A survey of human cancer-germline genes: linking X chromosome localization, DNA methylation and sex-biased expression in early embryos

Human cancer-germline (CG) genes are a group of testis-specific genes that become aberrantly activated in various tumors. Ongoing studies aim to understand their functions in order to evaluate their potential as anti-cancer therapeutic targets. Evidence suggests the existence of subcategories of CG genes, depending on location on autosomal or sex chromosomes, reliance on DNA methylation for transcriptional regulation, and profile of expression during gametogenesis and early embryogenesis. To clarify this issue, we developed CTexploreR, a R/Bioconductor package that integrates an up-to-date reference list of human CG genes (n=146) with multiple bulk and single-cell methylomic and transcriptomic datasets. Based on promoter methylation profiles and responsiveness to a DNA methylation inhibitor, 74% of the CG genes were classified as DNA methylation dependent (Methdep). Intriguingly, most X-linked CG genes (69/70) fell into this category, thereby implicating DNA methylation dependency in the well-documented over-representation of testis-specific genes on the X chromosome. We further observed that, whereas X-linked Methdep CG genes become demethylated and activated in pre-spermatogonia in the fetal testis, most of them resist DNA demethylation in female germ cells and remain therefore silent in fetal and adult oocytes. Importantly, a number of X-linked Methdep CG genes (e.g. FMR1NB, GAGE2A, MAGEB2/C2, PAGE2, VCX3A/B) maintained this maternal-specific imprinting after fertilization, and were expressed exclusively in female preimplantation embryos, which inherit a paternal X chromosome. Together, our study using the CTexploreR package has allowed us to show that X-linked CG genes undergo transient maternal imprinting and contribute therefore to transcriptional sexual dimorphism in early embryos. Author SummaryCancer-germline (CG) genes include a set of genes that are normally active only in the testis but become aberrantly switched on in different types of tumor, making them potential targets for new anti-cancer treatments. We developed a new analytical tool called CTexploreR, to study these genes, and observed that a large subset of CG genes reside on the X chromosome and use DNA methylation as a mechanism of repression in non-testicular tissues. In fetal testis, these genes lose methylation and become activated in early spermatogenic cells, while in female ovaries they stay methylated and remain silent. Notably, we demonstrate that several CG genes keep this methylation pattern after fertilization, and are therefore expressed in female but not male embryos, which inherit only one X chromosome of maternal origin. CG genes carrying this transient maternal imprinting appear therefore as main contributors of sex-biased mRNA expression in preimplantation embryos. Our Jindings therefore open up new Jields of investigation into the functions of CG genes, the sexual dimorphism of early embryos, and the intergenerational transmission of epigenetic imprints.