Cloning and characterization of a novel Kruppel-associated box family transcriptional repressor that interacts with the retinoblastoma gene product, RB

The retinoblastoma gene product, RB, seems to function as a key tumor suppressor by repressing the expression of genes activated by members of the E2F family of transcription factors. In order to accomplish this, RB has been proposed to interact with a transcriptional repressor. However, no genuine transcriptional repressors have been identified by virtue of interaction with RB. By using the yeast two-hybrid system, we have identified a novel member of a known family of transcriptional repressors that contain zinc fingers of the Kruppel type and a portable transcriptional repressor motif known as the Kruppel-associated box (KRAB). The mouse and human forms of the novel RB- associated KRAB protein (RBaK) are widely expressed. The amino acid motif that links the KRAB domain and zinc fingers appears to be required for interaction with RB in vitro. Human RBaK ectopically expressed in fibroblasts is an 80-kDa protein that is localized to the nucleus. The expression of either RB or RBaK in 10T1/2 fibroblasts represses the activation of an E2F- dependent promoter and decreases DNA synthesis to a similar degree. However, a mutant form of RBaK that cannot interact with RB in vitro is unable to prevent DNA synthesis. We present a model in which RB physically interacts with the novel transcriptional repressor RBaK to repress E2F-dependent genes and prevent DNA synthesis.