Evolution of Sex Chromosome Dosage Compensation in Animals: A Beautiful Theory, Undermined by Facts and Bedeviled by Details

Many animals with genetic sex determination harbor heteromorphic sex chromosomes, where the heterogametic sex has half the gene dose of the homogametic sex. This imbalance, if reflected in the abundance of transcripts or proteins, has the potential to deleteriously disrupt interactions between X-linked and autosomal loci in the heterogametic sex. Classical theory predicts that molecular mechanisms will evolve to provide dosage compensation that recovers expression levels comparable to ancestral expression prior to sex chromosome divergence. Such dosage compensating mechanisms may also, secondarily, result in balanced sex-linked gene expression between males and females. However, numerous recent studies addressing sex chromosome dosage compensation (SCDC) in a diversity of animals have yielded a surprising array of patterns concerning dosage compensation in the heterogametic sex, as well as dosage balance between sexes. These results substantially contradict longstanding theory, catalyzing both novel perspectives and new approaches in dosage compensation research. In this review, we summarize the theory, analytical approaches, and recent results concerning evolutionary patterns of SCDC in animals. We also discuss methodological challenges and discrepancies encountered in this research, which often underlie conflicting results. Finally, we discuss what outstanding questions and opportunities exist for future research on SCDC

Chromosome-specific adaptations of RNA stability and the roles of the roX RNAs in dosage compensation

Sex chromosomes evolved from an ancient pair of autosomes and the Y chromosome lost most of its genetic information in the process. This created two kinds of genomic imbalances: the first one between males (XY) and females (XX) and the second one between the sex chromosomes and the autosomes (X:AA). In mammals, the male:female dosage compensation is achieved through the random inactivation of one of the two female X chromosomes. Through genome-wide studies of RNA stability, we show that one of the strategies used for the X:AA dosage compensation is to specifically increase the RNA stability of its X chromosome transcripts in both sexes. We also observe an increase in ribosome density on the X chromosome´s transcripts and propose that a large part of dosage compensation in mammals happens at the translational level. In D. melanogaster (fruit flies), dosage compensation is achieved through a two-fold upregulation of transcription from the male X chromosome. This solves the male:female and the X:AA imbalance at once. We did not find any evidence for RNA stability having a role in fly dosage compensation. However, our data allowed us to propose two new RNA stability mediated mechanisms for the general regulation of gene expression. The first one is a buffering mechanism that responds to detrimental changes in transcription by increasing RNA stability upon decrease in transcription and vice versa. The second mechanism enhances the adapted differential transcription between the sexes by shifting RNA stability accordingly.            The MSL complex is a nucleoprotein complex composed of at least 5 proteins and two non-coding RNAs (roX1 and roX2). It is only assembled in males and specifically targets their X chromosome, promoting upregulation of transcription. Each and every protein is essential for male viability, but each roX RNA can be deleted without exhibiting any phenotype. However, the deletion of both also kills males specifically. Despite this redundancy, the roX RNAs have been shown to be expressed at different times during development and they differ in size and sequence. We analyzed the differential expression in roX1, roX2 and roX1 roX2 double mutants in regard to distance to high affinity binding sites of the MSL complex, MSL binding strength and replication timing and showed that the roX RNAs fulfill separate functions in dosage compensation.  We also discovered and characterized two ectopic female specific high affinity binding sites for the protein POF (painting of fourth) which specifically targets and upregulates the transcription from the fourth chromosome of D. melanogaster. We named these sites PoX1 and PoX2 because they are situated in the vicinity of roX1 and roX2 loci and we postulate that they constitute molecular evolutionary links between dosage compensation and the autosome specific gene regulation of the fourth chromosome

Chromosome-specific adaptations of RNA stability and the roles of the roX RNAs in dosage compensation

