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1.  Condensin controls recruitment of RNA polymerase II to achieve nematode X-chromosome dosage compensation 
eLife  2013;2:e00808.
The X-chromosome gene regulatory process called dosage compensation ensures that males (1X) and females (2X) express equal levels of X-chromosome transcripts. The mechanism in Caenorhabditis elegans has been elusive due to improperly annotated transcription start sites (TSSs). Here we define TSSs and the distribution of transcriptionally engaged RNA polymerase II (Pol II) genome-wide in wild-type and dosage-compensation-defective animals to dissect this regulatory mechanism. Our TSS-mapping strategy integrates GRO-seq, which tracks nascent transcription, with a new derivative of this method, called GRO-cap, which recovers nascent RNAs with 5′ caps prior to their removal by co-transcriptional processing. Our analyses reveal that promoter-proximal pausing is rare, unlike in other metazoans, and promoters are unexpectedly far upstream from the 5′ ends of mature mRNAs. We find that C. elegans equalizes X-chromosome expression between the sexes, to a level equivalent to autosomes, by reducing Pol II recruitment to promoters of hermaphrodite X-linked genes using a chromosome-restructuring condensin complex.
DOI: http://dx.doi.org/10.7554/eLife.00808.001
eLife digest
In many species, including humans, females have two X chromosomes whereas males have only one. To ensure that females do not end up with a double dose of the proteins encoded by genes on the X chromosome, animals employ a strategy called dosage compensation to control the expression of X-linked genes.
The mechanisms underlying dosage compensation vary between species, but they typically involve a regulatory complex that binds to the X chromosomes of one sex to modify gene expression. In the nematode worm Caenorhabditis elegans—which consists of hermaphrodites (XX) and males (XO)—this regulatory complex, called the dosage compensation complex (DCC), binds to both X chromosomes of XX individuals, reducing gene expression from each by 50%. DCC shares many subunits with a protein complex called condensin, which regulates the structure of chromosomes to achieve proper chromosome segregation. However, it is unclear exactly how the DCC controls the expression of X-linked genes.
For a gene to be expressed, an enzyme called RNA polymerase II must bind to the gene’s promoter—a stretch of DNA upstream of the protein-coding part of the gene—so that it can begin transcribing the DNA into RNA. Promoters have been difficult to define in C. elegans, but Kruesi et al. devised a strategy to map transcription start sites, and hence promoters, throughout the worm genome. The strategy integrates the results of two methods: One measures the extent and orientation of each gene’s transcribed region, and the other locates the distinctive cap structures that mark the true 5′ ends of newly made RNAs.
Using this new promoter information, coupled with genome-wide measurements of the levels of newly synthesized transcripts from wild-type and dosage-compensation-defective animals, they showed that C. elegans achieves dosage compensation by reducing the recruitment of RNA polymerase II to the promoters of X-linked genes in XX individuals.
Kruesi et al. also identified a second regulatory mechanism that acts in both sexes to increase the level of transcription of genes on the X chromosome. This ensures that after dosage compensation, genes on the X chromosome are expressed at a similar level to those on the autosomes (all chromosomes other than X and Y).
As well as shedding light on the mechanism by which dosage compensation occurs in C. elegans, the study by Kruesi et al. provides a valuable data set on transcription start sites in the worm, and puts forward a general strategy that could be used to map these sites in other species.
DOI: http://dx.doi.org/10.7554/eLife.00808.002
doi:10.7554/eLife.00808
PMCID: PMC3687364  PMID: 23795297
dosage compensation; transcription; X-chromosome and autosome balance; transcription start site identification technology; X chromosome; C. elegans
2.  Three Distinct Condensin Complexes Control C. elegans Chromosome Dynamics 
Current biology : CB  2009;19(1):9-19.
Summary
Background
Condensin complexes organize chromosome structure and facilitate chromosome segregation. Higher eukaryotes have two complexes, condensin I and condensin II, each essential for chromosome segregation. The nematode Caenorhabditis elegans was considered an exception, because it has a mitotic condensin II complex but appeared to lack mitotic condensin I. Instead, its condensin I-like complex (here called condensin IDC) dampens gene expression along hermaphrodite X chromosomes during dosage compensation.
Results
Here we report the discovery of a third condensin complex, condensin I, in C. elegans. We identify new condensin subunits and show that each complex has a conserved five-subunit composition. Condensin I differs from condensin IDC by only a single subunit. Yet condensin I binds to autosomes and X chromosomes in both sexes to promote chromosome segregation, whereas condensin IDC binds specifically to X chromosomes in hermaphrodites to regulate transcript levels. Both condensin I and II promote chromosome segregation, but associate with different chromosomal regions during mitosis and meiosis. Unexpectedly, condensin I also localizes to regions of cohesion between meiotic chromosomes before their segregation.
Conclusions
We demonstrate that condensin subunits in C. elegans form three complexes, one that functions in dosage compensation and two that function in mitosis and meiosis. These results highlight how the duplication and divergence of condensin subunits during evolution may facilitate their adaptation to specialized chromosomal roles and illustrate the versatility of condensins to function in both gene regulation and chromosome segregation.
doi:10.1016/j.cub.2008.12.006
PMCID: PMC2682549  PMID: 19119011
3.  Condensins Regulate Meiotic DNA Break Distribution, thus Crossover Frequency, by Controlling Chromosome Structure 
Cell  2009;139(1):73-86.
SUMMARY
Meiotic crossover (CO) recombination facilitates evolution and accurate chromosome segregation. CO distribution is tightly regulated: homolog pairs receive at least one CO, CO spacing is nonrandom, and COs occur preferentially in short genomic intervals called hotspots. We show that CO number and distribution are controlled on a chromosome-wide basis at the level of DNA double-strand break (DSB) formation by a condensin complex composed of subunits from two known condensins: the C. elegans dosage compensation complex and mitotic condensin II. Disruption of any subunit of the CO-controlling condensin dominantly changes DSB distribution, and thereby COs, and extends meiotic chromosome axes. These phenotypes are cosuppressed by disruption of a chromosome axis element. Our data implicate higher-order chromosome structure in the regulation of CO recombination, provide a model for the rapid evolution of CO hotspots, and show that reshuffling of interchangeable molecular parts can create independent machines with similar architectures but distinct biological functions.
doi:10.1016/j.cell.2009.07.035
PMCID: PMC2785808  PMID: 19781752
4.  Condensin-mediated chromosome organization and gene regulation 
Frontiers in Genetics  2015;5:473.
