Methylation analysis of a marsupial X-linked CpG island by bisulfite genomic sequencing.

Abstract

Paternal X chromosome inactivation occurs in rodent extraembryonic membranes and in all tissues of marsupials. Methylation of CpG islands occurs on the inactive X in eutherians and is considered to be a stabilizing mechanism. The only previous study of a marsupial X-linked CpG island was of the G6PD gene of the Virginia opossum, in which the paternally derived allele is not completely repressed. We have cloned the 5' end of the G6PD gene from an Australian marsupial, the common wailaroo, and sequenced the associated CpG island. The paternally derived C,6PD allele is completely repressed in tissues of this species. Methylation analysis using Hpall and Cfol restriction enzymes and bisulfite genomic sequencing of 47 CpG dinucleotides in a 613-bp region reveals hypomethylation of male and female DNA from tissues, cultured fibroblasts (in which the paternal allele is partially expressed) and sperm. This suggests that methylation of CpG islands is not required for maintenance of X inactivation in marsupials even where repression of the paternal allele is complete.

The origin and evolution of vertebrate sex chromosomes and dosage compensation

“…Differential acetylation had been observed on the X chromosomes in female tammar wallaby (Wakefield et al, 1997), as was enrichment of an active histone methylation mark (presumably on the active X) (Wakefield et al, 1997;Koina et al, 2009), and no differential DNA methylation differences were detected (Piper et al, 1993;Loebel and Johnston, 1996). However, interesting observations have come from several recent studies of representative Australian and American marsupials, using immunofluorescence to examine histone marks that are associated with XCI in human and mouse.…”

Bisulfite genomic sequencing: systematic investigation of critical experimental parameters

“…Therefore, it is more appropriate to use a nested PCR approach, i.e. two subsequent rounds of PCR using nested or semi-nested primer pairs (4,7,8,26). Primers will in general be longer than usual PCR primers and the primer binding sites should not contain methylatable sites.…”

Unravelling the evolutionary origins of X chromosome inactivation in mammals: insights from marsupials and monotremes

“…More recently, Hornecker et al (2007) used bisulfite sequencing to demonstrate that 5′ CpG islands of G6PD and PGK1 were not differentially methylated in M. domestica. The methylation status of the wallaroo G6PD 5′ CpG island gene was also determined using bisulfite sequencing, but no differential methylation was detected (Loebel and Johnston 1996).…”