Abstract
Class I MAGE proteins (MAGE I) are normally expressed only in developing germ cells but are aberrantly expressed in many cancers. They have been shown to promote tumor survival, aggressive growth, and chemoresistance but the underlying mechanisms and MAGE I functions have not been fully elucidated. KRAB domain zinc finger transcription factors (KZNFs) are the largest group of vertebrate transcription factors and regulate neoplastic transformation, tumor suppression, cellular proliferation, and apoptosis. KZNFs bind the KAP1 protein and direct KAP1 to specific DNA sequences where it suppresses gene expression by inducing localized heterochromatin characterized by histone 3 lysine 9 trimethylation (H3me3K9). Discovery that MAGE I proteins also bind to KAP1 prompted us to investigate whether MAGE I can affect KZNF and KAP1 mediated gene regulation. We found that expression of MAGE I proteins, MAGE-A3 or MAGE-C2, relieved repression of a reporter gene by ZNF382, a KZNF with tumor suppressor activity. ChIP of MAGE I (-) HEK293T cells showed KAP1 and H3me3K9 are normally bound to the ID1 gene, a target of ZNF382, but that binding is greatly reduced in the presence of MAGE I proteins. MAGE I expression relieved KAP1 mediated ID1 repression, causing increased expression of ID1 mRNA and ID1 chromatin relaxation characterized by loss of H3me3K9. MAGE I binding to KAP1 also induced ZNF382 poly-ubiquitination and degradation, consistent with loss of ZNF382 leading to decreased KAP1 binding to ID1. In contrast, MAGE I expression caused increased KAP1 binding to Ki67, another KAP1 target gene, with increased H3me3K9 and decreased Ki67 mRNA expression. Since KZNFs are required to direct KAP1 to specific genes, these results show that MAGE I proteins can differentially regulate members of the KZNF family and KAP1 mediated gene repression.
Highlights
Class I MAGE proteins (MAGE I) are normally expressed only in developing germ cells, trophoblast and placenta because their expression is suppressed in somatic tissues by hypermethylation of promoter region CpG dinucleotide islands [1] [2,3]
We and others have found that MAGE I proteins bind to the RBCC region of KAP1[11,13], a scaffolding protein whose functions include: (1). suppression of p53 by increasing KAP1 ubiquitin E3 ligase activity and p53 ubiquitination [9,11,13]. (2). global induction and maintenance of transcriptionally inactive heterochromatin characterized by Histone 3 tri-methyl lysine 9 (H3me3K9), and (3). suppression of specific genes by localized induction of transcriptionally inactive heterchromatin, targeted by KRAB domain containing zinc finger transcription factors (KZNFs)
Confirmation of MAGE I and KAP1 binding sites MAGE I have a large terminal exon that usually encodes the entire protein and contains the MAGE homology domain (MHD), a conserved region of,170 amino acids that comprises about 70% of each MAGE protein and that recently has been implicated as the binding site for KAP1 [2,13]
Summary
Class I MAGE proteins (MAGE I) are normally expressed only in developing germ cells, trophoblast and placenta because their expression is suppressed in somatic tissues by hypermethylation of promoter region CpG dinucleotide islands [1] [2,3]. MAGE I may be aberrantly expressed because of global hypomethylation that often occurs during epigenetic reprogramming [2,3]. We have shown that suppression of MAGE I decreases the growth of melanoma and malignant mast cell lines in vitro and in vivo [10,11], and Liu et al [12] have shown that MAGE I expression causes accelerated cell cycle progression, increases the rate of cell migration and invasion in vitro, and increases lung metastases in an orthotopic mouse model of human thyroid cancer. We and others have found that MAGE I proteins bind to the RBCC region of KAP1[11,13], a scaffolding protein whose functions include: (1). Global induction and maintenance of transcriptionally inactive heterochromatin characterized by Histone 3 tri-methyl lysine 9 (H3me3K9), and (3). Suppression of specific genes by localized induction of transcriptionally inactive heterchromatin, targeted by KRAB domain containing zinc finger transcription factors (KZNFs) We and others have found that MAGE I proteins bind to the RBCC region of KAP1[11,13], a scaffolding protein whose functions include: (1). suppression of p53 by increasing KAP1 ubiquitin E3 ligase activity and p53 ubiquitination [9,11,13]. (2). global induction and maintenance of transcriptionally inactive heterochromatin characterized by Histone 3 tri-methyl lysine 9 (H3me3K9), and (3). suppression of specific genes by localized induction of transcriptionally inactive heterchromatin, targeted by KRAB domain containing zinc finger transcription factors (KZNFs)
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