Sex chromosomes evolved from an ancient pair of autosomes and the Y chromosome lost most of its genetic information in the process. This created two kinds of genomic imbalances: the first one between males (XY) and females (XX) and the second one between the sex chromosomes and the autosomes (X:AA). In mammals, the male:female dosage compensation is achieved through the random inactivation of one of the two female X chromosomes. Through genome-wide studies of RNA stability, we show that one of the strategies used for the X:AA dosage compensation is to specifically increase the RNA stability of its X chromosome transcripts in both sexes. We also observe an increase in ribosome density on the X chromosome´s transcripts and propose that a large part of dosage compensation in mammals happens at the translational level. In D. melanogaster (fruit flies), dosage compensation is achieved through a two-fold upregulation of transcription from the male X chromosome. This solves the male:female and the X:AA imbalance at once. We did not find any evidence for RNA stability having a role in fly dosage compensation. However, our data allowed us to propose two new RNA stability mediated mechanisms for the general regulation of gene expression. The first one is a buffering mechanism that responds to detrimental changes in transcription by increasing RNA stability upon decrease in transcription and vice versa. The second mechanism enhances the adapted differential transcription between the sexes by shifting RNA stability accordingly.            The MSL complex is a nucleoprotein complex composed of at least 5 proteins and two non-coding RNAs (roX1 and roX2). It is only assembled in males and specifically targets their X chromosome, promoting upregulation of transcription. Each and every protein is essential for male viability, but each roX RNA can be deleted without exhibiting any phenotype. However, the deletion of both also kills males specifically. Despite this redundancy, the roX RNAs have been shown to be expressed at different times during development and they differ in size and sequence. We analyzed the differential expression in roX1, roX2 and roX1 roX2 double mutants in regard to distance to high affinity binding sites of the MSL complex, MSL binding strength and replication timing and showed that the roX RNAs fulfill separate functions in dosage compensation.  We also discovered and characterized two ectopic female specific high affinity binding sites for the protein POF (painting of fourth) which specifically targets and upregulates the transcription from the fourth chromosome of D. melanogaster. We named these sites PoX1 and PoX2 because they are situated in the vicinity of roX1 and roX2 loci and we postulate that they constitute molecular evolutionary links between dosage compensation and the autosome specific gene regulation of the fourth chromosome

Increased expression of X-linked genes in mammals is associated with a higher stability of transcripts and an increased ribosome density

Mammalian sex chromosomes evolved from the degeneration of one homolog of a pair of ancestral autosomes, the proto-Y. This resulted in a gene dose imbalance that is believed to be restored (partially or fully) through up-regulation of gene expression from the single active X-chromosome in both sexes by a dosage compensatory mechanism. We analyzed multiple genome-wide RNA stability datasets and found significantly longer average half-lives for X-chromosome transcripts than for autosomal transcripts in various human cell lines, both male and female, and in mice. Analysis of ribosome profiling data shows that ribosome density is higher on X-chromosome transcripts than on autosomal transcripts in both humans and mice, suggesting that the higher stability is causally linked to a higher translation rate. Our results and observations are in accordance with a dosage compensatory upregulation of expressed X-linked genes. We therefore propose that differential mRNA stability and translation rates of the autosomes and sex chromosomes contribute to an evolutionarily conserved dosage compensation mechanism in mammals

Increased expression of X-linked genes in mammals is associated with a higher stability of transcripts and an increased ribosome density

Mammalian sex chromosomes evolved from the degeneration of one homolog of a pair of ancestral autosomes, the proto-Y. This resulted in a gene dose imbalance that is believed to be restored (partially or fully) through up-regulation of gene expression from the single active X-chromosome in both sexes by a dosage compensatory mechanism. We analyzed multiple genome-wide RNA stability datasets and found significantly longer average half-lives for X-chromosome transcripts than for autosomal transcripts in various human cell lines, both male and female, and in mice. Analysis of ribosome profiling data shows that ribosome density is higher on X-chromosome transcripts than on autosomal transcripts in both humans and mice, suggesting that the higher stability is causally linked to a higher translation rate. Our results and observations are in accordance with a dosage compensatory upregulation of expressed X-linked genes. We therefore propose that differential mRNA stability and translation rates of the autosomes and sex chromosomes contribute to an evolutionarily conserved dosage compensation mechanism in mammals

