BIOLOGICAL OVERVIEW

The ratio of sex chromosomes to autosomes in females (1:1) is different from the ratio in males (0.5:1). This is because males have not two but only one X chromosome. This presents a dosage problem. The ratio of gene products coded for by the sex chromosome will be different in males and females, unless some compensatory action is taken. Most genes of the sex chromosome come along for the ride, that is they have nothing to do with sex determination. For these genes, the creation of a dosage imbalance spells catastrophe for development. What can be done to compensate for the dosage imbalance?

Two alternatives are possible, both resulting in dosage compensation. In females, one of the two X chromosomes could be inactivated. This is the solution humans and other higher vertebrates have employed. A second option would be to heighten the activity of the single X chromosome in males. This is the route flies have taken.

In Drosophila, dosage compensation is regulated by male specific lethals (MSLs): four proteins that bind specifically to the X chromosome in the male, but not in females. The term "male specific lethals" is derived from the fact that mutations of these genes are lethal to male mutants, but not females. Sex lethal, the splicing factor that directs all sex determination in the fly, regulates the function of MSLs by regulating the splicing of pre-messenger RNA of male specific lethals. The immediate target of SXL is a transcription factor, male specific lethal-2 (MSL-2). The msl2 produces two transcripts that differ by an intron of 133 nucleotides in the 5' untranslated region. Most female transcripts retain the intron, whereas most male transcripts remove it. Unlike the previously described cases of SXL reglation, this sex-specific intron does not affect the mRNA open reading frame. It is thought that association of SXL protein with multiple sites in the 5' and 3' untranslated regions of MSL2 transcript represses its translation in females (Kelley, 1997). The other three MSLs are not differentially spliced, although putative SXL-binding sites are also found in the 3' UTR of a subset of Msl-1 transcripts. MSL-2, spliced into a functional form in males, serves to heighten transcriptional activation of the solitary X chromosome. MSL-2 acts in concert with three partners in this task: Maleless, Male-specific lethal-1 and Male specific lethal-3. They bind to about one hundred sites on the male chromosome, modifying the chromatin structure to permit heightened gene activation (Kelly, 1995 and Zhou, 1995). The action of these male specific transcription factors is very similar to proteins of the trithorax complex.

How do MSLs function to heighten the level of transcriptional activation? Development of an antiserum able to distinguish acetylation of lysine residues on histone H4 has provided the best clues as to a mechanism. Examination of the giant polytene chromosomes of larval salivary glands reveals that H4 molecules, acetylated at lysines 5 and 8, are distributed in islands throughout the euchromatic chromosome arms. ß-Heterochromatin in the chromocenter is depleted in these isoforms, but relatively enriched in H4 acetylated at lysine 12. H4 acetylated at lysine 16 is found at numerous sites along the transcriptionally hyperactive X chromosome in male larvae, but not in male autosomes or any chomosomes in female cells. Therefore it seems likely that H4 molecules acetylated at particular sites mediate higher transcriptional activation of the male X chromosome (Turner, 1992).

Subsequently it was shown that MLE and MSL-1 bind to the X chromosome in an identical pattern, and that the pattern of acetylated H4 is largely coincident with that of MLE and MSL-1, and that no acetylated histone H4 was detected on the X chromosome in mutant msl males. In addition acetylated H4 was detected on X chromosomes in females mutant for Sex lethal, coincident with an inappropriate increase in X chromosome transcription (Bone, 1994).

The coincident distribution of MLE proteins and acetylated Histone H4 is not limited to salivary gland chromosomes. MSL proteins first associate with the male X chromosome as early as the blastoderm stage, slightly earlier than the histone H4 isoform acetylated at lysine 16 is deteted on the X chromosome. MSL binding to the male X chromosome is observed in all somatic tissues of embryos and larvae. In male pole cells (those cells that give rise to the germline), MSL proteins do not show any subnuclear localization of MSL proteins. This is consistent with observations that germline cells in MSL mutants produce functional sperm. Dosage compensation in the male germline takes place by a mechanism that is, at least in part, distinct from that which functions in somatic cells.

The most likely model for MSL action is that the presence of MSL on the active male X chromosome affects deposition of specifically modified histones; this results in an enhancement in the transcriptional activity of the male X chromosome. There is good evidence that newly synthesized histones H3 and H4 of Drosophila are acetylated in an evolutionarily conserved pattern, that is, on specific amino acid residues (Sobel, 1995). In yeast, it is clear that changes in H4 acetylation causes changes in silencing of the mating type genes (Braunstein, 1993). For more information about assembly of newly replicated chromatin and the relationship between initiation of DNA replication and gene silencing see ORC2 and NAP-1 sites, as well as the DNA replication site.

