Determination of dosage compensation of the mammalian X chromosome by RNA-seq is dependent on analytical approach

Nathaniel K Jue 0 Michael B Murphy 0 Seth D Kasowitz Sohaib M Qureshi Craig J Obergfell Sahar Elsisi Robert J Foley Rachel J O'Neill Michael J O'Neill 0 Equal contributors Department of Molecular and Cell Biology, University of Connecticut , 354 Mansfield Rd. U-2131, Storrs, CT 06235 , USA Background: An enduring question surrounding sex chromosome evolution is whether effective hemizygosity in the heterogametic sex leads inevitably to dosage compensation of sex-linked genes, and whether this compensation has been observed in a variety of organisms. Incongruence in the conclusions reached in some recent reports has been attributed to different high-throughput approaches to transcriptome analysis. However, recent reports each utilizing RNA-seq to gauge X-linked gene expression relative to autosomal gene expression also arrived at diametrically opposed conclusions regarding X chromosome dosage compensation in mammals. Results: Here we analyze RNA-seq data from X-monosomic female human and mouse tissues, which are uncomplicated by genes that escape X-inactivation, as well as published RNA-seq data to describe relative X expression (RXE). We find that the determination of RXE is highly dependent upon a variety of computational, statistical and biological assumptions underlying RNA-seq analysis. Parameters implemented in short-read mapping programs, choice of reference genome annotation, expression data distribution, tissue source for RNA and RNA-seq library construction method have profound effects on comparing expression levels across chromosomes. Conclusions: Our analysis shows that the high number of paralogous gene families on the mammalian X chromosome relative to autosomes contributes to the ambiguity in RXE calculations, RNA-seq analysis that takes into account that single- and multi-copy genes are compensated differently supports the conclusion that, in many somatic tissues, the mammalian X is up-regulated compared to the autosomes. - Background Chromosome-based sex determination systems are most often characterized by heterotypic sex chromosomes, with one sex carrying at least one degenerate homolog [1-3]. Heterokaryotypy may result from differential gene loss or gain as the sex chromosome complement evolves from an ancestral homologous pair. Depending on the extent of the loss or gain, and the dosage sensitivity of genes on the incipient sex chromosomes, natural selection may favor the evolution of compensating mechanisms to balance expression between the sexes and between the sex chromosomes and autosomes. This can be accomplished either by up-regulating expression of sex-linked genes in the heterogametic sex or by down-regulating expression in the homogametic sex in relation to the autosomes. In Drosophila [4] and Sciara [5], genes on the single X chromosome in males are transcriptionally up-regulated, while in the nematode worm, Caenorhabditis elegans, the two X chromosomes in hermaphrodites are down-regulated to equal that of the XO males [6]. In contrast, for organisms displaying female heterogamety, such as birds, evidence of sex chromosome dosage compensation is lacking [7-10]. The differences in compensating mechanisms, or lack thereof, will likely reflect the relative content of haplosufficient vs. haploinsufficient genes on the sex chromosomes, but will also reflect early events of sex chromosome evolution, outcomes of sexual selection and sexual conflict, and the life history of the organism [11]. In eutherian mammals and marsupials, sex chromosome dosage compensation is achieved by global inactivation of one of the two X chromosomes in females. X chromosome inactivation (XCI) in eutherians is initiated by the expression of the XIST noncoding RNA just prior to implantation of the embryo, leading to heterochromatinization of one of either parental X chromosome in the fetus [12]. X-inactivation in marsupials also involves heterochromatinization of one X, governed by a non-coding RNA, RSX, with XIST-like properties, but the paternal X is exclusively chosen for inactivation [13,14]. Halving the apparent dosage of X-linked genes in female mammals via XCI presents an evolutionary conundrum: if sex chromosomes evolve from an ancestral autosomal pair, it is the heterogametic sex that would be impelled to compensate for the complete loss or degradation of the evolving Y. In other words, since female mammals never receive a Y chromosome, it is difficult to see how loss of gene dosage from the evolving Y would have any influence on regulation of X genes in females. The simplest compensating step in response to attritional gene loss from the incipient Y would be cis-regulatory change or cis-gene duplication, i.e. genetic mutation, of genes on the X. In Drosophila, a male-specific epigenetic mechanism of dosage compensation spares the homogametic female a potentially detrimental up-regulation of X-linked genes. If, however, compensation is achieved by genetic mutation, selection would favor epigenetic down regulation in females. Ohno recognized this and hypothesized that down-regulation of X-linked genes might evolve in response to regulatory changes to the X that are transmitted from father to daughter [15]. This would appear to be the scenario played out in C. elegans and mammals. Regardless of the eventual dosage compensation mechanism settled upon, the first step in compensating for gradual haploinsufficient gene loss on the Y must be an increase in transcription of surviving genes on the X in males. Ohnos hypothesis appeared to be borne out in three recent reports [16-18], which each showed by microarray-based transcriptome analysis that the single active X chromosome in both males and females in several eutherian species was expressed at or near a 1:1 ratio to the averaged expression of the diploid autosomal complement, termed the X:A ratio. However, this work was called into question by He and colleagues [19] who, through analysis of high throughput transcriptome sequence (RNA-seq) data from various tissues from human and mouse, concluded that the X:A ratio of gene expression was closer to 0.5, indicative of a lack of X-linked gene up-regulation. Xiong et al. report that the former studies were compromised by apparent compression of expression differences; a factor they argue is inherent to microarray expression analysis. Recently, Disteche and colleagues published a report re-analyzing RNA-seq data from [19] as well as new RNA-seq data from human cells and tissues and arrive at the conclusion that the mammalian X chromosome is upregulated in relation to autosomes [20]. Additionally, three other studies [21-23] following on the heels of [20] also report up-regulation of the mammalian X. However, in reply to these reports, He and colleagues maintain their conclusion that Ohnos hypothesis is invalid [24]. The widely divergent conclusions, i.e. compensation vs. no compensation, of these studies highlight the dramatic differences in biological conclusio (...truncated)