In many organisms sexual fate is determined by a chromosome-based method which entails a difference in sex chromosome-linked gene dosage. Consequently, a gene regulatory mechanism called dosage compensation equalizes X-linked gene expression between the sexes. Dosage compensation initiates as cells transition from pluripotency to differentiation. In Caenorhabditis elegans, dosage compensation is achieved by the dosage compensation complex (DCC) binding to both X chromosomes in hermaphrodites to downregulate gene expression by twofold. The DCC contains a subcomplex (condensin IDC) similar to the evolutionarily conserved condensin complexes which play a fundamental role in chromosome dynamics during mitosis. Therefore, mechanisms related to mitotic chromosome condensation are hypothesized to mediate dosage compensation. Consistent with this hypothesis, monomethylation of histone H4 lysine 20 is increased, whereas acetylation of histone H4 lysine 16 is decreased, both on mitotic chromosomes and on interphase dosage compensated X chromosomes in worms. These observations suggest that interphase dosage compensated X chromosomes maintain some characteristics associated with condensed mitotic chromosome. This chromosome state is stably propagated from one cell generation to the next. In this review we will speculate on how the biochemical activities of condensin can achieve both mitotic chromosome compaction and gene repression.
doi:10.3389/fgene.2014.00473
PMCID: PMC4292777  PMID: 25628648
Caenorhabditis elegans; condensin; dosage compensation; gene expression; chromosome condensation; chromatin; interphase chromosome; epigenetics
5.  Requirement of Male-Specific Dosage Compensation in Drosophila Females—Implications of Early X Chromosome Gene Expression 
PLoS Genetics  2010;6(7):e1001041.
Dosage compensation equates between the sexes the gene dose of sex chromosomes that carry substantially different gene content. In Drosophila, the single male X chromosome is hypertranscribed by approximately two-fold to effect this correction. The key genes are male lethal and appear not to be required in females, or affect their viability. Here, we show these male lethals do in fact have a role in females, and they participate in the very process which will eventually shut down their function—female determination. We find the male dosage compensation complex is required for upregulating transcription of the sex determination master switch, Sex-lethal, an X-linked gene which is specifically activated in females in response to their two X chromosomes. The levels of some X-linked genes are also affected, and some of these genes are used in the process of counting the number of X chromosomes early in development. Our data suggest that before the female state is set, the ground state is male and female X chromosome expression is elevated. Females thus utilize the male dosage compensation process to amplify the signal which determines their fate.
Author Summary
When substantially different, sex chromosomes present the challenge of not only gene dose inequity between the sexes, in the heterogametic sex where one chromosome (frequently the Y) carries few genes, but also an inequity relative to the autosomes which are diploid. Dosage compensation refers to the process which equates gene dose between the sexes. Recent results, however, indicate that the mammalian X chromosome avoids monosomy and has a level of expression that is two-fold relative to the autosomes. Hyperactive X chromosome expression in Caenorhabditis elegans has also been suggested, and dosage compensation in the hermaphrodite appears to lower expression of the X chromosomes to match autosome levels. We find that, before the female state is set in Drosophila, the X chromosomes may also express their genes at the two-fold male level and that this level of expression is used to female advantage to consolidate their sex determination. Together, the results suggest that elevated X chromosome expression may be the norm, and that the various dosage compensation processes different organisms utilize reflect a mechanism to counteract an initial hyperactive X chromosome state.
doi:10.1371/journal.pgen.1001041
PMCID: PMC2912388  PMID: 20686653
6.  H4K20me1 Contributes to Downregulation of X-Linked Genes for C. elegans Dosage Compensation 
PLoS Genetics  2012;8(9):e1002933.
The Caenorhabditis elegans dosage compensation complex (DCC) equalizes X-chromosome gene dosage between XO males and XX hermaphrodites by two-fold repression of X-linked gene expression in hermaphrodites. The DCC localizes to the X chromosomes in hermaphrodites but not in males, and some subunits form a complex homologous to condensin. The mechanism by which the DCC downregulates gene expression remains unclear. Here we show that the DCC controls the methylation state of lysine 20 of histone H4, leading to higher H4K20me1 and lower H4K20me3 levels on the X chromosomes of XX hermaphrodites relative to autosomes. We identify the PR-SET7 ortholog SET-1 and the Suv4-20 ortholog SET-4 as the major histone methyltransferases for monomethylation and di/trimethylation of H4K20, respectively, and provide evidence that X-chromosome enrichment of H4K20me1 involves inhibition of SET-4 activity on the X. RNAi knockdown of set-1 results in synthetic lethality with dosage compensation mutants and upregulation of X-linked gene expression, supporting a model whereby H4K20me1 functions with the condensin-like C. elegans DCC to repress transcription of X-linked genes. H4K20me1 is important for mitotic chromosome condensation in mammals, suggesting that increased H4K20me1 on the X may restrict access of the transcription machinery to X-linked genes via chromatin compaction.
Author Summary
In many animals, males have one X chromosome and females have two. However, the same amount of gene expression from X chromosomes is needed in the two sexes. The process of dosage compensation (DC) globally regulates X-chromosome gene expression to make it equal between the sexes, and it occurs in different ways in different animals. In mammals, one X chromosome in females is randomly inactivated, leaving one active X chromosome. In contrast, in the nematode worm C. elegans, the two X chromosomes in hermaphrodites are repressed two-fold to match gene expression to the single X chromosome in males. Previous work in C. elegans identified proteins required for DC that bind to the X chromosome, but their mode of action is not known. Here we show that DC proteins lead to higher levels of histone H4 lysine 20 monomethylation (H4K20me1) on hermaphrodite X chromosomes and that H4K20me1 functions in repressing X-chromosome gene expression. This shows that histone modification is an important aspect of the mechanism of dosage compensation. Together with previous work linking H4K20me1 to chromatin structure regulation, our results suggest that dosage compensation might lower gene expression on hermaphrodite X chromosomes by compacting them.
doi:10.1371/journal.pgen.1002933
PMCID: PMC3441679  PMID: 23028348
7.  The Epigenome of Evolving Drosophila Neo-Sex Chromosomes: Dosage Compensation and Heterochromatin Formation 
PLoS Biology  2013;11(11):e1001711.
This study shows how young sex chromosomes have altered their chromatin structure in Drosophila, and what genomic changes have led to silencing of the Y, and hyper-transcription of the X.
Sex chromosomes originated from autosomes but have evolved a highly specialized chromatin structure. Drosophila Y chromosomes are composed entirely of silent heterochromatin, while male X chromosomes have highly accessible chromatin and are hypertranscribed as a result of dosage compensation. Here, we dissect the molecular mechanisms and functional pressures driving heterochromatin formation and dosage compensation of the recently formed neo-sex chromosomes of Drosophila miranda. We show that the onset of heterochromatin formation on the neo-Y is triggered by an accumulation of repetitive DNA. The neo-X has evolved partial dosage compensation and we find that diverse mutational paths have been utilized to establish several dozen novel binding consensus motifs for the dosage compensation complex on the neo-X, including simple point mutations at pre-binding sites, insertion and deletion mutations, microsatellite expansions, or tandem amplification of weak binding sites. Spreading of these silencing or activating chromatin modifications to adjacent regions results in massive mis-expression of neo-sex linked genes, and little correspondence between functionality of genes and their silencing on the neo-Y or dosage compensation on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those becoming heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities along the ancestral chromosome that formed the sex chromosome to adopt active or repressive chromatin configurations. Our findings have broad implications for current models of sex chromosome evolution, and demonstrate how mechanistic constraints can limit evolutionary adaptations. Our study also highlights how evolution can follow predictable genetic trajectories, by repeatedly acquiring the same 21-bp consensus motif for recruitment of the dosage compensation complex, yet utilizing a diverse array of random mutational changes to attain the same phenotypic outcome.
