Dosage Compensation Proteins Research Articles

MSL2 Localization to Histone Loci in Drosophila Species

Nuclear bodies are membraneless structures containing concentrated regulatory factors that coordinate nuclear processes such as gene expression. The histone locus body (HLB) is a discrete nuclear body that is the main site of histone mRNA production. While many factors of the HLB are known, there are many unknown factors. In addition, while HLB function is highly conserved, it is also unknown how the HLB has changed and evolved in different species. The histone locus (HL) of the model organism Drosophila melanogaster contains ~100 tandem arrays of the five histone genes. While D. melanogaster has one HL, the related species Drosophila virilis, has two HL. We observe localization of the male‐specific dosage compensation protein MSL2 to one of the two D. virilishistone loci using polytene chromosome immunofluorescence, which we do not observe in other Drosophila species. In order to confirm our immunofluorescence observations, we mapped existing MSL2 ChIP‐seq data and discovered that when MSL2 from either species is overexpressed, it targets the H2a‐H2b promoter. We immunostained transgenic flies carrying an H2a‐H2b promoter transgene to determine if MSL2 targets this region when outside the context of the histone locus. We do not see the same HL localization when MSL2 is expressed at wild‐type levels. Finally, we performed RNA isolation and qPCR analysis on four Drosophila species to compare expression levels of msl2. Our results indicate that MSL2 may be playing a different role at the HL in different Drosophila species. Overall, this gives us better insight on histone gene regulation.

Inducible degradation of dosage compensation protein DPY-27 facilitates isolation of Caenorhabditis elegans males for molecular and biochemical analyses.

Biological sex affects numerous aspects of biology, yet how sex influences different biological processes have not been extensively studied at the molecular level. Caenorhabditis elegans, with both hermaphrodites (functionally females as adults) and males, is an excellent system to uncover how sex influences physiology. Here, we describe a method to isolate large quantities of C. elegans males by conditionally degrading DPY-27, a component of the dosage compensation complex essential for hermaphrodite, but not male, development. We show that germ cells from males isolated following DPY-27 degradation undergo meiosis and spermiogenesis like wild type and these males are competent to mate and sire viable offspring. We further demonstrate the efficacy of this system by analyzing gene expression and performing affinity pull-downs from male worm extracts.

Live-Cell Imaging and Functional Dissection of Xist RNA Reveal Mechanisms of X Chromosome Inactivation and Reactivation

SummaryWe double-tagged Xist (inactivated X chromosome-specific transcript), a prototype long non-coding RNA pivotal for X chromosome inactivation (XCI), using the programmable RNA sequence binding domain of Pumilio protein, one tag for live-cell imaging and the other replacing A-repeat (a critical domain of Xist) to generate “ΔA mutant” and to tether effector proteins for dissecting Xist functionality. Based on the observation in live cells that the induced XCI in undifferentiated embryonic stem (ES) cells is counteracted by the intrinsic X chromosome reactivation (XCR), we identified Kat8 and Msl2, homologs of Drosophila dosage compensation proteins, as players involved in mammalian XCR. Furthermore, live-cell imaging revealed the obviously undersized ΔA Xist cloud signals, clarifying an issue regarding the previous RNA fluorescence in situ hybridization results. Tethering candidate proteins onto the ΔA mutant reveals the significant roles of Ythdc1, Ezh2, and SPOC (Spen) in Xist-mediated gene silencing and the significant role of Ezh2 in Xist RNA spreading.

A histone H4 lysine 20 methyltransferase couples environmental cues to sensory neuron control of developmental plasticity.

Animals change developmental fates in response to external cues. In the nematode Caenorhabditis elegans, unfavorable environmental conditions induce a state of diapause known as dauer by inhibiting the conserved DAF-2 insulin-like signaling (ILS) pathway through incompletely understood mechanisms. We have previously established a role for the C. elegans dosage compensation protein DPY-21 in the control of dauer arrest and DAF-2 ILS. Here, we show that the histone H4 lysine 20 methyltransferase SET-4, which also influences dosage compensation, promotes dauer arrest in part by repressing the X-linked ins-9 gene, which encodes a new agonist insulin-like peptide (ILP) expressed specifically in the paired ASI sensory neurons that are required for dauer bypass. ins-9 repression in dauer-constitutive mutants requires DPY-21, SET-4 and the FoxO transcription factor DAF-16, which is the main target of DAF-2 ILS. By contrast, autosomal genes encoding major agonist ILPs that promote reproductive development are not repressed by DPY-21, SET-4 or DAF-16/FoxO. Our results implicate SET-4 as a sensory rheostat that reinforces developmental fates in response to environmental cues by modulating autocrine and paracrine DAF-2 ILS.

