Poster presentation, P146

Official XXIst International Pigment Cell Conference website - 21-24 Sept 2011, Bordeaux - France | updated: September 04 2011

MAGE Proteins Are Master Regulators of KAP1 and KRAB Domain Zinc Finger Transcription Factor Mediated Gene Suppression

BACKGROUND: Class I MAGE proteins are normally expressed only in developing germ cells but are aberrantly expressed in melanoma and many other cancers, making them ideal therapeutic targets. In primary melanomas, MAGE expression is a strong prognostic biomarker, negatively correlated with relapse free survival independent of thickness, ulceration, and mitoses. We have previously shown that MAGE proteins promote melanoma survival in vitro and in vivo by binding to the KAP1 scaffolding protein and enhancing KAP1 suppression of p53, but MAGE functions and the underlying mechanisms have not been fully elucidated. The KRAB domain zinc finger transcription factors (KZNFs) are the largest group of vertebrate transcription factors. KZNFs also bind to KAP1 and direct it to specific DNA sequences where it represses gene expression by inducing localized heterochromatin characterized by histone 3 lysine 9 trimethylation (H3me3K9). OBJECTIVE AND METHODS: To test the hypothesis that MAGE proteins can affect KZNF KAP1 interactions and function we used loss of function mutants, ChIP, ChIP-seq, RT-qPCR and immunoblotting to determine effects of MAGE expression on KAP1 binding, chromatin structure, and gene repression. RESULTS: We found that MAGE expression relieves repression of a reporter gene by ZNF382, a KZNF with tumor suppressor activity. ChIP of MAGE (-) HEK293T cells showed KAP1 and H3me3K9 are normally bound to the ID1 oncogene, a target of ZNF382, but that binding is greatly reduced in the presence of MAGE proteins. MAGE expression relieves KAP1 mediated ID1 suppression, causing increased expression of ID1 mRNA and ID1 chromatin relaxation characterized by loss of H3me3K9. MAGE binding also induces KAP1 mediated ZNF382 poly-ubiquitination and degradation, consistent with loss of ZNF382 leading to decreased KAP1 binding to ID1. In contrast, MAGE expression caused increased KAP1 binding to other genes with increased H3me3K9 and decreased mRNA expression. Chip-seq showed MAGE affects KAP1 binding to large numbers of genes including oncogenes and tumor suppressors. CONCLUSION: Since KZNFs are required to direct KAP1 to specific genes, we conclude that MAGE proteins can differentially regulate members of the KZNF family and KAP1 mediated gene repression. Global analysis suggests that MAGE may be master regulators of transcription, affecting genes that involved in neoplastic transformation, tumor suppression, cellular proliferation, and apoptosis.

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