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.

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).

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