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).

Tandem stem-loops in roX RNAs act together to mediate X chromosome dosage compensation in Drosophila

Dosage compensation in Drosophila is an epigenetic phenomenon utilizing proteins and long noncoding RNAs (lncRNAs) for transcriptional upregulation of the male X chromosome. By using UV crosslinking followed by deep sequencing, this study shows that two enzymes in the Male-Specific Lethal complex, MLE RNA helicase and MSL2 ubiquitin ligase, bind evolutionarily conserved domains containing tandem stem-loops in roX1 and roX2 RNAs in vivo. These domains constitute the minimal RNA unit present in multiple copies in diverse arrangements for nucleation of the MSL complex. MLE binds to these domains with distinct ATP-independent and ATP-dependent behavior. Importantly, different roX RNA domains were shown to have overlapping function, since only combinatorial mutations in the tandem stem-loops result in severe loss of dosage compensation and consequently male-specific lethality. It is proposed that repetitive structural motifs in lncRNAs could provide plasticity during multiprotein complex assemblies to ensure efficient targeting in cis or in trans along chromosomes (Ilik, 2014).

Identification of functional domains in lncRNAs is an important step toward understanding how they work in vivo. This study characterizes roX1 and roX2 as the most significant RNAs associated with MLE and MSL2. roX1 and roX2 contain common, conserved, and distinct structural domains, which form the binding platform for these proteins. Interestingly, regions of lncRNAs that lie outside of these MLE/MSL2 interaction domains appear to be unstructured and not conserved. These data also provide evidence on how roX1 and roX2 RNAs can be functionally redundant by showing that MLE-MSL2-interacting regions are present in multiple copies in both RNAs (Ilik, 2014).

Earlier work on roX RNAs has identified short stretches of RNA that are shared between these lncRNAs that are conserved throughout evolution, called the roX boxes. This study shows that all three roX boxes at the 3' end of roX2 RNA and one of the three roX boxes in roX1 form stable helices in vitro. Moreover, these elements represent binding sites for MLE and MSL2 proteins in vivo. Detailed analysis of MLE individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) data revealed that there are other RNA elements in both roX1 and roX2 that resemble the roX box sequence, and also serve as binding sites for MLE. These elements were named roX box-like sequences. Together roX box and roX box-like sequences uncover a consensus binding site for MLE in roX RNAs. No other RNAs were identified in the Drosophila transcriptome that contained the roX box/roX box-like motif other than the roX RNAs, adding to the evidence that it is likely that there are no more roX-like RNAs that can function in Drosophila dosage compensation (Ilik, 2014).

The results expose the logic of a roX RNA: stem-loops containing RBL elements at the 5' end and roX boxe (RB) containing helical structures joined by a flexible, single-stranded spacer region. In roX2, these elements are repeated on a very small scale with perfect copies of RBL/RB elements. In contrast, in roX1, these sequence motifs deviate from the ideal consensus more than roX2, but roX1 probably compensates for this by containing multiple, autonomous interaction domains that form a much larger RNA, which is about six times as big as roX2 (Ilik, 2014).

Endogenously expressed MLE and affinity-tagged MLE proteins have the ability to differentiate between the two stem-loop clusters in the two halves of roX2 exon-3 (1-280 versus 281-504). Interestingly, addition of ATP led to the specific interaction of endogenously expressed MLE with the second stem-loop cluster of roX2 exon-3 that contains roX box-containing helical structures. Furthermore, it was observed that the N-terminal dsRBDs are not only required for the ATP-independent interactions with the 5' end of roX2 exon-3 but are also required for the ATP-dependent interactions of MLE with the 3' end of roX2 exon-3. A similar dichotomy was observed in High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) and iCLIP experiments. In HITS-CLIP, the reads accumulated around the first stem-loop cluster of roX2 exon-3, concentrating on R2H1, whereas in iCLIP the balance was shifted toward the second cluster (Ilik, 2014).

MLE is a member of the RHA/DEAH family of RNA helicases, which can remodel RNA and RNPs. Interestingly, biochemical work on the closely related DEAD box RNA helicases shows that these enzymes have varying degrees of affinity toward their RNA substrates during their ATPase cycles. Since the GRNA chromatography experiments reveal that the second half of roX2 gains MLE binding when supplemented with ATP in vitro, it is possible that the quaternary complex between MLE, roX2, and ADP-Pi is being scored for in these experiments, and enzyme is being caught on its way to eventually remodeling of this part of the RNA (Ilik, 2014).

