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

Gene name - male-specific lethal 2

Synonyms - Male-specific lethal 2

Cytological map position - 23E1-F6

Function - Dosage compensation

Keywords - Sex determination - Modification of chromatin

Symbol - msl-2

FlyBase ID:FBgn0005616

Genetic map position - 2-9.0

Classification - Ring finger motif - metallothionein motif

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Haussmann, I. U., Bodi, Z., Sanchez-Moran, E., Mongan, N. P., Archer, N., Fray, R. G. and Soller, M. (2016). m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination. Nature 540(7632): 301-304. PubMed ID: 27919081
Summary:
N6-methyladenosine (m6A) is the most common internal modification of eukaryotic messenger RNA (mRNA) and is decoded by YTH domain proteins. Drosophila mRNA m6A methylosome consists of Ime4 and KAR4 (Inducer of meiosis 4 and Karyogamy protein 4), and Female-lethal (2)d (Fl(2)d) and Virilizer (Vir). In Drosophila, fl(2)d and vir are required for sex-dependent regulation of alternative splicing of the sex determination factor Sex lethal (Sxl). However, the functions of m6A in introns in the regulation of alternative splicing remain uncertain. This study shows that m6A is absent in the mRNA of Drosophila lacking Ime4. In contrast to mouse and plant knockout models, Drosophila Ime4-null mutants remain viable, though flightless, and show a sex bias towards maleness. This is because m6A is required for female-specific alternative splicing of Sxl, which determines female physiognomy, but also translationally represses male-specific lethal 2 (msl-2) to prevent dosage compensation in females. The m6A reader protein YT521-B decodes m6A in the sex-specifically spliced intron of Sxl, as its absence phenocopies Ime4 mutants. Loss of m6A also affects alternative splicing of additional genes, predominantly in the 5' untranslated region, and has global effects on the expression of metabolic genes. The requirement of m6A and its reader YT521-B for female-specific Sxl alternative splicing reveals that this hitherto enigmatic mRNA modification constitutes an ancient and specific mechanism to adjust levels of gene expression.
Szostak, E., Garcia-Beyaert, M., Guitart, T., Graindorge, A., Coll, O. and Gebauer, F. (2018). Hrp48 and eIF3d contribute to msl-2 mRNA translational repression. Nucleic Acids Res 46(8):4099-4113. PubMed ID: 29635389
Summary:
Translational repression of msl-2 mRNA in females of Drosophila melanogaster is an essential step in the regulation of X-chromosome dosage compensation. Repression is orchestrated by Sex-lethal (SXL), which binds to both untranslated regions (UTRs) of msl-2 and inhibits translation initiation by poorly understood mechanisms. This study identified Heterogeneous nuclear ribonucleoprotein at 27C as a SXL co-factor. Hrp48 binds to the 3' UTR of msl-2 and is required for optimal repression by SXL. Hrp48 interacts with eIF3d, a subunit of the eIF3 translation initiation complex. Reporter and RNA chromatography assays showed that eIF3d binds to msl-2 5' UTR, and is required for efficient translation and translational repression of msl-2 mRNA. In line with these results, eIF3d depletion -but not depletion of other eIF3 subunits- de-represses msl-2 expression in female flies. These data are consistent with a model where Hrp48 inhibits msl-2 translation by targeting eIF3d. These results uncover an important step in the mechanism of msl-2 translation regulation, and illustrate how general translation initiation factors can be co-opted by RNA binding proteins to achieve mRNA-specific control.
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).


GENE STRUCTURE

cDNA clone length - 3.6 kb

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).


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

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