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Title: Dosage Regulation of the Active X Chromosome in Human Triploid Cells}
Article:
Abstract:
In mammals, dosage compensation is achieved by doubling expression of X-linked genes in both sexes, together with X inactivation in females. Up-regulation of the active X chromosome may be controlled by DNA sequence–based and/or epigenetic mechanisms that double the X output potentially in response to autosomal factor( s). To determine whether X expression is adjusted depending on ploidy, we used expression arrays to compare X-linked and autosomal gene expression in human triploid cells. While the average X:autosome expression ratio was about 1 in normal diploid cells, this ratio was lower( 0. 81–0. 84) in triploid cells with one active X and higher( 1. 32–1. 4) in triploid cells with two active X's. Thus, overall X-linked gene expression in triploid cells does not strictly respond to an autosomal factor, nor is it adjusted to achieve a perfect balance. The unbalanced X:autosome expression ratios that we observed could contribute to the abnormal phenotypes associated with triploidy. Absolute autosomal expression levels per gene copy were similar in triploid versus diploid cells, indicating no apparent global effect on autosomal expression. In triploid cells with two active X's our data support a basic doubling of X-linked gene expression. However, in triploid cells with a single active X, X-linked gene expression is adjusted upward presumably by an epigenetic mechanism that senses the ratio between the number of active X chromosomes and autosomal sets. Such a mechanism may act on a subset of genes whose expression dosage in relation to autosomal expression may be critical. Indeed, we found that there was a range of individual X-linked gene expression in relation to ploidy and that a small subset( ∼7%) of genes had expression levels apparently proportional to the number of autosomal sets.
Introduction:
Dosage compensation restores a balanced network of gene expression between autosomes and sex chromosomes in males( XY) and females( XX)[1]. Strategies to achieve this vary among species[2]. In Drosophila, a male-specific ribonucleoprotein complex binds to the X chromosome and modifies chromatin structure to increase expression of most X-linked genes by two fold. X up-regulation also occurs in C. elegans and in mammals but in both sexes[3],[4]. Silencing of one X chromosome in mammalian females and repression of both X chromosomes in C. elegans hermaphrodites have been adapted to avoid hyper-expression in the homogametic sex. While these repressive processes have been well studied, the mechanisms of X up-regulation in mammals and worms remain to be determined. Human triploids occur in about 1% of conceptions. Depending on their sex chromosome composition( XXX, XXY, XYY), triploid cells show a variety of X inactivation patterns[5]–[8]. Female triploid( XXX) fibroblast clones with either one or two active X chromosomes( Xa) have been successfully established[5]. These cloned lines with a defined number of Xa's as well as XYY triploid cell cultures provide a mean to test X chromosome expression in the presence of three sets of autosomes, which could help understand the underlying mechanisms of X up-regulation and test the potential existence of an autosomal counting factor. If DNA sequence changes that affect promoters, enhancers, and/or regions that alter mRNA elongation and stability were sufficient to mediate the two-fold elevated expression of X-linked genes in normal diploid cells, we would predict that the X:autosome( X:A) expression ratio would be 0. 67( i. e. 2/3) in triploid cells with a single Xa and 1. 33 with two Xa's, assuming that autosomal genes have a steady expression level per gene copy and thus produce approximately 1. 5-fold more products in triploid versus diploid cells. A second possibility is that epigenetic chromatin remodeling of the X chromosome and/or of the autosomes may modulate the balance of gene expression, in which case the X:A expression ratio in triploid cells with one Xa or two Xa's would differ from 0. 67 and 1. 33, respectively. If this epigenetic regulation of the active X chromosome in triploid cells was based on a counting mechanism mediated by autosomal factor( s) that would triple the output from the Xa, the predicted X:A expression ratio in triploid cells with one Xa would be 1( i. e. 3/3), similar to the situation in diploid cells[4], whereas triploid cells with two Xa's would have a ratio of 2( i. e. 6/3). Alternatively, both genetic and epigenetic mechanisms could be involved in regulation of X up-regulation, in which case the predicted ratios would be further adjusted. A perfect adjustment to a ratio of 1 could possibly be observed if X-linked and/or autosomal gene expression was adjusted in response to the number of active X chromosomes in relation to autosomes. Studies in Drosophila have shown that both X-linked and autosomal gene expression can respond to abnormal genotypes[9]. In the present study, we used expression array analyses to determine the global expression of X-linked genes versus autosomal genes in triploid cell cultures with either one or two Xa's. We found no evidence of a global change in absolute autosomal expression levels per gene copy in triploid versus diploid cells. Our results are consistent with a doubling of expression of most X-linked genes in triploid cells with two Xa's. However, X expression was further increased in triploid cells with one Xa, suggesting an epigenetic adjustment in response to the Xa/autosome ratio. The expression of genes with copies on both sex chromosomes and of genes that escape X inactivation was investigated in triploid cells. We determined that XYY triploid cells had a expression deficiency in genes that escape X inactivation compared to XXX triploid cells. We also found that expression of a small subset of X-linked genes apparently responds to the cell ploidy, suggesting that dosage of these genes is critical.