Author Summary
Sex chromosomes differ from non-sex chromosomes (“autosomes”) at the genomic, transcriptomic, and epigenomic level, yet the X and Y share a common evolutionary origin. The Drosophila Y chromosome is gene-poor and associated with a compact and transcriptionally inactive form of genetic material called heterochromatin. The X, in contrast, is enriched for activating chromatin marks and is consequently hyper-transcribed, a process thought to be an adaptation to decay and silencing of genes on the Y, resulting in “dosage compensation.” How sex chromosomes have altered their chromatin structure, and what genomic changes led to this dramatically different epigenetic makeup, however, has remained a mystery. By studying the genome, epigenome, and transcriptome of a species with a very recently evolved pair of sex chromosomes (the neo-X and neo-Y of a fruit fly, Drosophila miranda), we here recapitulate how both dosage compensation and heterochromatin formation evolve in Drosophila and establish several novel and important principles governing the evolution of chromatin structure. We dissect the evolutionary history of over 60 novel binding sites for the dosage compensation complex that evolved by natural selection on the neo-X within the last one million years. We show that the 21-bp consensus motifs for recruiting the dosage compensation complex were acquired by diverse molecular mechanisms along the neo-X, while the onset of heterochromatin formation is triggered by the accumulation of transposable elements, leading to silencing of adjacent neo-Y genes. We find that spreading of these chromatin modifications results in massive mis-expression of neo-sex linked genes, and that little correspondence exists between functional activity of genes on the neo-Y and whether they are dosage-compensated on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those that are heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities of the ancestral chromosome that formed the sex chromosome to evolve active versus repressive chromatin configurations. These findings have broad implications for current models of sex chromosome evolution.
doi:10.1371/journal.pbio.1001711
PMCID: PMC3825665  PMID: 24265597
8.  Genome-wide analysis of condensin binding in Caenorhabditis elegans 
Genome Biology  2013;14(10):R112.
Background
Condensins are multi-subunit protein complexes that are essential for chromosome condensation during mitosis and meiosis, and play key roles in transcription regulation during interphase. Metazoans contain two condensins, I and II, which perform different functions and localize to different chromosomal regions. Caenorhabditis elegans contains a third condensin, IDC, that is targeted to and represses transcription of the X chromosome for dosage compensation.
Results
To understand condensin binding and function, we performed ChIP-seq analysis of C. elegans condensins in mixed developmental stage embryos, which contain predominantly interphase nuclei. Condensins bind to a subset of active promoters, tRNA genes and putative enhancers. Expression analysis in kle-2-mutant larvae suggests that the primary effect of condensin II on transcription is repression. A DNA sequence motif, GCGC, is enriched at condensin II binding sites. A sequence extension of this core motif, AGGG, creates the condensin IDC motif. In addition to differences in recruitment that result in X-enrichment of condensin IDC and condensin II binding to all chromosomes, we provide evidence for a shared recruitment mechanism, as condensin IDC recruiter SDC-2 also recruits condensin II to the condensin IDC recruitment sites on the X. In addition, we found that condensin sites overlap extensively with the cohesin loader SCC-2, and that SDC-2 also recruits SCC-2 to the condensin IDC recruitment sites.
Conclusions
Our results provide the first genome-wide view of metazoan condensin II binding in interphase, define putative recruitment motifs, and illustrate shared loading mechanisms for condensin IDC and condensin II.
doi:10.1186/gb-2013-14-10-r112
PMCID: PMC3983662  PMID: 24125077
9.  The C. elegans dosage compensation complex mediates interphase X chromosome compaction 
Epigenetics & Chromatin  2014;7(1):31.
Background
Dosage compensation is a specialized gene regulatory mechanism which equalizes X-linked gene expression between sexes. In Caenorhabditis elegans, dosage compensation is achieved by the activity of the dosage compensation complex (DCC). The DCC localizes to both X chromosomes in hermaphrodites to downregulate gene expression by half. The DCC contains a subcomplex (condensin IDC) similar to the evolutionarily conserved condensin complexes which play fundamental roles in chromosome dynamics during mitosis and meiosis. Therefore, mechanisms related to mitotic chromosome condensation have been long hypothesized to mediate dosage compensation. However experimental evidence was lacking.
Results
Using 3D FISH microscopy to measure the volumes of X and chromosome I territories and to measure distances between individual loci, we show that hermaphrodite worms deficient in DCC proteins have enlarged interphase X chromosomes when compared to wild type. By contrast, chromosome I is unaffected. Interestingly, hermaphrodite worms depleted of condensin I or II show no phenotype. Therefore X chromosome compaction is specific to condensin IDC. In addition, we show that SET-1, SET-4, and SIR-2.1, histone modifiers whose activity is regulated by the DCC, need to be present for the compaction of the X chromosome territory.
Conclusion
These results support the idea that condensin IDC, and the histone modifications regulated by the DCC, mediate interphase X chromosome compaction. Our results link condensin-mediated chromosome compaction, an activity connected to mitotic chromosome condensation, to chromosome-wide repression of gene expression in interphase.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-8935-7-31) contains supplementary material, which is available to authorized users.
doi:10.1186/1756-8935-7-31
PMCID: PMC4232692  PMID: 25400696
Caenorhabditis elegans; Dosage compensation; Gene expression; Condensin; Chromosome condensation; Chromatin; Interphase chromosome; Epigenetics
10.  Restricting Dosage Compensation Complex Binding to the X Chromosomes by H2A.Z/HTZ-1 
PLoS Genetics  2009;5(10):e1000699.
Dosage compensation ensures similar levels of X-linked gene products in males (XY or XO) and females (XX), despite their different numbers of X chromosomes. In mammals, flies, and worms, dosage compensation is mediated by a specialized machinery that localizes to one or both of the X chromosomes in one sex resulting in a change in gene expression from the affected X chromosome(s). In mammals and flies, dosage compensation is associated with specific histone posttranslational modifications and replacement with variant histones. Until now, no specific histone modifications or histone variants have been implicated in Caenorhabditis elegans dosage compensation. Taking a candidate approach, we have looked at specific histone modifications and variants on the C. elegans dosage compensated X chromosomes. Using RNAi-based assays, we show that reducing levels of the histone H2A variant, H2A.Z (HTZ-1 in C. elegans), leads to partial disruption of dosage compensation. By immunofluorescence, we have observed that HTZ-1 is under-represented on the dosage compensated X chromosomes, but not on the non-dosage compensated male X chromosome. We find that reduction of HTZ-1 levels by RNA interference (RNAi) and mutation results in only a very modest change in dosage compensation complex protein levels. However, in these animals, the X chromosome–specific localization of the complex is partially disrupted, with some nuclei displaying DCC localization beyond the X chromosome territory. We propose a model in which HTZ-1, directly or indirectly, serves to restrict the dosage compensation complex to the X chromosome by acting as or regulating the activity of an autosomal repellant.
