Dosage Compensation and the Distribution of Sex-Biased Gene Expression in Drosophila: Considerations and Genomic Constraints
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Several studies in Drosophila have shown a paucity of male-biased genes (i.e., genes that express higher in males than in females) on the X chromosome. Dosage compensation (DC) is a regulatory mechanism of gene expression triggered in males that hypertranscribes the X-linked genes to the level of transcription in females. There are currently two different hypotheses about the effects of DC on the distribution of male-biased genes: (1) it might limit male-expression level, or (2) it might interfere with the male upregulation of gene expression. Here, we used previously published gene expression datasets to reevaluate both hypotheses and introduce a mutually exclusive prediction that helped us to reject the hypothesis that the paucity of male-biased genes in the X chromosome is due to a limit in the male-expression level. Our analysis also uncovers unanticipated details about how DC interferes with the genomic distribution of both, male-biased and female-biased genes. We suggest that DC actually interferes with female downregulation of gene expression and not male upregulation, as previously suggested.
KeywordsDosage compensation X chromosome Sex-biased gene expression Drosophila
In Drosophila (fruit flies), females carry two X chromosomes (XX) while males carry one X chromosome and one Y chromosome (XY). Because the Y chromosome is highly degenerated and nearly devoid of genes (Carvalho et al. 2009), males require a molecular system to compensate the hemizygosity at the X chromosome (Baker et al. 1994). Such a regulatory system of gene expression is known as dosage compensation (DC). DC is achieved in Drosophila males by a set of chromatin modifications on the X chromosome that enhance the processivity of the RNA polymerase during the transcription of the X-linked genes (Lucchesi et al. 2005; Larschan et al. 2011). These modifications are triggered by a ribonucleoprotein complex known as dosage compensation complex (DCC). How DC may affect the genome-wide distribution of male-biased genes is a matter of debate (Vicoso and Charlesworth 2006, 2009; Bachtrog et al. 2010; Vensko and Stone 2014; Huylmans and Parsch 2015).
The differential expression of genes between males and females is known as sex-biased gene expression. There are different ways for a gene to achieve sex-biased expression from an unbiased ancestral state. However, it has been shown that most male-biased gene expression that originated in the D. melanogaster lineage occurred by upregulation of gene expression in males (Connallon and Knowles 2005; Vicoso and Charlesworth 2009; Gallach and Betran, under review). According to some studies, the upregulation of gene expression in males as the main mechanism to evolve male-biased gene expression would be incompatible with DC (Vicoso and Charlesworth 2009; Bachtrog et al. 2010). Such a conflict would explain the paucity of male-biased genes on the X chromosome of Drosophila (a. k. a. demasculinization of the X chromosome; Parisi et al. 2003; Ranz et al. 2003; Zhang et al. 2007; Meisel et al. 2012; but see Meiklejohn and Presgraves 2012). Because DC is an X-specific phenomenon occurring in males, two hypotheses have been suggested about the influence of DC on the distribution of male-biased genes (Vicoso and Charlesworth 2009; Bachtrog et al. 2010). A DC hypothesis of the distribution of the male-biased genes was first suggested by Vicoso and Charlesworth (2006, 2009). The authors suggest that if there is a limit in gene expression level that can be attained, dosage compensated genes in males will be closer to such a limit than non-dosage compensated genes. Therefore, evolving male-biased gene expression by means of an increase in transcription rate should be harder for X-linked genes than for autosomal genes. We will refer to this hypothesis as the “DC limiting hypothesis.” An alternative hypothesis suggests that there is a direct “interference” of chromatin remodeling complexes and the DCC on the X chromosome, which impedes upregulation in males beyond that induced by DC (Bachtrog et al. 2010). We will refer to this hypothesis as the “DC interference hypothesis.” The DC limiting hypothesis predicts that there will be a deficit of male-biased genes on the X chromosome compared to the autosomes, and this deficit will be stronger for genes with high expression than for genes with low expression. The DC interference hypothesis predicts that male-biased genes will be scarce in regions bound by the DCC compared to unbound regions, regardless of the expression level. These predictions have not been tested yet.
