Figure 4.

Yeast two-hybrid analysis of the interaction between different KRAB domains and the TIF1 family proteins. (A) Schematic representation of the yeast two-hybrid system used in this study. The DBD of the ERα (amino acids 176–282) and the acidic activation domain (AAD) of VP16 (amino acids 411–490) unfused or fused to the proteins tested for interaction (white boxes) are shown. The URA3 reporter gene, which is regulated by three estrogen response elements (ERE3X) in the yeast reporter strain PL3, is represented below. (B–G) Selective interaction of KRAB(AB), KRAB(Ab), and KRAB(A) domains with TIF1β. Plasmids expressing the indicated DBD-KRAB fusions were introduced into PL3 together with either the VP16 AAD (as a control) or the VP16 AAD fused to TIF1α, TIF1β, or TIF1γ. OMPdecase activities determined for each cell-free extract are expressed in nanomolar substrate/minute/milligram protein. The values (±20%) represent the averages of at least three independent experiments. Note that expression of all fusion proteins was confirmed by Western blotting by using antibodies F3 against the F region tag of the ERα DBD and 2GV4 against VP16, respectively (data not shown).