Author Summary
In organisms where females have two X chromosomes and males only have one, a mechanism called dosage compensation ensures that both sexes receive the same amount of information from their X chromosomes. Disruption of dosage compensation leads to lethality in the affected sex. While the precise mechanisms of dosage compensation differ between organisms, changes to the structure of the X chromosomes are involved in each case. The DNA of all chromosomes is packaged into a complex protein–DNA structure called chromatin. The most basic level of packaging involves wrapping DNA around a group of small proteins called histones. In both mammals and flies, dosage compensation is associated with specific changes to the histones on the dosage compensated X chromosome. Until now, no such change has been associated with dosage compensation in worms. Here we present evidence that the histone variant HTZ-1/H2A.Z plays a role in dosage compensation in the worm. Specifically, we suggest that HTZ-1 functions to ensure that only the X chromosomes, and not the other chromosomes, are subjected to dosage compensation. This suggests that, despite different mechanisms, one common theme of dosage compensation is a change at the level of the histones associated with the chromosomal DNA.
doi:10.1371/journal.pgen.1000699
PMCID: PMC2760203  PMID: 19851459
11.  Mechanisms and Evolutionary Patterns of Mammalian and Avian Dosage Compensation 
PLoS Biology  2012;10(5):e1001328.
A large-scale comparative gene expression study reveals the different ways in which the chromosome-wide gene dosage reductions resulting from sex chromosome differentiation events were compensated during mammalian and avian evolution.
As a result of sex chromosome differentiation from ancestral autosomes, male mammalian cells only contain one X chromosome. It has long been hypothesized that X-linked gene expression levels have become doubled in males to restore the original transcriptional output, and that the resulting X overexpression in females then drove the evolution of X inactivation (XCI). However, this model has never been directly tested and patterns and mechanisms of dosage compensation across different mammals and birds generally remain little understood. Here we trace the evolution of dosage compensation using extensive transcriptome data from males and females representing all major mammalian lineages and birds. Our analyses suggest that the X has become globally upregulated in marsupials, whereas we do not detect a global upregulation of this chromosome in placental mammals. However, we find that a subset of autosomal genes interacting with X-linked genes have become downregulated in placentals upon the emergence of sex chromosomes. Thus, different driving forces may underlie the evolution of XCI and the highly efficient equilibration of X expression levels between the sexes observed for both of these lineages. In the egg-laying monotremes and birds, which have partially homologous sex chromosome systems, partial upregulation of the X (Z in birds) evolved but is largely restricted to the heterogametic sex, which provides an explanation for the partially sex-biased X (Z) expression and lack of global inactivation mechanisms in these lineages. Our findings suggest that dosage reductions imposed by sex chromosome differentiation events in amniotes were resolved in strikingly different ways.
Author Summary
Mammalian sex chromosomes (the X and Y) evolved from an ordinary pair of ancestral somatic chromosomes (the proto-sex chromosomes). The process that led to emergence of distinct sex chromosomes involved the degeneration of the Y chromosome, leaving males with only one copy of most proto-sex chromosomal genes on their single X chromosome. It has remained unclear whether mechanisms evolved that compensate for this dosage reduction. Here we trace the evolution of sex chromosomal expression levels in all major mammalian lineages and in birds. We find that the X has become globally upregulated in response to the dosage reduction in marsupials, whereas in placental mammals, genes resident on autosomal (non-sex) chromosomes that interact with X-linked genes have instead become downregulated. These mechanisms restore ancestral gene expression balances and also presumably drove the evolution of secondary compensation mechanisms (i.e., female X-inactivation) in these mammalian lineages. In egg-laying mammals and birds, sex chromosomes have become partially upregulated specifically in the heterogametic sex, i.e., in male monotremes (which are XY) and female birds (which are WZ). This probably explains why the evolution of inactivation mechanisms in the homogametic sexes in these lineages (XX and ZZ, respectively) was not necessary. Our findings suggest that gene dosage alterations associated with the emergence of sex chromosome systems can be compensated in various different ways.
doi:10.1371/journal.pbio.1001328
PMCID: PMC3352821  PMID: 22615540
12.  Unexpected Role for Dosage Compensation in the Control of Dauer Arrest, Insulin-Like Signaling, and FoxO Transcription Factor Activity in Caenorhabditis elegans 
Genetics  2013;194(3):619-629.
During embryogenesis, an essential process known as dosage compensation is initiated to equalize gene expression from sex chromosomes. Although much is known about how dosage compensation is established, the consequences of modulating the stability of dosage compensation postembryonically are not known. Here we define a role for the Caenorhabditis elegans dosage compensation complex (DCC) in the regulation of DAF-2 insulin-like signaling. In a screen for dauer regulatory genes that control the activity of the FoxO transcription factor DAF-16, we isolated three mutant alleles of dpy-21, which encodes a conserved DCC component. Knockdown of multiple DCC components in hermaphrodite and male animals indicates that the dauer suppression phenotype of dpy-21 mutants is due to a defect in dosage compensation per se. In dpy-21 mutants, expression of several X-linked genes that promote dauer bypass is elevated, including four genes encoding components of the DAF-2 insulin-like pathway that antagonize DAF-16/FoxO activity. Accordingly, dpy-21 mutation reduced the expression of DAF-16/FoxO target genes by promoting the exclusion of DAF-16/FoxO from nuclei. Thus, dosage compensation enhances dauer arrest by repressing X-linked genes that promote reproductive development through the inhibition of DAF-16/FoxO nuclear translocation. This work is the first to establish a specific postembryonic function for dosage compensation in any organism. The influence of dosage compensation on dauer arrest, a larval developmental fate governed by the integration of multiple environmental inputs and signaling outputs, suggests that the dosage compensation machinery may respond to external cues by modulating signaling pathways through chromosome-wide regulation of gene expression.
doi:10.1534/genetics.113.149948
PMCID: PMC3697968  PMID: 23733789
Caenorhabditis elegans; dosage compensation; dauer; insulin signaling; DAF-16/FoxO
13.  The worm solution: a chromosome-full of condensin helps gene expression go down 
Dosage compensation in the nematode C. elegans is achieved by the binding of a condensin-like dosage compensation complex (DCC) to both X chromosomes in hermaphrodites to downregulate gene expression two-fold. Condensin IDC, a sub-part of the DCC, differs from the mitotic condensin I complex by a single subunit, strengthening the connection between dosage compensation and mitotic chromosome condensation. The DCC is targeted to X chromosomes by initial binding to a number of recruiting elements, followed by dispersal or spreading to secondary sites. While the complex is greatly enriched on the X chromosomes, many sites on autosomes also bind the complex. DCC binding does not correlate with DCC-mediated repression, suggesting that the complex acts in a chromosome-wide manner, rather than on a gene-by-gene basis. Worm dosage compensation represents an excellent model system to study how condensin-mediated changes in higher order chromatin organization affect gene expression.
doi:10.1007/s10577-009-9061-y
PMCID: PMC2992697  PMID: 19802703
14.  Sex Chromosome-Specific Regulation in the Drosophila Male Germline But Little Evidence for Chromosomal Dosage Compensation or Meiotic Inactivation 
PLoS Biology  2011;9(8):e1001126.
