Generation of a cancer testis antigen mCherry reporter HCT116 colorectal carcinoma cell line.

In the context of cancer immunotherapy, agents that target the immune system to cancer cells need to fulfil two criteria: 1) that they are only expressed on the desired target cell and 2) that they can elicit a potent immunological response. Cancer Testis Antigens are a large disparate family of factors ordinarily expressed in the germ-line but aberrantly expressed across multiple types of cancer. The ability to enforce their expression on tumour cells is an attractive strategy that could render such cells potent targets of the immune system, but very little is known about their regulation. We describe the generation of an mCherry reporter cell line using HCT116 colorectal carcinoma cells that we anticipate will be useful for screen-based approaches to identify novel regulators of CTA expression. Discoveries arising from their use could in future be exploited to enhance tumour cell immunogenicity and improve cancer immuno-therapy.

A novel requirement for DROSHA in maintenance of mammalian CG methylation.

In mammals, faithful inheritance of genomic methylation patterns ensures proper gene regulation and cell behaviour, impacting normal development and fertility. Following establishment, genomic methylation patterns are transmitted through S-phase by the maintenance methyltransferase Dnmt1. Using a protein interaction screen, we identify Microprocessor component DROSHA as a novel DNMT1-interactor. Drosha-deficient embryonic stem (ES) cells display genomic hypomethylation that is not accounted for by changes in the levels of DNMT proteins. DNMT1-mediated methyltransferase activity is also reduced in these cells. We identify two transcripts that are specifically upregulated in Drosha- but not Dicer-deficient ES cells. Regions within these transcripts predicted to form stem-loop structures are processed by Microprocessor and can inhibit DNMT1-mediated methylation in vitro. Our results highlight DROSHA as a novel regulator of mammalian DNA methylation and we propose that DROSHA-mediated processing of RNA is necessary to ensure full DNMT1 activity. This adds to the DROSHA repertoire of non-miRNA dependent functions as well as implicating RNA in regulating DNMT1 activity and correct levels of genomic methylation.