Landscape of DNA Methylation on the Marsupial X.

Shafagh A Waters,Jennifer A Marshall Graves,Alexandra M Livernois,Hardip Patel,Catherine M Suter,Paul D Waters,Jeff M Craig,Denis O’Meally

doi:10.1093/molbev/msx297

Abstract

DNA methylation plays a key role in maintaining transcriptional silence on the inactive X chromosome of eutherian mammals. Beyond eutherians, there are limited genome wide data on DNA methylation from other vertebrates. Previous studies of X borne genes in various marsupial models revealed no differential DNA methylation of promoters between the sexes, leading to the conclusion that CpG methylation plays no role in marsupial X-inactivation. Using reduced representation bisulfite sequencing, we generated male and female CpG methylation profiles in four representative vertebrates (mouse, gray short-tailed opossum, platypus, and chicken). A variety of DNA methylation patterns were observed. Platypus and chicken displayed no large-scale differential DNA methylation between the sexes on the autosomes or the sex chromosomes. As expected, a metagene analysis revealed hypermethylation at transcription start sites (TSS) of genes subject to X-inactivation in female mice. This contrasted with the opossum, in which metagene analysis did not detect differential DNA methylation between the sexes at TSSs of genes subject to X-inactivation. However, regions flanking TSSs of these genes were hypomethylated. Our data are the first to demonstrate that, for genes subject to X-inactivation in both eutherian and marsupial mammals, there is a consistent difference between DNA methylation levels at TSSs and immediate flanking regions, which we propose has a silencing effect in both groups.

Highlights

In therian mammals, females have two copies of the X chromosome, one of which is transcriptionally silenced in somatic cells by an epigenetic process called X chromosome inactivation (XCI)
We looked for regions of differential DNA methylation between the sexes, on the X or Z, which would indicate a role in sex chromosome gene silencing
Large Scale Differential DNA Methylation between the Sexes Only CpG dinucleotides sampled in both sexes were retained for analysis, which were classified as mapping to an autosome or sex chromosome

Summary

Introduction

In therian (eutherian and marsupial) mammals, females have two copies of the X chromosome, one of which is transcriptionally silenced in somatic cells by an epigenetic process called X chromosome inactivation (XCI). Methylation of cytosine (5-methylcytosine—5mC) is an epigenetic modification associated with gene silencing on the X chromosome in eutherian mammals, where it is established relatively late on the inactive X chromosome (Xi) (Lock et al 1987) and is essential for long-term maintenance of the XCI state (Mohandas et al 1981; Graves 1982). Eutherian XCI is facilitated by a noncoding RNA (ncRNA), called XIST, which is expressed from and spreads in cis along the nascent Xi (reviewed in Augui et al 2011). The relevance of DNA methylation in marsupial XCI is poorly understood, a consequence of the limited number of genes analyzed and disparate species examined (Kaslow and Migeon 1987; Loebel and Johnston 1996; Wang et al 2014)

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Results

Conclusion