Sex-chromosome dosage compensation
Dosage compensation is the process by which organisms equalize the expression of genes between members of different biological sexes. Across species, different sexes are often characterized by different types and numbers of sex chromosomes. In order to neutralize the large difference in gene dosage produced by differing numbers of sex chromosomes among the sexes, various evolutionary branches have acquired various methods to equalize gene expression among the sexes. Because sex chromosomes contain different numbers of genes, different species of organisms have developed different mechanisms to cope with this inequality. Replicating the actual gene is impossible; thus organisms instead equalize the expression from each gene. For example, in humans, female cells randomly silence the transcription of one X chromosome, and transcribe all information from the other, expressed X chromosome. Thus, human females have the same number of expressed X-linked genes per cell as do human males, both sexes having essentially one X chromosome per cell, from which to transcribe and express genes.
Different lineages have evolved different mechanisms to cope with the differences in gene copy numbers between the sexes that are observed on sex chromosomes. Some lineages have evolved dosage compensation, an epigenetic mechanism which restores expression of X or Z specific genes in the heterogametic sex to the same levels observed in the ancestor prior to the evolution of the sex chromosome. Other lineages equalize the expression of the X- or Z- specific genes between the sexes, but not to the ancestral levels, i.e. they possess incomplete compensation with "dosage balance". One example of this is X-inactivation which occurs in humans. The third documented type of gene dose regulatory mechanism is incomplete compensation without balance. In this system gene expression of sex-specific loci is reduced in the heterogametic sex i.e. the females in ZZ/ZW systems and males in XX/XY systems.
There are three main mechanisms of achieving dosage compensation which are widely documented in the literature and which are common to most species. These include random inactivation of one female X chromosome, a two-fold increase in the transcription of a single male X chromosome, and decreased transcription by half in both of the X chromosomes of a hermaphroditic organism. These mechanisms have been widely studied and manipulated in model organisms commonly used in the laboratory research setting. A summary of these forms of dosage compensation is illustrated below. However, there are also other less common forms of dosage compensation, which are not as widely researched and are sometimes specific to only one species.
Random inactivation of one ♀ X
One logical way to equalize gene expression amongst males and females that follow a XX/XY sex differentiation scheme would be to decrease or altogether eliminate the expression of one of the X chromosomes in an XX, or female, homogametic individual, such that both males and females then express only one X chromosome. This is the case in many mammalian organisms, including humans and mice.The evidence for this mechanism of dosage compensation was discovered prior to scientists' understanding of what its implications were. In 1949, Murray Barr and Ewert Bertram published data describing the presence of "nucleolar satellites, which they observed were present in the mature somatic tissue of different female species. Further characterization of these satellites revealed that they were actually packages of condensed heterochromatin, but a decade would pass before scientists grasped the significance of this specialized DNA.
Then, in 1959 Susumu Ohno proved that these satellite-like structures found exclusively in female cells were actually derived from female X chromosomes. He called these structures Barr bodies after one of the investigators who originally documented their existence. Ohno's studies of Barr bodies in female mammals with multiple X chromosomes revealed that such females used Barr bodies to inactivate all but one of their X chromosomes. Thus, Ohno described the "n-1" rule to predict the number of Barr bodies in a female with n number of X chromosomes in her karyotype.
Simultaneously, Mary F. Lyon began investigating manipulations of X-linked traits that had phenotypically visible consequences, particularly in mice, whose fur color is a trait intimately linked to the X chromosome. Building on work done by Ohno and his colleagues, Lyon eventually proved that either the maternal or paternal X chromosome is randomly inactivated in every cell of the female body in the species she was studying, which explained the heterogeneous fur patterns she observed in her mosaic mice. This process is known as X-inactivation, and is sometimes referred to as "lyonization". This discovery can be easily extrapolated to explain the mixed color patterns observed in the coats of tortoiseshell cats. The fur patterns characteristic of tortoiseshell cats are found almost exclusively in females, because only they randomly inactivate one X chromosome in every somatic hair cell. Thus, presuming that hair color determining genes are X-linked, it makes sense that whether the maternal or paternal X chromosome is inactivated in a particular hair cell can result in differential fur color expression.
Compounding on Lyon's discoveries, in 1962 Ernest Beutler used female fibroblast cell lineages grown in culture to demonstrate the heritability of lyonization or random X-inactivation. By analyzing the differential expression of two existing, viable alleles for the X-linked enzyme glucose-6-phosphate dehydrogenase gene, Beutler observed that the inactivation of the gene was heritable across passaged generations of the cells.
This pattern of dosage compensation, caused by random X-inactivation, is regulated across development in female mammals, following concerted patterns throughout development; for example, at the beginning of most female mammal development, both X chromosomes are initially expressed, but gradually undergo epigenetic processes to eventually achieve random inactivation of one X. In germ cells, inactivated X chromosomes are then once again activated to ensure their expression in gametes produced by female mammals.