Suppression of X-linked transgene reporters versus normal expression of endogenous X-linked genes suggest a novel form of X chromosome-specific regulation in Drosophila testes, instead of sex chromosome dosage compensation or meiotic inactivation.
The evolution of heteromorphic sex chromosomes (e.g., XY in males or ZW in females) has repeatedly elicited the evolution of two kinds of chromosome-specific regulation: dosage compensation—the equalization of X chromosome gene expression in males and females— and meiotic sex chromosome inactivation (MSCI)—the transcriptional silencing and heterochromatinization of the X during meiosis in the male (or Z in the female) germline. How the X chromosome is regulated in the Drosophila melanogaster male germline is unclear. Here we report three new findings concerning gene expression from the X in Drosophila testes. First, X chromosome-wide dosage compensation appears to be absent from most of the Drosophila male germline. Second, microarray analysis provides no evidence for X chromosome-specific inactivation during meiosis. Third, we confirm the previous discovery that the expression of transgene reporters driven by autosomal spermatogenesis-specific promoters is strongly reduced when inserted on the X chromosome versus the autosomes; but we show that this chromosomal difference in expression is established in premeiotic cells and persists in meiotic cells. The magnitude of the X-autosome difference in transgene expression cannot be explained by the absence of dosage compensation, suggesting that a previously unrecognized mechanism limits expression from the X during spermatogenesis in Drosophila. These findings help to resolve several previously conflicting reports and have implications for patterns of genome evolution and speciation in Drosophila.
Author Summary
Many species have heteromorphic sex chromosomes (XY males or ZW females) where one sex chromosome (the Y or W) has degenerated. In the somatic cells of mammals, worms, and flies, the X-to-autosome ratio of expression is equalized between the sexes by dedicated sex chromosome-specific dosage compensation systems. In the germline cells of male mammals and worms, however, the X chromosome is transcriptionally silenced early in meiosis. Here we have analyzed gene expression in Drosophila testes and show that the X chromosome lacks both of these types of chromosomal regulation. We find that X chromosome-wide dosage compensation is absent from most cells in the Drosophila male germline, and there is little or no evidence for X chromosome-specific inactivation during meiosis. However, another kind of sex-chromosome-specific regulation occurs. Testes-specific transgene reporters show much weaker expression when inserted on the X chromosome versus the autosomes, suggesting that some other, uncharacterized mechanism limits their expression from the X during spermatogenesis. The strong suppression of X-linked transgenes—but not X-linked endogenous genes—suggests that endogenous X-linked testes-specific promoters might have adapted to a suppressive X chromosome environment in the Drosophila male germline.
doi:10.1371/journal.pbio.1001126
PMCID: PMC3156688  PMID: 21857805
15.  Sex-Specific Embryonic Gene Expression in Species with Newly Evolved Sex Chromosomes 
PLoS Genetics  2014;10(2):e1004159.
Sex chromosome dosage differences between females and males are a significant form of natural genetic variation in many species. Like many species with chromosomal sex determination, Drosophila females have two X chromosomes, while males have one X and one Y. Fusions of sex chromosomes with autosomes have occurred along the lineage leading to D. pseudoobscura and D. miranda. The resulting neo-sex chromosomes are gradually evolving the properties of sex chromosomes, and neo-X chromosomes are becoming targets for the molecular mechanisms that compensate for differences in X chromosome dose between sexes. We have previously shown that D. melanogaster possess at least two dosage compensation mechanisms: the well- characterized MSL-mediated dosage compensation active in most somatic tissues, and another system active during early embryogenesis prior to the onset of MSL-mediated dosage compensation. To better understand the developmental constraints on sex chromosome gene expression and evolution, we sequenced mRNA from individual male and female embryos of D. pseudoobscura and D. miranda, from ∼0.5 to 8 hours of development. Autosomal expression levels are highly conserved between these species. But, unlike D. melanogaster, we observe a general lack of dosage compensation in D. pseudoobscura and D. miranda prior to the onset of MSL-mediated dosage compensation. Thus, either there has been a lineage-specific gain or loss in early dosage compensation mechanism(s) or increasing X chromosome dose may strain dosage compensation systems and make them less effective. The extent of female bias on the X chromosomes decreases through developmental time with the establishment of MSL-mediated dosage compensation, but may do so more slowly in D. miranda than D. pseudoobscura. These results also prompt a number of questions about whether species with more sex-linked genes have more sex-specific phenotypes, and how much transcript level variance is tolerable during critical stages of development.
Author Summary
Many animals have sex-specific combinations of chromosomes. In humans, for example, females have two X chromosomes while males have one X and one Y. In most species with XX:XY systems, the Y chromosome is degenerate and gene-poor while the X encodes a large number of functional genes. A variety of systems have evolved to ensure that males with one X chromosome and females with two X chromosomes have the same gene expression level for X-linked genes. The vinegar fly D. melanogaster has at least two dosage compensation systems: one that acts early in development, and another active in later stages. In this paper, we determine expression levels for thousands of genes in male and female embryos at different developmental stages in two species, D. pseudoobscura and D. miranda, that have unusually large fractions of their genomes in X or X-like chromosomes. We show that dosage compensation is established slowly during embryogenesis, and that in these species, dosage compensation appears to be absent in early development. This may be due to a lineage-specific loss or gain of compensation mechanism, or possibly because the machinery of dosage compensation cannot effectively handle the increased demand in these species.
doi:10.1371/journal.pgen.1004159
PMCID: PMC3923672  PMID: 24550743
16.  The role of LINEs and CpG islands in dosage compensation on the chicken Z chromosome 
Chromosome Research  2009;17(6):727-736.
Most avian Z genes are expressed more highly in ZZ males than ZW females, suggesting that chromosome-wide mechanisms of dosage compensation have not evolved. Nevertheless, a small percentage of Z genes are expressed at similar levels in males and females, an indication that a yet unidentified mechanism compensates for the sex difference in copy number. Primary DNA sequences are thought to have a role in determining chromosome gene inactivation status on the mammalian X chromosome. However, it is currently unknown whether primary DNA sequences also mediate chicken Z gene compensation status. Using a combination of chicken DNA sequences and Z gene compensation profiles of 310 genes, we explored the relationship between Z gene compensation status and primary DNA sequence features. Statistical analysis of different Z chromosomal features revealed that long interspersed nuclear elements (LINEs) and CpG islands are enriched on the Z chromosome compared with 329 other DNA features. Linear support vector machine (SVM) classifiers, using primary DNA sequences, correctly predict the Z compensation status for >60% of all Z-linked genes. CpG islands appear to be the most accurate classifier and alone can correctly predict compensation of 63% of Z genes. We also show that LINE CR1 elements are enriched 2.7-fold on the chicken Z chromosome compared with autosomes and that chicken chromosomal length is highly correlated with percentage LINE content. However, the position of LINE elements is not significantly associated with dosage compensation status of Z genes. We also find a trend for a higher proportion of CpG islands in the region of the Z chromosome with the fewest dosage-compensated genes compared with the region containing the greatest concentration of compensated genes. Comparison between chicken and platypus genomes shows that LINE elements are not enriched on sex chromosomes in platypus, indicating that LINE accumulation is not a feature of all sex chromosomes. Our results suggest that CpG islands are not randomly distributed on the Z chromosome and may influence Z gene dosage compensation status.