Genetic Testing of the Hypothesis That Hybrid Male Lethality Results From a Failure in Dosage Compensation

Several recent studies have suggested that F(1) hybrid male lethality in crosses between Drosophila melanogaster and D. simulans is due to a failure in dosage compensation, caused by incompatibilities between D. simulans dosage compensation proteins and the D. melanogaster X chromosome. Contrary to the predictions of this hypothesis, mutations in four essential D. melanogaster dosage compensation genes are shown here to moderately increase rather than decrease hybrid male viability.

HMOF, a KAT(8) with Many Lives

Dynamic lysine acetylation regulates proteins involved in diverse cellular processes, with individual enzymes often acetylating multiple substrates. Here, Li et al. (2009) show that the substrate specificity of hMOF/MYST1/KAT8 is controlled by differential interaction with two mutually exclusive partners.

Meiotic crossover number and distribution are regulated by a dosage compensation protein that resembles a condensin subunit

Biological processes that function chromosome-wide are not well understood. Here, we show that the Caenorhabditis elegans protein DPY-28 controls two such processes, X-chromosome dosage compensation in somatic cells and meiotic crossover number and distribution in germ cells. DPY-28 resembles a subunit of condensin, a conserved complex required for chromosome compaction and segregation. In the soma, DPY-28 associates with the dosage compensation complex on hermaphrodite X chromosomes to repress transcript levels. In the germline, DPY-28 restricts crossovers. In many organisms, one crossover decreases the likelihood of another crossover nearby, an enigmatic process called crossover interference. In C. elegans, interference is complete: Only one crossover occurs per homolog pair. dpy-28 mutations increase crossovers, disrupt crossover interference, and alter crossover distribution. Early recombination intermediates (RAD-51 foci) increase concomitantly, suggesting that DPY-28 acts to limit double-strand breaks (DSBs). Reinforcing this view, dpy-28 mutations partially restore DSBs in mutants lacking HIM-17, a chromatin-associated protein required for DSB formation. Our work further links dosage compensation to condensin and establishes a new role for condensin components in regulating crossover number and distribution. We propose that both processes utilize a related mechanism involving changes in higher-order chromosome structure to achieve chromosome-wide effects.

A human protein complex homologous to the Drosophila MSL complex is responsible for the majority of histone H4 acetylation at lysine 16.

We describe a stable, multisubunit human histone acetyltransferase complex (hMSL) that contains homologs of the Drosophila dosage compensation proteins MOF, MSL1, MSL2, and MSL3. This complex shows strong specificity for histone H4 lysine 16 in chromatin in vitro, and RNA interference-mediated knockdown experiments reveal that it is responsible for the majority of H4 acetylation at lysine 16 in the cell. We also find that hMOF is a component of additional complexes, forming associations with host cell factor 1 and a protein distantly related to MSL1 (hMSL1v1). We find two versions of hMSL3 in the hMSL complex that differ by the presence of the chromodomain. Lastly, we find that reduction in the levels of hMSLs and acetylation of H4 at lysine 16 are correlated with reduced transcription of some genes and with a G(2)/M cell cycle arrest. This is of particular interest given the recent correlation of global loss of acetylation of lysine 16 in histone H4 with tumorigenesis.

Painting of fourth in genus Drosophila suggests autosome-specific gene regulation.

Painting of fourth (POF) is a chromosome-specific protein in Drosophila and represents the first example of an autosome-specific protein. POF binds to chromosome 4 in Drosophila melanogaster, initiating at the proximal region, followed by a spreading dependent on chromosome 4-specific sequences or structures. Chromosome-specific gene regulation is known thus far only as a mechanism to equalize the transcriptional activity of the single male X chromosome with that of the two female X chromosomes. In Drosophila, a complex including the male-specific lethal proteins, "paints" the male X chromosome, mediating its hypertranscription, explained to some extent by the acetylation of lysine 16 on histone H4. Here, we show that Pof is essential for viability in both sexes and for female fertility. POF binding to an autosome, the F element, is conserved in genus Drosophila, indicating functional conservation of the autosome specificity. In three of nine studied species, POF binds to the male X chromosome. When bound to the male X, it also colocalizes with the dosage compensation protein male-specific lethal 3, suggesting a relationship to dosage compensation. The chromosome specificity is determined at the species level and not by the amino acid sequence. We argue that POF is involved in a chromosome-specific regulatory function.

