NOVEL MUTANTS OF NAB COREPRESSORS ENHANCE ACTIVATION BY EGR TRANSACTIVATORS

7The NGFI-A binding corepressors NAB1 and NAB2 interact with a conserv ed domain (R1 domain) within the Egr1/NGFI-A and Egr2/Krox20 transacti vators, and repress the transcription of Egr target promoters. Using a novel adaptation of the yeast two-hybrid screen, we have identified s everal point mutations in NAB corepressors that interfere with their a bility to bind to the Egr1 R1 domain. Surprisingly, NAB proteins beari ng some of these mutations increased Egr1 activity dramatically. The m echanism underlying the unexpected behavior of these mutants was eluci dated by the discovery that NAB conserved domain 1 (NCD1) not only bin ds to Egr proteins but also mediates multimerization of NAB molecules. The activating mutants exert a dominant negative effect on NAB repres sion by multimerizing with native NAB proteins and preventing binding of endogenous NAB proteins with Egr transactivators. To examine NAB re pression of a native Egr target gene, we show that NAB2 represses Egr2 /Krox20-mediated activation of the bFGF/FGF-2 promoter, and that repre ssion is reversed by coexpression of dominant negative NAB2. Because o f their specific ability to alleviate NAB repression of Egr target gen es, the dominant negative NAB mutants will be useful in elucidating th e mechanism and function of NAB corepressors.