Abstract
BackgroundDespite DNA methylation being one of the most widely studied epigenetic modifications in eukaryotes, only a few studies have examined the global methylation status of marsupial chromosomes. The emergence of devil facial tumour disease (DFTD), a clonally transmissible cancer spreading through the Tasmanian devil population, makes it a particularly pertinent time to determine the methylation status of marsupial and devil facial tumour chromosomes. DNA methylation perturbations are known to play a role in genome instability in human tumours. One of the interesting features of the devil facial tumour is its remarkable karyotypic stability over time as only four strains with minor karyotypic differences having been reported.The cytogenetic monitoring of devil facial tumour (DFT) samples collected over an eight year period and detailed molecular cytogenetic analysis performed on the different DFT strains enables chromosome rearrangements to be correlated with methylation status as the tumour evolves.ResultsWe used immunofluorescent staining with an antibody to 5-methylcytosine on metaphase chromosomes prepared from fibroblast cells of three distantly related marsupials, including the Tasmanian devil, as well as DFTD chromosomes prepared from samples collected from different years and representing different karyotypic strains. Staining of chromosomes from male and female marsupial cell lines indicate species-specific differences in global methylation patterns but with the most intense staining regions corresponding to telomeric and/or centromeric regions of autosomes. In males, the X chromosome was hypermethylated as was one X in females. Similarly, telomeric regions on DFTD chromosomes and regions corresponding to material from one of the two X chromosomes were hypermethylated. No difference in global methylation in samples of the same strain taken in different years was observed.ConclusionsThe methylation patterns on DFTD chromosomes suggests that the hypermethylated active X was shattered in the formation of the tumour chromosomes, with atypical areas of methylation on DFTD chromosomes corresponding to locations of X chromosome material from the shattered X. The incredibly stable broad methylation patterns observed between strains and over time may reflect the overall genomic stability of the devil facial tumour.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-015-0176-x) contains supplementary material, which is available to authorized users.
Highlights
Despite DNA methylation being one of the most widely studied epigenetic modifications in eukaryotes, only a few studies have examined the global methylation status of marsupial chromosomes
The cytogenetic monitoring of devil facial tumour (DFT) samples collected from 2005 to 2012 [9] and detailed molecular cytogenetic analysis performed on the different DFT strains [8], enables chromosome rearrangements to be correlated with changes in methylation status as the tumour evolves
We show speciesspecific methylation patterns on metaphase chromosomes prepared from marsupial fibroblast cells, distinct differences in the level of methylation on sex chromosomes, an association of X chromosome material with hypermethylation on devil facial tumour disease (DFTD) chromosomes, and stability of global methylation patterns on DFTD chromosomes over time
Summary
Despite DNA methylation being one of the most widely studied epigenetic modifications in eukaryotes, only a few studies have examined the global methylation status of marsupial chromosomes. The cytogenetic monitoring of devil facial tumour (DFT) samples collected over an eight year period and detailed molecular cytogenetic analysis performed on the different DFT strains enables chromosome rearrangements to be correlated with methylation status as the tumour evolves. The Tasmanian devil (Sarcophilus harrisii) is an interesting species in which to study methylation patterns on chromosomes. It has a 2n = 14 karyotype similar to the predicted ancestral marsupial karyotype [5] (Fig. 1). The cytogenetic monitoring of DFT samples collected from 2005 to 2012 [9] and detailed molecular cytogenetic analysis performed on the different DFT strains [8], enables chromosome rearrangements to be correlated with changes in methylation status as the tumour evolves
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