title: Histone methylation is a critical regulator of the abnormal expression of POU5F1 and RASSF1A in testis cancer cell lines.
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.
authors.affiliation: Department of Animal Science, McGill University, Montreal, Quebec, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Lambrot
authors.forename: S
authors.initials: S
authors.lastname: Kimmins
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D000077209:Decitabine; D020868:Gene Silencing / Q000187:drug effects; D006657:Histones / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008297:Male; D008745:Methylation; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis*; D000090063:Proto-Oncogene Mas; D013736:Testicular Neoplasms; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis*
chemical_list: D006657:Histones; C000715470:MAS1 protein, human; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D000090063:Proto-Oncogene Mas; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; D000077209:Decitabine; D008239:Lysine; D001374:Azacitidine