Electronic supplementary material
The online version of this article (doi:10.1007/s10577-009-9068-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s10577-009-9068-4
PMCID: PMC2759020  PMID: 19672682
dosage compensation; Z chromosome; DNA sequence; LINEs; CpG; chicken; sex chromosome; X chromosome
17.  Noncanonical Compensation of Zygotic X Transcription in Early Drosophila melanogaster Development Revealed through Single-Embryo RNA-Seq 
PLoS Biology  2011;9(2):e1000590.
Mmany genes from the X chromosome are expressed at the same level in female and male embryos during early Drosophila development, prior to the establishment of MSL-mediated dosage compensation, suggesting the existence of a novel mechanism.
When Drosophila melanogaster embryos initiate zygotic transcription around mitotic cycle 10, the dose-sensitive expression of specialized genes on the X chromosome triggers a sex-determination cascade that, among other things, compensates for differences in sex chromosome dose by hypertranscribing the single X chromosome in males. However, there is an approximately 1 hour delay between the onset of zygotic transcription and the establishment of canonical dosage compensation near the end of mitotic cycle 14. During this time, zygotic transcription drives segmentation, cellularization, and other important developmental events. Since many of the genes involved in these processes are on the X chromosome, we wondered whether they are transcribed at higher levels in females and whether this might lead to sex-specific early embryonic patterning. To investigate this possibility, we developed methods to precisely stage, sex, and characterize the transcriptomes of individual embryos. We measured genome-wide mRNA abundance in male and female embryos at eight timepoints, spanning mitotic cycle 10 through late cycle 14, using polymorphisms between parental lines to distinguish maternal and zygotic transcription. We found limited sex-specific zygotic transcription, with a weak tendency for genes on the X to be expressed at higher levels in females. However, transcripts derived from the single X chromosome in males were more abundant that those derived from either X chromosome in females, demonstrating that there is widespread dosage compensation prior to the activation of the canonical MSL-mediated dosage compensation system. Crucially, this new system of early zygotic dosage compensation results in nearly identical transcript levels for key X-linked developmental regulators, including giant (gt), brinker (brk), buttonhead (btd), and short gastrulation (sog), in male and female embryos.
Author Summary
Variation in gene dose can have profound effects on animal development. Yet every generation, animals must cope with differences in sex chromosome numbers. Drosophila compensate for the difference in X chromosome dosage (two in females, one in males) with a mechanism that allows for more transcription of the single X chromosome in males. But this mechanism is not established until over an hour after the embryo begins transcription, during which time a number of important events in development occur such as cellularization and segmentation. Here we use an mRNA sequencing method to characterize gene expression in individual female and male embryos before the onset of the previously characterized dosage compensation system. While we find more transcripts from X chromosomal genes in females, we also find many genes with equal transcript levels in males and females. These results indicate that there is an alternate mechanism to compensate for dosage acting earlier in development, prior to the onset of the previously characterized dosage compensation system.
doi:10.1371/journal.pbio.1000590
PMCID: PMC3035605  PMID: 21346796
18.  Rapid De Novo Evolution of X Chromosome Dosage Compensation in Silene latifolia, a Plant with Young Sex Chromosomes 
PLoS Biology  2012;10(4):e1001308.
Evidence for dosage compensation in Silene latifolia, a plant with 10-million-year-old sex chromosomes, reveals that dosage compensation can evolve rapidly in young XY systems and is not an animal-specific phenomenon.
Silene latifolia is a dioecious plant with heteromorphic sex chromosomes that have originated only ∼10 million years ago and is a promising model organism to study sex chromosome evolution in plants. Previous work suggests that S. latifolia XY chromosomes have gradually stopped recombining and the Y chromosome is undergoing degeneration as in animal sex chromosomes. However, this work has been limited by the paucity of sex-linked genes available. Here, we used 35 Gb of RNA-seq data from multiple males (XY) and females (XX) of an S. latifolia inbred line to detect sex-linked SNPs and identified more than 1,700 sex-linked contigs (with X-linked and Y-linked alleles). Analyses using known sex-linked and autosomal genes, together with simulations indicate that these newly identified sex-linked contigs are reliable. Using read numbers, we then estimated expression levels of X-linked and Y-linked alleles in males and found an overall trend of reduced expression of Y-linked alleles, consistent with a widespread ongoing degeneration of the S. latifolia Y chromosome. By comparing expression intensities of X-linked alleles in males and females, we found that X-linked allele expression increases as Y-linked allele expression decreases in males, which makes expression of sex-linked contigs similar in both sexes. This phenomenon is known as dosage compensation and has so far only been observed in evolutionary old animal sex chromosome systems. Our results suggest that dosage compensation has evolved in plants and that it can quickly evolve de novo after the origin of sex chromosomes.
Author Summary
The mammalian sex chromosomes originated from an ancestral pair of autosomes about 150 million years ago and the Y chromosome subsequently degenerated, losing most of its genes. During this process, a phenomenon called dosage compensation evolved to compensate for the gene loss on the Y chromosome and to equalize expression of X-linked genes in the two sexes. In humans, this is achieved by inactivating one of the two X chromosomes in females. Dosage compensation has also been reported in other animal XY systems such as fruit flies and worms, each 100 million years old or more. Here we studied dosage compensation in plants. We used high-throughput RNA sequencing in male and female Silene latifolia (white campion)—a dioecious plant whose XY chromosomes originated only about 10 million years ago—to identify hundreds of sex-linked genes. Analysis of their expression patterns in males and females revealed equal doses of sex-linked transcripts in both sexes, regardless of the degree of reduction of Y expression due to degeneration. Our results thus show that dosage compensation occurs in plants and is thus not an animal-specific phenomenon. They also reveal that proportionate dosage compensation can evolve rapidly de novo after the origin of sex chromosomes.
doi:10.1371/journal.pbio.1001308
PMCID: PMC3328428  PMID: 22529744
19.  Dosage Compensation of Sex Chromosome Genes in Eukaryotes  
Acta Naturae  2010;2(4):36-43.
