Epigenetic regulation of cancer-testis gene expression

Abstract

Cancer-testis (CT) genes are composed of mostly X-linked gene families that are widely expressed in cancer, in a coordinate manner, with ideally no expression in normal tissues except spermatogonia, oogonia and trophoblasts. Exact mechanisms reactivating their expression during carcinogenesis are not known yet. Epigenetic factors emerge as key controllers of CT expression. Selective DNA demethylation of the promoter regions of CT genes has been demonstrated to correlate closely with CT expression in all examples studied so far. Tumorsuppressor genes (TSGs), on the other hand, are known to be frequently downregulated in cancer by hypermethylation of DNA. In order to elucidate mechanisms that could help explain tumor-specific CT gene up-regulation, we aimed to understand how these seemingly opposite effects could exist in close proximity. For this purpose, we identified eight CT-proximal X-linked putative TSGs (pTSGs) down-regulated in tumors, by screening the SAGE and EST libraries of the Cancer Genome Anatomy Project. By conventional and real-time RT-PCR, we verified that two such genes, ALAS2 and CDR1, were significantly down-regulated in almost all cancers tested, while three others were down-regulated at least in some cancers, and by bisulfite sequencing we demonstrated that the promoter DNA of these pTSGs were hyper-methylated in correlation with their expression levels. Our search for the presence of insulators between CT and TSG genes did not yield a consensus site. However, we hypothesized that the dynamic organization of CT genes into inverted repeats (identified by the Inverted Repeats Finder program) throughout the X chromosome could be a candidate regulator of CT expression. By using the chromosome conformation capture (3C) assay, we have shown the alteration in higher-order chromosomal structure of the CT gene NY-ESO-1- bearing inverted repeat region in SK-LC-17 as well as in 5-Aza-2’-deoxycytidinetreated HT29 cell lines, correlating with CT as well as ncRNA expression from within the repeat. As it is known that many CT genes are embedded in inverted repeats, our results suggest a general mechanism regarding epigenetic regulation of CT gene expression.