Recruitment of C. elegans dosage compensation proteins for gene-specific versus chromosome-wide repression.

In C. elegans, an X-chromosome-wide regulatory process compensates for the difference in X-linked gene dose between males (XO) and hermaphrodites (XX) by equalizing levels of X-chromosome transcripts between the sexes. To achieve dosage compensation, a large protein complex is targeted to the X chromosomes of hermaphrodites to reduce their expression by half. This repression complex is also targeted to a single autosomal gene, her-1. By silencing this male-specific gene, the complex induces hermaphrodite sexual development. Our analysis of the atypical dosage compensation gene dpy-21 revealed the first molecular differences in the complex that achieves gene-specific versus chromosome-wide repression. dpy-21 mutations, shown here to be null, cause elevated X-linked gene expression in XX animals, but unlike mutations in other dosage compensation genes, they do not cause extensive XX-specific lethality or disrupt the stability or targeting of the dosage compensation complex to X. Nonetheless, DPY-21 is a member of the dosage compensation complex and localizes to X chromosomes in a hermaphrodite-specific manner. However, DPY-21 is the first member of the dosage compensation complex that does not also associate with her-1. In addition to a difference in the composition of the complex at her-1 versus X, we also found differences in the targeting of the complex to these sites. Within the complex, SDC-2 plays the lead role in recognizing X-chromosome targets, while SDC-3 plays the lead in recognizing her-1 targets.

The Yeast NuA4 and Drosophila MSL Complexes Contain Homologous Subunits Important for Transcription Regulation

In Drosophila, the MSL complex is required for the dosage compensation of X-linked genes in males and contains a histone acetyltransferase, MOF. A point mutation in the MOF acetyl-CoA-binding site results in male-specific lethality. Yeast Esa1p, a MOF homolog, is essential for cell cycle progression and is the catalytic subunit of the NuA4 acetyltransferase complex. Here we report that NuA4 purified from yeast with a point mutation in the acetyl-CoA-binding domain of Esa1p exhibits a strong decrease in histone acetyltransferase activity, yet has no effect on growth. We demonstrate that Eaf3p (Esa1p-associated factor-3 protein), a yeast protein homologous to the Drosophila dosage compensation protein MSL3, is also a stable component of the NuA4 complex. Unlike other subunits of the complex, it is not essential, and the deletion mutant has no growth phenotype. NuA4 purified from the mutant strain has a decreased apparent molecular mass, but retains wild-type levels of histone H4 acetyltransferase activity. The EAF3 deletion and the ESA1 mutation lead to a decrease in PHO5 gene expression; the EAF3 deletion also significantly reduces HIS4 and TRP4 expressions. These results, together with those previously obtained with both the MSL and NuA4 complexes, underscore the importance of targeted histone H4 acetylation for the gene-specific activation of transcription.

X-Ray Microscopic Studies of the Drosophila Dosage Compensation Complex

X-raymicroscopy is applied to detect specific proteins in whole cell nuclei of Drosophila melanogaster using immunogold labeling and silver enhancement. As a model for a small subnuclear structure the Drosophila dosage compensation protein MSL-1 was chosen. It associates with a number of other proteins to form a hetero-multiprotein complex, which elevates the transcriptional activity of the single X chromosome in males. This phenomenon is known as dosage compensation and is essential for the survival of male flies. The distribution of the Drosophila dosage compensation complex was studied by X-ray microscopy, because though the complex is expected to function by remodeling the structureof chromatin, its exact mode of action is not yet known. Many similar protein complexes are associated with different aspects of chromatin-mediated gene regulation in all eucaryotic organisms and can also be studied with the approach presented in this work. The distribution of MSL-1 protein in the nuclei of fixed D. melanogaster culture cells is visualized using the Göttingen X-ray microscope at the electron storage ring BESSY I. In addition to conventional and confocal laserscan fluorescence microscopy, X-ray microscopic investigations were performed at room as well as at cryogenic temperatures. The label can clearly be identified in the X-ray micrographs and shows detailed structure in the cell nuclei. Currently, X-ray micrographs show details in the cell nuclei about five times smaller than those in visible light micrographs.

