicted by sequence conservation. A variety of roles have been reported for Set2 in several organisms. In Neurospora, S. pombe, and NIH 3T3 cells, Set2 is required for optimal growth rate. The S. cerevisiae set2Δ mutant suppresses the loss of positive elongation factors. In Drosophila, mutants lacking zygotic Set2 function fail to proceed through the developmental transitions from late larval to adult stages. The cause(s) of inviability in Drosophila set2 mutants remains to be determined, but eyes composed entirely of homozygous set2 mutant tissue were small and rough, indicating defects in cell proliferation (Larschan, 2007).

In vitro studies using recombinant MSL3 produced in baculovirus revealed preferential interaction with nucleosomes that were trimethylated at H3K36, suggesting that a direct interaction may occur between MSL complex and H3K36me3 chromatin on the X chromosome. In S. cerevisiae, an MSL3 homolog, Eaf3, mediates an interaction between the Rpd3(S) complex and H3K36me3 at active genes. If conserved, this function in Drosophila presumably would be played by another MSL3 family member, MRG15. In S. cerevisiae, Rpd3(S) is thought to deacetylate histones in the wake of RNA polymerase II to prevent uncontrolled activation and transcription initiation from cryptic start sites within genes. This raises the possibility that, on the X chromosome, MSL complex might compete for binding to H3K36me3 with the repressive deacetylation function of Rpd3(S). Alternatively, H3K36me3 may simply be a mark utilized by MSL complex to regulate target genes by a mechanism independent of Rpd3(S) (Larschan, 2007).

H3K36me3 marks transcribed genes independent of transcript level but is a weak modulator of endogenous transcript and RNA polymerase II levels. In S. cerevisiae, where its role is best understood, Set2 functions to suppress formation of aberrant internal transcripts by facilitating histone deacetylation yet has only small effects on endogenous transcript levels. In Drosophila, small but reproducible changes were detected in transcript levels at MSL complex target genes in set2 mutant larvae. Also, minimal changes were observed in RNA Pol II levels as previously reported for the set2Δ mutant in S. cerevisiae. Also, changes in transcription level due to loss of dosage compensation are small, with a maximal 50% decrease predicted. Thus, the combined loss of the Set2 protein and reduction in MSL complex recruitment did not cause dramatic changes in transcript level. Furthermore, levels of H4Ac16 were decreased but not eliminated at target genes, consistent with residual MSL function that can explain why more dramatic changes in transcription of MSL complex target genes were not observed (Larschan, 2007).

A defined mechanism for MSL complex targeting to hundreds of sites along the male X chromosome has remained elusive. Previous reports have posited two highly related models for MSL complex recruitment: a 'spreading' model and an 'affinities' model. Both models are based on the idea that specific MSL interaction occurs at high-affinity sites that mark the X chromosome. These sites have been mapped on polytene chromosomes, but most are not yet defined at the molecular level. roX genes and other high-affinity sites are thought to concentrate MSL complex within an X chromosome domain. In the spreading model, MSL complex creates the full MSL binding pattern by searching the X chromosome for general characteristics of active genes without necessarily requiring a specific DNA sequence at each gene. This could occur either by scanning along the chromosome in a linear manner or by releasing and rebinding chromosomal regions in close physical proximity. It has been demonstrated that roX RNAs can move in trans from one DNA molecule to another, so linear scanning is possible but not obligatory. The affinities model proposes that there is a continuum of affinity sites for MSL complex, ranging from high to low. Only when high-affinity sites are locally concentrated can low-affinity sites be recognized, similar to the spreading model. The major difference is that even low-affinity sites are predicted to contain sequence elements that direct MSL binding. It is thought that the results documenting the pattern of ectopic MSL binding on chromosome 2L surrounding a roX transgene make the existence of sequence elements at every MSL binding site on the X chromosome unlikely. That the 2L pattern was analogous to that normally found on the X chromosome, targeting transcribed genes marked by H3K36me3 and binding with a 3' bias, is strong evidence that MSL complex recognizes target genes marked by transcription. This does not exclude the possibility that transcribed genes carry common sequence elements but makes it unlikely that such sequence elements differ between autosomal genes and the majority of MSL target genes on the X chromosome (Larschan, 2007).

In summary, the data are consistent with a model in which MSL complex first recognizes nascent roX transcripts and a series of high-affinity sequences along the male X chromosome and then scans the X for target genes that exhibit H3K36 trimethylation and other marks of active transcription. Recognition may involve the MSL3 chromodomain and additional factors. Trimethylation of H3K36 marks the middle and 3' ends of transcription units, independent of absolute transcript levels in Drosophila, consistent with S. cerevisiae and mammalian systems. Thus, MSL complex recognition of H3K36me3 provides an important mechanism for identification of transcribed genes and avoidance of silenced regions (Larschan, 2007).

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

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

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

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

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

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

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

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

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

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

Functional domains of Drosophila UNR in translational control

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

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

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

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

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

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

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

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

The essential Drosophila CLAMP protein differentially regulates non-coding roX RNAs in male and females

Heterogametic species require chromosome-wide gene regulation to compensate for differences in sex chromosome gene dosage. In Drosophila melanogaster, transcriptional output from the single male X-chromosome is equalized to that of XX females by recruitment of the male-specific lethal (MSL) complex, which increases transcript levels of active genes 2-fold. The MSL complex contains several protein components and two non-coding RNA on the X (roX) RNAs that are transcriptionally activated by the MSL complex. Targeting of the MSL complex to the X-chromosome has been shown to be dependent on the chromatin-linked adapter for MSL proteins (CLAMP) zinc finger protein. To better understand CLAMP function, the CRISPR/Cas9 genome editing system was used to generate a frameshift mutation in the clamp gene that eliminates expression of the CLAMP protein. clamp null females were found to die at the third instar larval stage, while almost all clamp null males die at earlier developmental stages. Moreover, it was found that in clamp null females roX gene expression is activated, whereas in clamp null males roX gene expression is reduced. Therefore, CLAMP regulates roX abundance in a sex-specific manner. These results provide new insights into sex-specific gene regulation by an essential transcription factor (Urban, 2017).

