Abstract
Ohno proposed that dosage compensation in mammals evolved as a two-step mechanism involving X-inactivation and X-upregulation. While X-inactivation is well characterized, it remains to further analysis whether upregulation of the single activated X chromosome in mammals occurs. We obtained RNA-seq data, including single-cell RNA-seq data, from cells undergoing inactivation/reactivation in both germ cell development and early embryogenesis stages in mouse and calculated the X: A ratio from the gene expression. Our results showed that the X: A ratio is always 1, regardless of the number of X chromosomes being transcribed for expressed genes. Furthermore, the single-cell RNA-seq data across individual cells of mouse preimplantation embryos of mixed backgrounds indicated that strain-specific SNPs could be used to distinguish transcription from maternal and paternal chromosomes and further showed that when the paternal was inactivated, the average gene dosage of the active maternal X chromosome was increased to restore the balance between the X chromosome and autosomes. In conclusion, our analysis of RNA-seq data (particularly single-cell RNA-seq) from cells undergoing the process of inactivation/reactivation provides direct evidence that the average gene dosage of the single active X chromosome is upregulated to achieve a similar level to that of two active X chromosomes and autosomes present in two copies.
Highlights
The formation of sex was one of the most important events in evolution
To examine whether types with inactive X chromosomes (the X) chromosome undergoes a process of reactivation in mouse female germ cells at the different stages examined in this study, we isolated primordial germ cell (PGC) (E12.5, E13.5 and E16.5) and female germline stem cells (FGSCs) using two-step enzymatic digestion, as previously described[27], and purified them using fluorescence-activated cell sorting (FACS) (Fig. 1A–F,H,I)
The germ cells collected at different stages were analyzed using single-cell reverse-transcriptase PCR (RT-PCR) for Xist, Oct[4] and Mvh
Summary
The formation of sex was one of the most important events in evolution. Sex in mammals is determined by the sex chromosomes. An analysis of the genes that encode components of large protein complexes (≥7 members) revealed that their expressions, which were expected to be dosage-sensitive, were similar to those of autosomal genes within the complex These results support Ohno’s hypothesis that X chromosome inactivation acts as a dosage-compensation mechanism[19]. Marks et al found that gene expression in the active X chromosome (Xa) is upregulated, resulting in complete dosage compensation between X-linked and autosomal genes in the early stages of XCI during the differentiation of female embryonic stem cells (ESCs) to embryoid bodies (EBs)[20]. We used RNA-seq data from mouse germ cells at different developmental stages and female embryonic stem cells to compare the expression levels of X-linked and autosomal genes. When the paternal X chromosome was inactivated, the average gene dosage in the active maternal X chromosome increased to achieve a balance between the X chromosome and autosomes
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