The ATP-independent binding of MLE to the first half of roX2 RNA via its N-terminal dsRBDs could be an initial regulatory step as roX RNAs constitute important tethers for MLE within the MSL complex. However, the in vivo analysis revealed that at high expression levels this cluster 'A' is dispensable for dosage compensation. The second hairpin cluster appears to recruit MLE through the helicase domain in a way that still requires N-terminal dsRBDs, thus providing a dynamic platform for rerecruitment and spreading of MLE along the roX RNA. The function of this cluster 'B' can only be partially compensated by higher expression, suggesting that it is functionally distinct from 'A.' It is proposed that the dynamic interaction of MLE with roX RNAs may ensure that different regions of roX RNAs are exposed such that they can be used in a redundant or cooperative manner for the interaction with the MSL complex members providing plasticity, as clearly detected for MSL2 binding on roX RNAs in the iCLIP data in vivo. However, it can be excluded that integration of roX RNAs with the 'core' MSL complex may also follow a more direct route involving independent MSL-RNA interactions that need not require MLE as a mediator (Ilik, 2014).

roX genes have a dual function in Drosophila dosage compensation: they are sites of roX transcription, but they also contain two high-affinity sites (HASs) that can recruit the MSL complex independent of the roX RNAs. Moreover, roX RNAs can travel from their sites of synthesis to the X chromosome when placed as a transgene to an autosomal site. This suggests that the holo- MSL complex, containing the core components (MSL1-3 and MOF) and MLE together with roX RNAs, can form on chromatin at roX transcription sites and spread in cis on the X chromosome, but it may also form in solution and be targeted to X-chromosomal sites in trans. It has been recently shown that MSL2 interacts with a dimer of MSL1 and is itself present as a dimer within the MSL complex. Interestingly, this study found that roX RNAs do not interact with each other in vivo, suggesting that there could be one roX RNA species per holo-MSL complex (Ilik, 2014).

Notably, iCLIP methodology utilizing UV crosslinking provides a snapshot of a pool of interactions that are present at the instant of irradiation. Although iCLIP data clearly show that MLE and MSL2 bind to the same domains, it is possible that they occupy different stem-loops on different molecules of roX RNAs rather than occupying the same structure at the same time. roX RNAs, by evolving multiple interaction platforms, can indeed support such combinatorial binding events, thus facilitating spreading along the X chromosome. It is proposed that roX RNAs, by virtue of being able to interact with MLE and the MSL complex, play a central role in the assembly of the holo- MSL complex containing the 'core' (MSL1, MSL2, MSL3, MOF) and MLE. These complexes may form in solution or on chromatin such as on sites of roX transcription. Such configuration thus brings different enzymatic activities together (ATPase/ helicase activity of MLE; acetyltransferase activity of MOF and ubiquitin-ligase activity of MSL2) for the X chromosome-specific dosage compensation process. Directing the assembly of such a complex and by providing plasticity through its multiple stem-loops, roX RNAs could thus facilitate local spreading of the MSL complex from HAS into neighboring chromatin including low-affinity sites (LASs) (Ilik, 2014).

Domains identified in roX1 and roX2 could be defined as 'information units' within these lncRNAs, which organizationally resemble stem-loops strung together like 'beads on a string'. It is tempting to hypothesize that one could compare the 'information unit' within roX lncRNAs to be the counterpart of codons which provide the basic information units in mRNAs. For roX lncRNAs, the information unit is a bit larger (11-15 nt) and involves both primary sequence and secondary structure. Another important aspect is that the domains are repeated in the lncRNA but do not appear to have a strict requirement of spacing or order relative to each other (i.e., no polarity and reading frame), so functional versions may be relatively easy to evolve during evolution (Ilik, 2014).

In summary, these data suggest that lncRNAs may utilize discrete repetitive motifs for distinct protein-RNA or even RNADNA interactions to achieve functional specificity in vivo. Such a mechanism may also be used for other global epigenetic phenomenon such as the X-inactivation in mammalian cells. Future analysis of other lncRNAs that combine CLIP methods and RNA structural analysis will be crucial in identifying structural domains within these RNAs and understanding their precise roles in the regulation of gene expression (Ilik, 2014).