Sex chromosome evolution is accompanied by significant divergence in morphology and gene content and results in most genes of one of the sex chromosomes being present in two dosages in one sex and in one dosage in the other. To eliminate the difference in the expression levels of these genes between sexes and to restore equal expression levels of the genes between sex chromosomes and autosomes, mechanisms of dosage compensation have appeared. Studies of three classical objects,Drosophila melanogaster,Caenorhabditis elegans, and mammals, have shown that dosage compensation of X-linked genes can be achieved through completely different chromosome-wide mechanisms. New data on sex chromosome gene expression demonstrating that many sex chromosome genes can be expressed at different levels in males and females were recently obtained from birds and butterflies. In this review, dosage compensation mechanisms inD. melanogaster,C. elegans, and mammals are considered and the data on sex chromosome gene expression in birds and butterflies, and their influence on our view of dosage compensation, are discussed.
PMCID: PMC3347590  PMID: 22649662
dosage compensation; sex chromosomes; gene expression; X-chromosome inactivation
20.  SU(VAR)3-7 Links Heterochromatin and Dosage Compensation in Drosophila 
PLoS Genetics  2008;4(5):e1000066.
In Drosophila, dosage compensation augments X chromosome-linked transcription in males relative to females. This process is achieved by the Dosage Compensation Complex (DCC), which associates specifically with the male X chromosome. We previously found that the morphology of this chromosome is sensitive to the amounts of the heterochromatin-associated protein SU(VAR)3-7. In this study, we examine the impact of change in levels of SU(VAR)3-7 on dosage compensation. We first demonstrate that the DCC makes the X chromosome a preferential target for heterochromatic markers. In addition, reduced or increased amounts of SU(VAR)3-7 result in redistribution of the DCC proteins MSL1 and MSL2, and of Histone 4 acetylation of lysine 16, indicating that a wild-type dose of SU(VAR)3-7 is required for X-restricted DCC targeting. SU(VAR)3-7 is also involved in the dosage compensated expression of the X-linked white gene. Finally, we show that absence of maternally provided SU(VAR)3-7 renders dosage compensation toxic in males, and that global amounts of heterochromatin affect viability of ectopic MSL2-expressing females. Taken together, these results bring to light a link between heterochromatin and dosage compensation.
Author Summary
In Drosophila, females have two X chromosomes and males only one. The difference in the dose of X-associated genes is compensated by male-specific protein machinery, the Dosage Compensation Complex (DCC), which augments the activity of genes of the single male X. We report that the specific targeting of the DCC on the male X chromosome depends critically on the correct dose of the SU(VAR)3-7 protein. This protein was previously known to associate with condensed and silenced regions of the chromosomes called heterochromatin by contrast with the active form of chromatin called euchromatin. Loss of SU(VAR)3-7 in males causes displacement of the DCC to heterochromatin and bloating of the X chromosome. In contrast, excess of SU(VAR)3-7 leads to a delocalization of the DCC to other chromosomes and to massive shrinking of the X chromosome. We show that SU(VAR)3-7 is involved in the dosage compensated expression of the X-linked white gene and in the viability of dosage compensated flies. Altogether, these results bring to light a link between silencing mechanisms of heterochromatin and mechanisms controlling the balance of sex-chromosome activity (dosage compensation). This opens new perspectives on how complexes that control the global chromosome organisation impact the fine tuning of gene expression.
doi:10.1371/journal.pgen.1000066
PMCID: PMC2320979  PMID: 18451980
21.  Progress and prospects toward our understanding of the evolution of dosage compensation 
Chromosome Research  2009;17(5):585-602.
In many eukaryotic organisms, gender is determined by a pair of heteromorphic sex chromosomes. Degeneration of the non-recombining Y chromosome is a general facet of sex chromosome evolution. Selective pressure to restore expression levels of X-linked genes relative to autosomes accompanies Y-chromosome degeneration, thus driving the evolution of dosage compensation mechanisms. This review focuses on evolutionary aspects of dosage compensation, in light of recent advances in comparative and functional genomics that have substantially increased our understanding of the molecular mechanisms of dosage compensation and how it evolved. We review processes involved in sex chromosome evolution, and discuss the dynamic interaction between Y degeneration and the acquisition of dosage compensation. We compare mechanisms of dosage compensation and the origin of dosage compensation genes between different taxa and comment on sex chromosomes that apparently lack compensation mechanisms. Finally, we discuss how dosage compensation systems can also influence the evolution of well-established sex chromosomes.
doi:10.1007/s10577-009-9053-y
PMCID: PMC2758192  PMID: 19626444
Dosage compensation; Sex chromosomes; Evolution; Chromatin; Epigenetics
22.  Functional Dissection of the Drosophila melanogaster Condensin Subunit Cap-G Reveals Its Exclusive Association with Condensin I 
PLoS Genetics  2013;9(4):e1003463.
The heteropentameric condensin complexes have been shown to participate in mitotic chromosome condensation and to be required for unperturbed chromatid segregation in nuclear divisions. Vertebrates have two condensin complexes, condensin I and condensin II, which contain the same structural maintenance of chromosomes (SMC) subunits SMC2 and SMC4, but differ in their composition of non–SMC subunits. While a clear biochemical and functional distinction between condensin I and condensin II has been established in vertebrates, the situation in Drosophila melanogaster is less defined. Since Drosophila lacks a clear homolog for the condensin II–specific subunit Cap-G2, the condensin I subunit Cap-G has been hypothesized to be part of both complexes. In vivo microscopy revealed that a functional Cap-G-EGFP variant shows a distinct nuclear enrichment during interphase, which is reminiscent of condensin II localization in vertebrates and contrasts with the cytoplasmic enrichment observed for the other EGFP-fused condensin I subunits. However, we show that this nuclear localization is dispensable for Cap-G chromatin association, for its assembly into the condensin I complex and, importantly, for development into a viable and fertile adult animal. Immunoprecipitation analyses and complex formation studies provide evidence that Cap-G does not associate with condensin II–specific subunits, while it can be readily detected in complexes with condensin I–specific proteins in vitro and in vivo. Mass-spectrometric analyses of proteins associated with the condensin II–specific subunit Cap-H2 not only fail to identify Cap-G but also the other known condensin II–specific homolog Cap-D3. As condensin II–specific subunits are also not found associated with SMC2, our results question the existence of a soluble condensin II complex in Drosophila.
Author Summary
The accurate duplication and segregation of chromosomes during cell divisions are prerequisites for ensuring genetic stability within an individual organism and in entire populations. Among the many components involved in regulating these processes, a protein complex called condensin plays a crucial role in shaping mitotic chromosomes, so that they can be faithfully distributed. Many organisms contain two of these condensin complexes (condensin I and II), which both have been shown to be required for accurate chromosome distribution. In the fly Drosophila melanogaster, condensin II appears to lack one of its components, called Cap-G2. We have tested the hypothesis whether the corresponding component of condensin I (Cap-G) might also participate in the assembly of condensin II. Careful analyses of complexes formed in the living organism or in the test tube argue against Cap-G being part of condensin II. Moreover, our results question the very existence of a soluble condensin II complex in flies, as opposed to other organisms. Surprisingly, a substantially truncated variant of the essential Cap-G still supports development of living and fertile flies. As this variant localizes within the cell differently from full-length Cap-G, our results show that localization of a protein does not always determine its function.
doi:10.1371/journal.pgen.1003463
PMCID: PMC3630105  PMID: 23637630
23.  The Status of Dosage Compensation in the Multiple X Chromosomes of the Platypus 
PLoS Genetics  2008;4(7):e1000140.