Thus, dosage compensation in mammals is largely achieved through the silencing of one of two female X chromosomes via X-inactivation. This process involves histone tail modifications, DNA methylation patterns, and reorganization of large-scale chromatin structure encoded by the X-ist gene. In spite of these extensive modifications, not all genes along the X chromosome are subject to X-inactivation; active expression at some loci is required for homologous recombination with the pseudo-autosomal region of the Y chromosome during meiosis. Additionally, 10-25% of human X chromosome genes, and 3-7% of mouse X chromosome genes outside of the PARs show weak expression from the inactive X chromosome.
Random X-inactivation demands that the cell can determine if it contains more than one active X-chromosome before acting to silence any extraneous X-chromosome. This process is known as "counting". The exact molecular mechanism of counting is still unknown, but a popular model posits that autosomes produce factors that repress X-inactivation, while X-chromosome products that promote X-inactivation. These two conflicting forces are balanced such that if there is more than one X-chromosome X-inactivation will occur, but if there is only one, the autosomal products will successfully prevent the process.
Not all random X-inactivation is entirely random. Some alleles, generally mutations in the X-inactivation center on the X-chromosome have been demonstrated to confer a bias towards inactivation for the chromosome on which they sit. Truly random X-inactivation may also appear to be non-random if one X-chromosome carries a deleterious mutation. This can result in fewer cells which express the lower-fitness X-chromosome to be present in the body as these cells are selected against.
Two-fold increased transcription of a single ♂ X
Another mechanism common for achieving equal X-related genetic expression between males and females involves two-fold increased transcription of a single male X chromosome. Thus, heterogametic male organisms with one X chromosome may match the level of expression achieved in homogametic females with two active X chromosomes. This mechanism is observed in Drosophila.The concept of dosage compensation actually originated from an understanding of organisms in which males upregulated X-linked genes two-fold, and was much later extended to account for the observation of the once mysterious Barr bodies. As early as 1932, H.J. Muller carried out a set of experiments which allowed him to track the expression of eye color in flies, which is an X-linked gene. Muller introduced a mutant gene that caused loss of pigmentation in fly eyes, and subsequently noted that males with only one copy of the mutant gene had similar pigmentation to females with two copies of the mutant gene. This led Muller to coin the phrase "dosage compensation" to describe the observed phenomenon of gene expression equalization.
Despite these advances, it was not until Ardhendu Mukherjee and W. Beermann performed more advanced autoradiography experiments in 1965 that scientists could confirm that transcription of genes in the single male X chromosome was double that observed in the two female X chromosomes. Mukherjee and Beermann confirmed this by designing a cellular autoradiography experiment that allowed them to visualize incorporation of into ribonucleic acid of the X chromosomes. Their studies showed equal levels of incorporation in the single male X chromosome and the two female X chromosomes. Thus, the investigators concluded that the two-fold increase in the rate of RNA synthesis in the X chromosome of the male relative to those of the female could account for Muller's hypothesized dosage compensation.
In the case of two-fold increased transcription of a single male X chromosome, there is no use for a Barr body, and the male organism must use different genetic machinery to increase the transcriptional output of their single X chromosome. It is common in such organisms for the Y chromosome to be necessary for male fertility, but not for it to play an explicit role in sex determination. In Drosophila, for example, the sex lethal gene acts as a key regulator of sexual differentiation and maturation in somatic tissue; in XX animals, SXL is activated to repress increased transcription, while in XY animals SXL is inactive and allows male development to proceed via increased transcription of the single X. Several binding sites exist on the Drosophila X chromosome for the dosage compensation complex, a ribonucleoprotein complex; these binding sites have varying levels of affinity, presumably for varying expression of specific genes. The Male Specific Lethal complex, composed of protein and RNA binds and selectively modifies hundreds of X-linked genes, increasing their transcription to levels comparable to female D. melanogaster.
In organisms that use this method of dosage compensation, the presence of one or more X chromosomes must be detected early on in development, as failure to initiate the appropriate dosage compensation mechanisms is lethal. Male specific lethal proteins are a family of four proteins that bind to the X chromosome exclusively in males. The name "MSL" is used because mutations in these genes cause inability to effectively upregulate X-linked genes appropriately, and are thus lethal to males only and not their female counterparts. SXL regulates pre-messenger RNA in males to differentially splice MSLs and result in the appropriate increase in X chromosome transcription observed in male Drosophila. The immediate target of SXL is male specific lethal-2. Current dogma suggests that the binding of MSL-2 at multiple sites along the SXL gene in females prevents proper MSL-2 translation, and thus, as previously stated, represses the possibility for X-linked genetic upregulation in females. However, all other transcription factors in the MSL family—maleless, MSL-1, and MSL-3—are able to act when SXL is not expressed, as in the case in males. These factors act to increase male X chromosome transcriptional activity. Histone acetylation and the consequent upregulation of X-linked genes in males is dictated by the MSL complex. Specifically, special roX non-coding RNAs on the MSL complexes facilitate binding to the single male X chromosome, and dictate acetylation of specific loci along the X chromosome as well as the formation of euchromatin. Though these RNAs bind at specific sites along the male X chromosome, their effects spread along the length of the chromosome and have the ability to influence large-scale chromatin modifications. The implications of this spreading epigenetic regulation along the male X chromosome is thought to have implications for understanding the transfer of epigenetic activity along long genomic stretches.