Global DNA Methylation patterns on marsupial and devil facial tumour chromosomes

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.

Identification of candidate genes for devil facial tumour disease tumourigenesis

“…The event leading to DFT1 tumourigenesis is proposed to be the fusion of chromosome 1 with an X chromosome, which led to a series of breakage-fusion-bridge cycles and extensive rearrangement of chromosome 1 material on M1 13 , 18 . With this idea in mind, along with the now seemingly stable nature of the extensively rearranged chromosome 1 material in DFT1, chromosome 1 is a likely source for DFT1 driver mutations.…”

“…7 ). Previous research has suggested the loss of telomeres led to the fusion of chromosomes 1 and X 13 , 18 . Our mapping data suggest the initial fusion most likely occurred close to the NF2 region at the distal end of 1q and the distal end of Xq.…”

“…In this context, a copy gain for the NF1 region rather than a deletion, make it less likely that NF1 is a driver of DFT1 tumourigenesis. In additional, the extra copy of this region is also located on chromosome M4, a highly methylated chromosome compared to other DFT1 chromosomes 18 , which may be an indicator of transcriptional silencing of this chromosome and therefore, may not alter the overall gene expression of genes on the NF1 scaffold. The copy number gains observed for NGF and KCNH6 also have a low probability of being driver mutations of DFT1 as they are not on a chromosome posited to be initially involved in the formation of the tumour.…”

“…DFT1 is proposed to have arisen from telomeres on one homologue of chromosomes 1 and X becoming critically short, permitting the fusion of these two chromosomes, followed by a series of breakage-fusion-bridge cycles to ultimately result in their shattered appearance in the DFT1 karyotype 13 , 18 . The transmissible tumour is evolving as it as spreads through the devil population, accumulating mutations resulting in visible, karyotypic differences 12 .…”

“…As an alternative approach, we have exploited our understanding of the structural changes and the Schwann cell origin of the tumour to predict prime candidate genes that may be affected in DFT1, in order to identify potentially perturbed pathways for further investigation. The fusion of chromosome 1 and X is predicted to be the event leading to tumourigenesis in DFT1 13 , 18 , making the large chromosome 1 a logical place to start a search for potential driver mutations, with strong candidate genes being those involved in tumourigenesis in peripheral nerve sheath tumours in humans. Firstly, we decided to test the stability of this chromosome through further mapping of bacterial artificial chromosome (BAC) clones on DFT1 chromosomes from three different samples for which there is already mapping data available 13 .…”