Dosage compensation has been thought to be a ubiquitous property of sex chromosomes that are represented differently in males and females. The expression of most X-borne genes is equalized between XX females and XY males in therian mammals (marsupials and “placentals”) by inactivating one X chromosome in female somatic cells. However, compensation seems not to be strictly required to equalize the expression of most Z-borne genes between ZZ male and ZW female birds. Whether dosage compensation operates in the third mammal lineage, the egg-laying monotremes, is of considerable interest, since the platypus has a complex sex chromosome system in which five X and five Y chromosomes share considerable genetic homology with the chicken ZW sex chromosome pair, but not with therian XY chromosomes. The assignment of genes to four platypus X chromosomes allowed us to examine X dosage compensation in this unique species. Quantitative PCR showed a range of compensation, but SNP analysis of several X-borne genes showed that both alleles are transcribed in a heterozygous female. Transcription of 14 BACs representing 19 X-borne genes was examined by RNA-FISH in female and male fibroblasts. An autosomal control gene was expressed from both alleles in nearly all nuclei, and four pseudoautosomal BACs were usually expressed from both alleles in male as well as female nuclei, showing that their Y loci are active. However, nine X-specific BACs were usually transcribed from only one allele. This suggests that while some genes on the platypus X are not dosage compensated, other genes do show some form of compensation via stochastic transcriptional inhibition, perhaps representing an ancestral system that evolved to be more tightly controlled in placental mammals such as human and mouse.
Author Summary
Dosage compensation equalizes the expression of genes found on sex chromosomes so that they are equally expressed in females and males. In placental and marsupial mammals, this is accomplished by silencing one of the two X chromosomes in female cells. In birds, dosage compensation seems not to be strictly required to balance the expression of most genes on the Z chromosome between ZZ males and ZW females. Whether dosage compensation exists in the third group of mammals, the egg-laying monotremes, is of considerable interest, particularly since the platypus has five different X and five different Y chromosomes. As part of the platypus genome project, genes have now been assigned to four of the five X chromosomes. We have shown that there is some evidence for dosage compensation, but it is variable between genes. Most interesting are our results showing that there is a difference in the probability of expression for X-specific genes, with about 50% of female cells having two active copies of an X gene while the remainder have only one. This means that, although the platypus has the variable compensation characteristic of birds, it also has some level of inactivation, which is characteristic of dosage compensation in other mammals.
doi:10.1371/journal.pgen.1000140
PMCID: PMC2453332  PMID: 18654631
24.  Faster-X Evolution of Gene Expression in Drosophila 
PLoS Genetics  2012;8(10):e1003013.
DNA sequences on X chromosomes often have a faster rate of evolution when compared to similar loci on the autosomes, and well articulated models provide reasons why the X-linked mode of inheritance may be responsible for the faster evolution of X-linked genes. We analyzed microarray and RNA–seq data collected from females and males of six Drosophila species and found that the expression levels of X-linked genes also diverge faster than autosomal gene expression, similar to the “faster-X” effect often observed in DNA sequence evolution. Faster-X evolution of gene expression was recently described in mammals, but it was limited to the evolutionary lineages shortly following the creation of the therian X chromosome. In contrast, we detect a faster-X effect along both deep lineages and those on the tips of the Drosophila phylogeny. In Drosophila males, the dosage compensation complex (DCC) binds the X chromosome, creating a unique chromatin environment that promotes the hyper-expression of X-linked genes. We find that DCC binding, chromatin environment, and breadth of expression are all predictive of the rate of gene expression evolution. In addition, estimates of the intraspecific genetic polymorphism underlying gene expression variation suggest that X-linked expression levels are not under relaxed selective constraints. We therefore hypothesize that the faster-X evolution of gene expression is the result of the adaptive fixation of beneficial mutations at X-linked loci that change expression level in cis. This adaptive faster-X evolution of gene expression is limited to genes that are narrowly expressed in a single tissue, suggesting that relaxed pleiotropic constraints permit a faster response to selection. Finally, we present a conceptional framework to explain faster-X expression evolution, and we use this framework to examine differences in the faster-X effect between Drosophila and mammals.
Author Summary
As species diverge over evolutionary time, they accumulate differences in the sequences of their genes and how those genes are expressed. We show that gene expression changes accumulate faster for genes on the X chromosome than for genes on the other chromosomes (autosomes) in Drosophila (the “faster-X” effect). The X chromosome is only found in a single copy in males, whereas the autosomes are found in two copies in both sexes. To compensate for the reduced dosage of X-linked genes in males, a molecular complex binds the Drosophila X chromosome to upregulate gene expression in males. We demonstrate that genes that escape this dosage compensation process have faster evolving expression levels. X-linked genes are inherited in a unique manner, and we hypothesize that this permits a faster rate of adaptive evolution, thereby driving the faster-X evolution of gene expression. We compare these observations with the recently described faster-X evolution of gene expression in mammals, and we explain how differences in dosage compensation, mutation rate, and population size could affect the extent of the faster-X effect.
doi:10.1371/journal.pgen.1003013
PMCID: PMC3469423  PMID: 23071459
25.  Clustered DNA motifs mark X chromosomes for repression by a dosage compensation complex 
Nature  2006;444(7119):614-618.
Gene expression in metazoans is regulated not only at the level of individual genes but also in a coordinated manner across large chromosomal domains (for example centromeres, telomeres and imprinted gene clusters1-3) and along entire chromosomes (for example X-chromosome dosage compensation4-6). The primary DNA sequence usually specifies the regulation of individual genes, but the nature of cis-acting information that controls genes over large regions has been elusive: higher-order DNA structure, specific histone modifications, subnuclear compartmentalization and primary DNA sequence are possibilities. One paradigm of chromosome-wide gene regulation is Caenorhabditis elegans dosage compensation in which a large dosage compensation complex (DCC) is targeted to both X chromosomes of hermaphrodites to repress transcript levels by half6. This essential process equalizes X-linked gene expression between the sexes (XO males and XX hermaphrodites). Here we report the discovery and dissection of cis-acting sites that mark nematode X chromosomes as targets for gene repression by the DCC. These rex (recruitment element on X) sites are widely dispersed along X and reside in promoters, exons and intergenic regions. rex sites share at least two distinct motifs that act in combination to recruit the DCC. Mutating these motifs severely reduces or abolishes DCC binding in vivo, demonstrating the importance of primary DNA sequence in chromosome-wide regulation. Unexpectedly, the motifs are not enriched on X, but altering motif numbers within rex sites demonstrates that motif co-occurrence in unusually high densities is essential for optimal DCC recruitment. Thus, X-specific repression is established through sequences not specific to X. The distribution of common motifs provides the foundation for repression along an entire chromosome.
doi:10.1038/nature05338
PMCID: PMC2693371  PMID: 17122774

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