In an attempt to better understand the constraints that DC imposes on changes in gene expression, we tested whether the distribution of male-biased genes across bound and unbound regions depends on the gene expression level. We also uncover unanticipated details about how DC interferes with the genomic distribution of both, male-biased and female-biased genes.
Materials and Methods
We used the data from Bachtrog et al. (2010) as the main database for our analyses. The database included two gene expression profiles from whole adult (Parisi et al. 2003; Zhang et al. 2007), one from fly gonads (Parisi et al. 2003) and one from gonadectomized flies (Parisi et al. 2003), as well as the chromosomal coordinates of the DCC binding regions (D. melanogaster release 5.5). These four gene expression profiles were obtained using microarrays and the expression level measured as hybridization signal intensity. The DCC binding regions were previously identified from high-resolution ChIP-chip mapping data in MSL3 mutant male embryos. According to the original study, a gene was classified as “bound” if it overlaps with a DCC binding region and as “unbound” otherwise (Alekseyenko et al. 2006). High affinity sites for the DCC (Alekseyenko et al. 2008) were not considered in this study. We also included in our analysis RNA-sequencing (RNA-Seq) data from two recent studies, which measured gene expression in whole adult as reads per kilobase per million reads (RPKM; Daines et al. 2011) and fragments per kilobase per million reads (FPKM; Graveley et al. 2011). The RNA-Seq data were integrated in our database based on the FlyBase identifier (FBgn number) associated with each gene. We classified genes as male-biased and female-biased when the differences in expression level between males and females were significant at a false discovery rate of 5 %, as computed in the original studies. Otherwise, genes were classified as unbiased.
Data manipulation and statistical analysis were performed in R (http://www.r-project.org).
DC Interferes with the Genomic Distribution of Male-Biased and Female-Biased Genes
Typical chromatin modifications associated with DC, as well as chromatin modifier proteins interacting with the DCC in males, are also enriched on the X chromosome in females, indicating that the chromatin structure of the X chromosomes also differs from the autosomes in this sex (Jin et al. 2000; Kind et al. 2008; Zhang and Oliver 2010; Sala et al. 2011; Brown and Bachtrog 2014). This prompted us to investigate whether DC also “interferes” with the distribution of female-biased genes. To do so, we classified female-biased genes into three groups of equal size according to their expression level, as we did for male-biased genes. Interestingly, our analysis reveals that female-biased genes are significantly enriched in bound regions, regardless of their expression level (X 2 = 162.17; df = 5; P = 3.42 × 10−33; Fig. 1b). Therefore, DC seems to interfere with the distribution of female-biased genes, but in the opposite direction of that found for male-biased genes. This effect is observable in the supplementary information published by Bachtrog and colleagues, yet overlooked in the text (Bachtrog et al. 2010).
Number and fraction (in brackets) of male-biased and female-biased genes located in bound and unbound regions
4.69 × 10−8
<2.20 × 10−16
<2.20 × 10−16
<2.20 × 10−16
<2.20 × 10−16
4.27 × 10−13
5.32 × 10−6
5.22 × 10−5
5.09 × 10−7
To gain a better insight about the differences between male-biased genes and female-biased genes, we extended our analysis to published gene expression data from gonads and gonadectomized adult flies (Parisi et al. 2003). Consistently, we found that, in gonads, there is an underrepresentation of male-biased genes in bound regions and an overrepresentation of female-biased genes in those regions, regardless of the expression level (X 2 = 118.34; df = 5; P = 7.03 × 10−24 for male-biased genes and X 2 = 130.48; df = 5; P = 1.88 × 10−26 for female-biased genes; Fig. 1g, h). However, this pattern was not observed in somatic tissues (i.e., gonadectomized flies; Fig. 1d, e), suggesting that a different selective pressure for sex-biased genes is at work in gonads compared to the somatic tissues. The difference between gonads (Fig. 1g, h) and gonadectomized samples (Fig. 1d, e) suggests that, even if excision of germline tissue from the adult carcass was incomplete, this contamination is not enough as to replicate the pattern found in gonads. Alternatively, the absence of a clear pattern in gonadectomized flies may reflect the heterogeneity among somatic tissues in their sex bias expression patterns (Meisel et al. 2012; Huylmans and Parsch 2015).