GENE STRUCTURE

Bases in 5' UTR - 343

Base pairs in 3' UTR - 1091

PROTEIN STRUCTURE

Amino Acids - 769

Structural Domains

The amino terminus (residues 39-82) contains a cysteine-rich C3HC4 zinc finger sequence motif known as the RING finger, characterized by a pattern of conserved Cys, His, and hydrophobic residues. Several family members are likely to interact with DNA, including Posterior sex combs and Suppressor 2 of zeste, both regulators of repressive chromatin structure in Drosophila. A 55 amino acid synthetic peptide corresponding to the motif from RING1 ( a human gene of unknown function) has a general DNA binding activity that is dependent on the presence of zinc. An alternative potential function for this zinc finger-like motif is the mediation of protein-protein interactions. In the center of the MSL-2 protein is a large domain consisting of a putative coiled coil. A metallothionein-like domain is located towards the C-terminus of MSL-2. MSL-2 has eight of the 20 cysteines that are typical of metallothioneins. The cysteines found in the metallothionein-like cluster of MSL-2 are probably sufficient to bind at least three divalent cations. The sequence of the 3' UTR of MSL-2 mRNA reveals a cluster of four poly U stretches containing optimal target sequences for the binding of Sex Lethal. Two additional poly U stretches are found in the 5'UTR. Four putative SXL-binding sites are also found in the 3' UTR of a subset of MSL-1 transcripts. The presence of SXL-binding sites in MSL-1 and MSL-2 transcripts suggests that they are direct targets of Sxl regulation (Brashaw, 1995, Kelly, 1995, Zhou, 1995 and references).

The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for the Zinc finger, C3HC4 type (RING finger) signature.

EVOLUTIONARY HOMOLOGS

MSL-2 is the first protein that has been shown to contain both a zinc finger and metallothionein domain. However, interactions between these two types of metal binding domains on different proteins have been reported in the case of transcription factors SP1 and TFIIIA: thionein, the apo-form of metallothionein, abstracts zinc cations from the fingers of these factors and abrogates their DNA binding activity. It is tempting to speculate that the affinity of the metallothionein-like domain for zinc ions may serve as a biochemical 'governor' for the activity of the RING finger portion of the MSL-2 protein, thereby preventing the dosage compensation complex from enhancing transcription beyond the observed 2-fold range (Zhou, 1995).

The RING finger controls protein-protein interaction. Human KAP-1 (KRAB-associated protein-1) functions as a universal corepressor for a large family of KRAB (Krüppel-associate box) domain-containing transcription factors. A multifunctional protein, KAP-1 contains a RING finger motif. The amino-terminal RING finger motif is identified by the signature C3HC4 spacing of cysteine and histidine residues. The RING finger region of KAP-1 is required for binding to the KRAB motif. RING finger-containing proteins have been implicated in cell growth regulation and transcripion. Proteins with RING finger motifs include the tumor suppressor BRCA-1, and the proto-oncogene PML, that is fused to the retinoic acid receptor-alpha in promyelocytic leukemia. Immediately C-terminal to the Ring finger are B1 and B2 boxes, often conserved in Ring finger proteins. Also present is a Cys/His-rich structure identified as a PHD finger. The extreme C-terminus displays significant similarity to the bromodomain. The KRAB motif has not been identified in Drosophila (Friedman, 1996 and references).

Dosage compensation in Drosophila is mediated by a complex, called compensasome, composed of at least five proteins and two noncoding RNAs. Genes encoding compensasome proteins have been collectively named male-specific lethals or msls. Recent work shows that three of the Drosophila msls (msl-3, mof, and mle) have an ancient origin. This study describes likely orthologues of the two remaining msls, msl-1 and msl-2, in several invertebrates and vertebrates. The MSL-2 protein is the only one found in Drosophila and vertebrate genomes that contains both a RING finger and a peculiar type of CXC domain, related to the one present in Enhancer of Zeste proteins. MSL-1 also contains two evolutionarily conserved domains: a leucine zipper and a second characteristic region, described here for the first time, which is called here the PEHE domain. These two domains are present in the likely orthologues of MSL-1 as well as in other genes in several invertebrate and vertebrate species. Although it cannot be excluded that the compensasome complex is a recent evolutionary novelty, these results shows that all msls are found in mammals, suggesting that protein complexes related to the compensasome may be present in mammalian species. Metazoans that lack several of the msls, such as Caenorhabditis elegans, cannot contain compensasomes. The evolutionary relationships of the compensasome and the NuA4 complex, another chromatin-remodeling complex that contains related subunits, are discussed (Marin, 2003).

date revised: 20 June 2024

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