SDC-3 coordinates the assembly of a dosage compensation complex on the nematode X chromosome.

X chromosome expression in C. elegans is controlled by a chromosome-wide regulatory process called dosage compensation that specifically reduces by half the level of transcripts made from each hermaphrodite X chromosome. This process equalizes X expression between the sexes (XX hermaphrodites and XO males), despite their two-fold difference in X chromosome dose, and thereby prevents sex-specific lethality. Dosage compensation is achieved by a protein complex that associates with X in a sex-specific fashion to modulate gene expression. SDC-3, a protein that coordinately controls both sex determination and dosage compensation, activates dosage compensation by directing the dosage compensation protein complex to the hermaphrodite X chromosomes. We show that SDC-3 coordinates this assembly through its own sex-specific association with X. SDC-3 in turn requires other members of the dosage compensation gene hierarchy for its stability and its X localization. In addition, SDC-3 requires its own zinc finger motifs and an amino-terminal region for its X association. Our experiments suggest the possible involvement of zinc finger motifs in X chromosome recognition and the amino-terminal region in interactions with other dosage compensation proteins.

Genes Expressed in Neurons of Adult Male Drosophila

We have identified two genes, roX1 and roX2, whose expression in the adult fly is restricted to neurons of males. The two genes reside on the X chromosome, and each encodes an RNA with no apparent open reading frame. Both genes are physically linked to female-specific genes that encode proteins expressed in the ovary: opt1, a novel peptide transporter, and nod, a member of the kinesin family. The male-specific transcripts are positively regulated by the dosage compensation pathway in an all-or-none fashion. Our data suggest that the multimeric complex of dosage compensation proteins may operate in different ways on different sets of X-linked genes.

DPY-26, a link between dosage compensation and meiotic chromosome segregation in the nematode.

The DPY-26 protein is required in the nematode Caenorhabditis elegans for X-chromosome dosage compensation as well as for proper meiotic chromosome segregation. DPY-26 was shown to mediate both processes through its association with chromosomes. In somatic cells, DPY-26 associates specifically with hermaphrodite X chromosomes to reduce their transcript levels. In germ cells, DPY-26 associates with all meiotic chromosomes to mediate its role in chromosome segregation. The X-specific localization of DPY-26 requires two dosage compensation proteins (DPY-27 and DPY-30) and two proteins that coordinately control both sex determination and dosage compensation (SDC-2 and SDC-3).

Expression of Msl-2 causes assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila

Male-specific lethal-2 (msl-2) is a RING finger protein that is required for X chromosome dosage compensation in Drosophila males. Consistent with the formation of a dosage compensation protein complex, msl-2 colocalizes with the other MSL proteins on the male X chromosome and coimmunoprecipitates with msl-1 from male larval extracts. Ectopic expression of msl-2 in females results in the appearance of the other MSL dosage compensation regulators on the female X chromosomes and decreased female viability. We suggest that msl-2 RNA is the primary target of Sx1 regulation in the dosage compensation pathway and present a speculative model for the regulation of two distinct modes of dosage compensation by Sxl.

Regulation of the sex-specific binding of the maleless dosage compensation protein to the male X chromosome in Drosophila

In Drosophila, the single male X chromosome is transcribed at twice the rate of a single female X chromosome. This hypertranscription requires the functions of at least four autosomal male-specific lethal genes ( msls) and is under the control of the Sex-lethal ( S×I) gene. One of the msls, the maleless ( mle) gene, encodes a protein that is associated with the male X chromosome. To investigate how dosage compensation is regulated, we have determined whether S×I and the other msls are required for mle X chromosome binding. We have found that in females, S×I functions to prevent mle from binding to the two X chromosomes. Additionally, we have found that mle X chromosome binding requires wild-type msl1, msl2, and msl3 functions. These data support a model whereby the activity of the mle protein is regulated through its association with one or more of the other msl proteins.