Many species employ a sex determination system that generates an inherent imbalance in sex chromosome copy number, such as the XX/XY system in most mammals and some insects. In this system, one sex has twice the number of X-chromosome-encoded genes compared to the other. Therefore, a mechanism of dosage compensation is required to equalize levels of X-linked transcripts, both between the sexes and between the X-chromosome and autosomes. Dosage compensation is an essential mechanism that corrects for this imbalance by coordinately regulating the gene expression of most X-linked genes (Urban, 2017).

In Drosophila melanogaster, transcription from the single male X-chromosome is increased 2-fold by recruitment of the male-specific lethal (MSL) complex. The MSL complex is composed of two structural proteins, MSL1 and MSL2, three accessory proteins, MSL3, males absent on the first (MOF), and maleless (MLE), and two functionally redundant non-coding RNAs, RNA on the X (roX1) and roX2. Previous work has shown that recruitment of the MSL complex to the X-chromosome requires the zinc finger protein chromatin-linked adapter for MSL proteins (CLAMP) (Soruco, 2013; Urban, 2017 and references therein).

In addition to its role in male MSL complex recruitment, it was suggested that CLAMP has an additional non-sex-specific essential function because targeting of the clamp transcript by RNA interference results in a pupal lethal phenotype in both males and females (Soruco, 2013). Further understanding of CLAMP function in the context of the whole organism required a null mutant. However, due to the pericentric location of the clamp gene, no deficiencies or null mutations were available. Using the CRISPR/Cas9 system, a frameshift mutation was introduced in the clamp gene, leading to an early termination codon before the major zinc finger binding domain. This frameshift mutation generated the clamp2 allele, which eliminates detectable CLAMP protein production and is therefore a protein null allele. The majority of clamp2 mutant males die prior to the third instar stage. On the other hand, females die at the third instar stage, suggesting sex-specific functions for CLAMP. Furthermore, CLAMP regulates the roX genes in a sex-specific manner, activating their accumulation in males and repressing their accumulation in females. Overall, we present a new tool for studying dosage compensation and suggest that CLAMP functions to assure that roX RNA accumulation is sex specific (Urban, 2017).

Previous work demonstrated that CLAMP has an essential role in MSL complex recruitment to the male X-chromosome (Soruco, 2013). However, it was not possible to perform in vivo studies to further investigate CLAMP function because there was no available null mutant line. The current work present a CLAMP protein null mutant and determine that this protein is essential in both sexes. This allele will provide a key tool for future in vivo studies on the role of CLAMP in dosage compensation, as well as identification of the essential function of CLAMP in both sexes (Urban, 2017).

The initial characterization of the clamp2 protein null allele revealed sexually dimorphic roles for CLAMP in regulation of the roX genes. CLAMP was seen to promotes roX2 transcription in males but represses transcription of both roX genes in females. It is likely that recruitment of the MSL complex to the roX2 locus by CLAMP promotes roX2 expression in males. In females, where the MSL complex is not present, CLAMP may function to repress these loci as an additional mechanism to ensure that dosage compensation is male-specific. Additionally, it was determined that most clamp2 homozygous males die earlier in development than clamp2 homozygous females. Earlier lethality in males is likely due to a misregulation of the dosage compensation process as a result of the loss of CLAMP-mediated MSL complex recruitment. However, CLAMP is enriched at the 5' regulatory regions of thousands of genes across the genome. Therefore, it is likely that other non-sex-specific regulatory pathways are disrupted resulting in female lethality (Urban, 2017).

Furthermore, CLAMP is an essential protein because our CRISPR/Cas9-generated protein null clamp allele is homozygous lethal in both males and females. These results indicate that CLAMP has a previously unstudied non-sex-specific role that is essential to the viability of both males and females. An interesting observation that arose from this characterization is that polytene chromosome organization is disrupted in clamp2 mutant females, suggesting that CLAMP may play a role in regulation of genome-wide chromatin organization of interphase chromosomes. A function in regulating chromatin organization provides one possible explanation for how CLAMP performs sexually dimorphic functions. For example, CLAMP may repress roX expression in females by promoting the recruitment of a repressive chromatin-modifying factor in the absence of the MSL complex. In contrast, CLAMP may activate roX2 in males by creating a chromatin environment permissive for MSL complex recruitment in males. Although roX1 and roX2 are functionally redundant, the results suggest that CLAMP specifically activates roX2 but not roX1 in males. Interestingly, Villa (2016) recently reported that roX2, but not roX1, is likely to be an early site of MSL complex recruitment (Villa, 2016), suggesting that CLAMP may function early in the process of dosage compensation (Urban, 2017).

Overall, the newly generated clamp2 protein null allele provides an important tool to study how the essential CLAMP protein regulates its many target genes in vivo. The generation of the clamp2 allele will facilitate future studies that will reveal a mechanistic understanding of how a single transcription factor can promote different sex-specific functions within an organism (Urban, 2017).

RNA on the X-1: Biological Overview | Developmental Biology | Effects of Mutation | References

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