Abstract
DNA and histone methylation are epigenetic modifications functioning in transcriptional control and have been implicated in the deregulation of gene expression in cancer. As a first step to determine if histone methylation could be involved in testis cancer pathogenesis, we performed immunofluorescent localization of histone H3 methylation at lysine 4 (H3-K4; gene activating) and lysine 9 (H3-K9; gene silencing) in healthy testis tissue and in samples of non-seminoma germ-cell tumours. In healthy testis, the distribution of histone H3 methylation was dependent on the developmental stage of spermatogenic cells and in non-seminoma, histone H3-K4 and K9 methylation was detected in all histological subtypes. This suggested that histone H3-K4 and K9 methylation could be associated with abnormal gene expression in non-seminoma. To determine the gene-specific function of histone H3 methylation, we proceeded to define the epigenetic status of key genes implicated in the pathogenesis of non-seminoma, namely the proto-oncogene POU5F1, which is overexpressed in testis cancer, and the tumour suppressor RASSF1A, which is aberrantly silenced. Cell lines representative of non-seminoma were treated with the chromatin-modifying drug, 5-aza-2'-deoxycytidine (5-aza-dC). Chromatin immunoprecipitation and real-time polymerase chain reaction analyses revealed that treatment with 5-aza-dC restored RASSF1A expression through a loss of gene silencing H3-K9 methylation and by retention of gene activating H3-K4 tri-methylation in the promoter region. In contrast, the expression of POU5F1 was reduced by 5-aza-dC and was associated with a loss of gene activating H3-K4 di-methylation in the promoter region. Analysis of DNA methylation revealed a slight reduction in DNA hypermethylation at the RASSF1A promoter, whereas the POU5F1 promoter remained mostly unmethylated and unaffected. Our results indicate that the effects of 5-aza-dC on histone methylation profiles are gene-specific and that aberrant histone modifications may serve as a principal means of misregulation of RASSF1A and POU5F1 expression in testis cancer.