Histone methylation is a critical regulator of the abnormal expression of POU5F1 and RASSF1A in testis cancer cell...

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.