MAGE I Transcription Factors Regulate KAP1 and KRAB Domain Zinc Finger Transcription Factor Mediated Gene Repression
Figure 1
MAGE-KAP1 binding decreases reporter gene repression by ZNF382.
A. Full length MAGE-A3, full length MAGE-C2, or corresponding MAGE homology domain constructs, bind to full length KAP1 and RBCC-KAP1 detected by a Mammalian Two-Hybrid Assay. CHO cells transfected with the indicated plasmids were assayed for SEAP (secreted alkaline phosphatase) activity 48 h after transfection, using a chemiluminescent substrate CSPD. pM3, pM53 and pVP16-T are positive control vectors. pM and pVP16 are negative control vectors. pVP16-KAP1 alone shows minimal light units. MHD = MAGE homology domain. B. MAGE-A3 and MAGE-C2 decrease KAP1 repression of a ZNF382 responsive reporter gene. Luciferase activities were measured in CHO-5xGal4-UAS-TK-Luc-2p cells transfected with (+) control vector (Gal4-DBD), with vector expressing full length (FL) MAGE-A3 or C2, or with their corresponding MAGE homology domains (MHDs). Maximum repression occurs in the presence of ZNF382 alone. Addition of MAGE expression plasmids causes dose dependent release of repression with increased luciferase activity. C. MAGE-C2 mutant L152A, L153A and MAGE-A3 mutant L120A, L121A, which cannot bind KAP1, fail to relieve repression mediated by ZNF382. D. MAGE-A3 and C2 mediated KAP1 repression of a ZNF382 responsive reporter gene is reversible. Addition of ZNF382 expression plasmids initiate dose dependent regaining of repression with decreased luciferase activity.