Selective elimination of long INterspersed element-1 expressing tumour cells by targeted expression of the HSV-TK suicide gene.

In gene therapy, effective and selective suicide gene expression is crucial. We exploited the endogenous Long INterspersed Element-1 (L1) machinery often reactivated in human cancers to integrate the Herpes Simplex Virus Thymidine Kinase (HSV-TK) suicide gene selectively into the genome of cancer cells. We developed a plasmid-based system directing HSV-TK expression only when reverse transcribed and integrated in the host genome via the endogenous L1 ORF1/2 proteins and an Alu element. Delivery of these new constructs into cells followed by Ganciclovir (GCV) treatment selectively induced mortality of L1 ORF1/2 protein expressing cancer cells, but had no effect on primary cells that do not express L1 ORF1/2. This novel strategy for selective targeting of tumour cells provides high tolerability as the HSV-TK gene cannot be expressed without reverse transcription and integration, and high selectivity as these processes take place only in cancer cells expressing high levels of functional L1 ORF1/2.

Disruption of the interaction between transcriptional intermediary factor 1{beta} and heterochromatin protein 1 leads to a switch from DNA hyper- to hypomethylation and H3K9 to H3K27 trimethylation on the MEST promoter correlating with gene reactivation.

Here, we identified the imprinted mesoderm-specific transcript (MEST) gene as an endogenous TIF1beta primary target gene and demonstrated that transcriptional intermediary factor (TIF) 1beta, through its interaction with heterochromatin protein (HP) 1, is essential in establishing and maintaining a local heterochromatin-like structure on MEST promoter region characterized by H3K9 trimethylation and hypoacetylation, H4K20 trimethylation, DNA hypermethylation, and enrichment in HP1 that correlates with preferential association to foci of pericentromeric heterochromatin and transcriptional repression. On disruption of the interaction between TIF1beta and HP1, TIF1beta is released from the promoter region, and there is a switch from DNA hypermethylation and histone H3K9 trimethylation to DNA hypomethylation and histone H3K27 trimethylation correlating with rapid reactivation of MEST expression. Interestingly, we provide evidence that the imprinted MEST allele DNA methylation is insensitive to TIF1beta loss of function, whereas the nonimprinted allele is regulated through a distinct TIF1beta-DNA methylation mechanism.