Dosage Compensation Does Not Impede Further Upregulation of Genes in Males
High Male Bias Level of Gene Expression Requires Low Gene Expression in Females
Our analyses indicate that male-biased genes are expressed higher in regions bound by the DCC than in unbound regions and in autosomes in somatic tissues (where DC takes place). This observation is incompatible with the hypothesis that DC and other chromatin remodeling proteins impede the upregulation of gene expression (Vicoso and Charlesworth 2009; Bachtrog et al. 2010). Is there any evidence supporting the hypothesis that chromatin remodeling complexes interacting with the DCC impede the upregulation of gene expression in males? Some chromatin remodeling proteins, such as ISWI, the ATAC complex, and SU(VAR)3-7, do certainly have specific roles in the X chromosomes or even interact genetically with DC (Corona et al. 2007; Carré et al. 2008; Spierer et al. 2008; Sala et al. 2011). If these proteins impeded upregulation of dosage compensated genes, we would expect dosage compensated genes to be upregulated in null flies for iswi, atac of su(var)3-7. However, mutations in these genes do not upregulate X-linked genes or even cause a generalized misregulation of the X-linked genes. Therefore, to our knowledge, there is no evidence to suggest that these proteins impede upregulation of X-linked genes in males (Bachtrog et al. 2010).
Chromatin modifications associated with DC and open chromatin structure still persist in females (Jin et al. 2000; Kind et al. 2008; Zhang and Oliver 2010; Sala et al. 2011; Brown and Bachtrog 2014). One important consequence that comes out from this feature is that silencing or downregulating gene expression may be a harder task for X-linked genes in females than for autosomal genes (Zhang and Oliver 2010). Consistently, autosomal testis-biased genes, as well as other autosomal tissue-specific genes, are enriched with repressors of gene expression in other tissues, while in the case of the X-linked genes, this trend is reduced or reversed in favor of activators of gene expression (Mikhaylova and Nurminsky 2011). In other words, because of DC, the X chromosome most likely provides an inadequate environment for genes that need repression in some tissues or females (Zhang and Oliver 2010; Mikhaylova and Nurminsky 2011). This simple model may explain why high male bias levels mainly occur on the autosomes or far from DCC (Bachtrog et al. 2010), where gene expression can be easily downregulated in females. This pattern is especially strong for testis-biased genes (Fig. 4), because testis represents the largest group of tissue-specific genes in Drosophila (Chintapalli et al. 2007) and it might extend to non-coding genes as well (Gao et al. 2014).
We are grateful to Doris Bachtrog and Beatriz Vicoso for kindly providing data. We also thank Anna Williford and Jeff Demuth for comments to this work. M.G. thanks Arndt von Haeseler for his support when finalizing the manuscript. E.B. is supported by a grant from the National Institute of General Medical Sciences of the National Institutes of Health (R01GM071813). The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank Jennifer Gage for kindly proofreading the manuscript.
- Vibranovski MD, Lopes HF, Karr TL, Long M (2009) Stage-specific expression profiling of Drosophila spermatogenesis suggests that meiotic sex chromosome inactivation drives genomic relocation of testis-expressed genes. PLoS Genet 5:e1000731. doi: 10.1371/journal.pgen.1000731 CrossRefPubMedPubMedCentralGoogle Scholar
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