RNA-on-X 1 and 2 in Drosophila melanogaster fulfill separate functions in dosage compensation

In Drosophila melanogaster, the male-specific lethal (MSL) complex plays a key role in dosage compensation by stimulating expression of male X-chromosome genes. It consists of MSL proteins and two long noncoding RNAs, roX1 and roX2, that are required for spreading of the complex on the chromosome and are redundant in the sense that loss of either does not affect male viability. However, despite rapid evolution, both roX species are present in diverse Drosophilidae species, raising doubts about their full functional redundancy. Thus, we have investigated consequences of deleting roX1 and/or roX2 to probe their specific roles and redundancies in D. melanogaster. We have created a new mutant allele of roX2 and show that roX1 and roX2 have partly separable functions in dosage compensation. In larvae, roX1 is the most abundant variant and the only variant present in the MSL complex when the complex is transmitted (physically associated with the X-chromosome) in mitosis. Loss of roX1 results in reduced expression of the genes on the X-chromosome, while loss of roX2 leads to MSL-independent upregulation of genes with male-biased testis-specific transcription. In roX1 roX2mutant, gene expression is strongly reduced in a manner that is not related to proximity to high-affinity sites. Our results suggest that high tolerance of mis-expression of the X-chromosome has evolved. We propose that this may be a common property of sex-chromosomes, that dosage compensation is a stochastic process and its precision for each individual gene is regulated by the density of high-affinity sites in the locus

Modulation of RNA stability regulates gene expression in two opposite ways : through buffering of RNA levels upon global perturbations and by supporting adapted differential expression

The steady state levels of RNAs, often referred to as expression levels, result from a well-balanced combination of RNA transcription and decay. Alterations in RNA levels will therefore result from tight regulation of transcription rates, decay rates or both. Here, we explore the role of RNA stability in achieving balanced gene expression and present genome-wide RNA stabilities in Drosophila melanogaster male and female cells as well as male cells depleted of proteins essential for dosage compensation. We identify two distinct RNA-stability mediated responses involved in regulation of gene expression. The first of these responds to acute and global changes in transcription and thus counteracts potentially harmful gene mis-expression by shifting the RNA stability in the direction opposite to the transcriptional change. The second response enhances inter-individual differential gene expression by adjusting the RNA stability in the same direction as a transcriptional change. Both mechanisms are global, act on housekeeping as well as non-housekeeping genes and were observed in both flies and mammals. Additionally, we show that, in contrast to mammals, modulation of RNA stability does not detectably contribute to dosage compensation of the sex-chromosomes in D. melanogaster

Targeting of painting of fourth to roX1 and roX2 proximal sites suggests evolutionary links between dosage compensation and the regulation of the 4th chromosome in Drosophila melanogaster

In Drosophila melanogaster, two chromosome-specific targeting and regulatory systems have been described. The male-specific lethal (MSL) complex supports dosage compensation by stimulating gene expression from the male X-chromosome and the protein Painting of fourth (POF) specifically targets and stimulates expression from the heterochromatic 4(th) chromosome. The targeting sites of both systems are well characterized, but the principles underlying the targeting mechanisms have remained elusive. Here we present an original observation, namely that POF specifically targets two loci on the X-chromosome, PoX1 and PoX2 (POF-on-X). PoX1 and PoX2 are located close to the roX1 and roX2 genes, which encode ncRNAs important for the correct targeting and spreading of the MSL-complex. We also found that the targeting of POF to PoX1 and PoX2 is largely dependent on roX expression and identified a high-affinity target region which ectopically recruits POF. The results presented support a model linking the MSL-complex to POF and dosage compensation to regulation of heterochromatin