Discussion:
Our results indicate that X-linked gene expression is up-regulated approximately two-fold in triploid cultures with two active X chromosomes like in diploid cultures[3],[4]. However, in triploid cultures with a single active X, X expression is apparently adjusted further upward, although not tripled. Thus, global X-linked gene expression does not strictly respond to an autosomal counting factor in triploid cells. However, comparisons between triploid and diploid cultures showed a range of individual gene expression, suggesting that not all genes respond to a change in ploidy in the same manner. We previously proposed two mechanisms for X up-regulation[4]:( 1) permanent DNA sequence changes during evolution that may affect promoters, enhancers, and/or regions that alter mRNA elongation and stability;( 2) epigenetic mechanisms that may control X expression similar to the situation in Drosophila[1]. A basic doubling of X expression in triploid cells with two Xa's could be mediated by DNA-sequence modifications and/or by epigenetic mechanisms that normally operate in diploid cells. The notion that the doubling cannot default even when X expression is too high in cells with two active X chromosomes strongly suggests that DNA sequence changes or highly stable epigenetic modifications are involved in X up-regulation. We found no evidence of a global change in absolute expression level per autosomal gene copy in triploid versus diploid cultures, suggesting that gene expression is strictly proportional to the number of autosomal sets. Hence, adjustments in gene expression in triploid cultures mostly affect X-linked genes. In triploid cells with one Xa, X expression is adjusted upward from a basic doubling. This further adjustment is presumably mediated by an epigenetic mechanism that senses X expression but does not respond to the simple X:A genomic ratio since this occurs both in XYY and XaXiXi cultures. Our data suggest that such a mechanism may act on a subset of genes( ∼7% of X-linked genes) whose expression dosage in relation to autosomal expression may be critical. Preliminary investigation in their function did not reveal a specific role for these genes. It is also possible that the triploid cultures with one Xa we examined survived because X expression was adjusted upward, thus reflecting a bias of ascertainment. Interestingly, a previous study in Drosophila showed that the normalized enzymatic activity of a protein encoded by an X-linked gene was 0. 87 in triploid flies with a single X relative to diploid flies, which suggests a more than doubled, but not tripled expression, similar to our results in human[20]. Previous studies have suggested that fold-changes in expression are often considerably less than differences in gene copy number[3]. It should be noted that we measured steady-state RNA and thus cannot rule out adjustments in protein levels at translation. We previously reported low X:A expression ratios( 0. 84–0. 89) in a subset of normal human diploid tissues[4], suggesting that ratios within this low range could be tolerated in a tissue-specific manner. Conversely, undifferentiated mouse female ES cells with two Xa's have an X:A expression ratio of 1. 39[21], indicating that such a high ratio is tolerated in normal diploid cells during a short time window prior to X inactivation in blastocysts. However, later stage mouse embryos with two Xa's due to failure of X inactivation have a severely abnormal phenotype similar to that of embryos with tetrasomy for an autosome[22]. A previous study of female mouse ES cells with two active X chromosomes showed genome-wide DNA methylation changes in repeated DNA sequences, perhaps in response to the high X expression[23]. However, in our study we did not detect an adjustment in global autosomal expression in triploid cells. Triploidy, which predominantly results from dispermy, causes lethality in human embryos due to multiple phenotypic abnormalities[8]. The unbalanced X:A expression ratios that we observed could contribute to this lethality. XYY fetuses are much rarer than XXY or XXX fetuses, in which the majority of cells often have two Xa's[5],[24]. We found that XYY cultures had a deficiency in expression of genes that escape X inactivation compared to XaXiXi cultures, which could contribute to the early lethality of XYY triploid fetuses. Relevant to this observation is the prevalent lethality in Turner syndrome fetuses with a single X chromosome, associated with haplo-insufficiency of genes that escape X inactivation. In XYY triploid fetus, Y paralog expression could partially compensate, provided that the function of the X and Y paralogs are similar. Deficiency in gene expression due to chromosomal abnormalities such as monosomy is less well tolerated than trisomy. Thus, lower than normal X expression may be less compatible with embryonic development than higher expression. A recent study of tetraploid mouse ES cells with a different number of Xa's also supports the notion that an excess of Xa's is more favorable to cell survival than a deficiency[25]. Live-born triploid fetuses have a mixture of cells with one or two Xa's, while early aborted fetuses tend to have a majority of cells with two Xa's[5],[24]. Interestingly, the uncloned culture we studied that consisted of a mixture of cells with one or two Xa's had a balanced overall X:A expression ratio close to 1. 0. Thus, mosaicism for cells with different X inactivation patterns may help achieve a more balanced proteome, excluding cell-autonomous protein products. Mechanisms involved in X up-regulation in mammals will need to be investigated at early embryonic stages to follow changes in the level of X-linked gene expression during normal development.
Many organisms have a single X chromosome in males and two in females , leading to a chromosome imbalance between autosomes and sex chromosomes and between the sexes . In mammals , this dosage imbalance is adjusted by doubling expression of X-linked genes in both sexes and by silencing one X chromosome in females . We used expression array analyses of human triploid cultures to test X chromosome expression in the presence of three sets of autosomes and address the question of an autosomal counting factor . We found that overall X-linked gene expression is not tripled in the presence of three sets of autosomes . However , in triploid cells with a single active X chromosome , its expression is adjusted upward presumably by an epigenetic mechanism that senses the active X/autosome ratio . Based on the range of individual gene expression we identified a subset of dosage-sensitive genes whose expression is apparently proportional to the ploidy . Our findings are important for understanding the regulation of the X chromosome and the role of ploidy in the balance of gene expression and associated phenotypes .