MAGE proteins regulate KRAB zinc finger transcription factors and KAP1 E3 ligase activity

• MAGE-A3 regulates genes via zinc finger transcription factor (ZNF) KRAB domains.
• MAGE-A3 enhances gene repression by ZNF KRAB domains with A or A+b box motifs.
• MAGE-A3 de-represses genes by ZNF KRAB domains with A+B box motifs.
• MAGE-A3 does not affect gene regulation by ZNFs with KRAB Scan motifs.
• MAGE-A3 gene regulation depends on the structure of ZNF KRAB domains.

Expression of Melanoma AntiGen Encoding (MAGE) genes, particularly MAGE-A3, has been correlated with aggressive clinical course, the acquisition of resistance to chemotherapy and poor clinical outcomes of melanoma and other malignancies. MAGE proteins bind to KAP1, a gene repressor and ubiquitin E3 ligase which also binds KRAB domain containing zinc finger transcription factors (KZNFs), and MAGE expression may affect KZNF mediated gene regulation. To investigate mechanisms for these effects, we tested the hypothesis that differences in KRAB domain composition affect KZNF poly-ubiquitination and determine whether MAGE expression increases, decreases, or has no effect on KZNFs mediated gene repression. Using an integrated reporter gene responsive to repression by KRAB domain fusion proteins, we found that MAGE-A3 relieved KZNF mediated repression and induced KZNF poly-ubiquitination and degradation in association with expression of the A+B box KRAB domain. In contrast, MAGE-A3 enhanced KAP1 mediated repression of KZNFs expressing A or A+b box KRAB domains but caused no increase in poly-ubiquitination or degradation. MAGE-A3 has no significant impact on KZNFs with KRAB domains containing the Scan box motif. These data support our hypothesis by showing that the effects of MAGE-A3 on gene repression depend on the type of KZNF KRAB domain involved.