An inducible dominant inhibitor of C/EBP-regulated genes.

(A) KRAB-C/EBPα-ER (KαER) and KRAB-C/EBPα-1,2Val-ER (KVER) are diagrammed. The KRAB segment is a transrepression domain derived from Kox-1. The C/EBPα segment contains the C/EBPα DNA-binding domain, but not its trans-activating domains. All C/EBP family members have a common DNA-binding consensus, and it is therefore expected that each will be inhibited by KαER. The ER segment is responsive to 4HT. In KVER, mutation of 2 leucines (L) to valine (V) prevents DNA binding. (B) Total cellular proteins corresponding to 1 × 10⁶ cells, from 32D cl3 cells or from subclones transduced with pBabe-KVER or pBabe-KαER, were subjected to Western blotting with the use of a murine ER antiserum. (C) First, 5 × 10⁶ 32D–KαER-1 and 32D–KVER-2 cells proliferating in IL-3 were transfected with 20 μg p(C/EBP)₂TKLUC and 1.0 μg pCMV-βGal with the use of diethylaminoethyl-dextran. One-half of each culture was then treated with 200 nM 4HT and the other half with 0.1% ethanol. Luciferase and βGal activities were assessed 2 days later. The ratio of reporter activity (activity with 4HT/activity without 4HT), normalized to the internal control, is shown for each condition (mean and SE from 2 experiments).