Abstract
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.
Highlights
Over the last decade, many studies of cancer susceptibility genes have yielded new insight into both the pathogenesis of malignancy and the normal biology of cellular and developmental processes
Our studies indicate that this RB-associated Kruppel-associated box (KRAB) (RBaK) protein can physically interact with RB and may contribute to RB-dependent suppression of E2F-mediated transcriptional activation and RB-mediated cell cycle arrest
Restriction enzyme and sequence analysis of pACT-M17.1 indicated that it contained a 1.4-kb cDNA that was predicted to encode a novel protein with Kruppel-type zinc fingers and a KRAB repressor motif
Summary
Plasmids and Subcloning Strategies—Plasmids used in the yeast two-hybrid screen included pAS1-RB (p56) (amino acids 295–928 of mouse RB), pAS1-RB⌬A (deletion of amino acids 434 – 476), and pAS1RB⌬B (deletion of amino acids 697–763). We generated pCMV-MT6hRBaK-K⌬L by using PCR to amplify the coding sequence of hRBaK from bp 523 to 1032, which corresponds to the protein sequence from the second amino acid through the first half of the linker motif (Fig. 1A). A cDNA probe was made by using PCR to amplify a 388-bp fragment from pACT-mM17.1 that corresponds to amino acids 126 –255 of mouse RBaK (Fig. 1B) (5Ј primer, 5Ј-tcatttcccccagctcaggt; 3Ј primer, 5Ј-cagttccatttgtgatctcggat) This PCR product was gel-purified, labeled with [32P]dCTP by using the Primit II random primer labeling kit (Stratagene), and used as a probe to screen cDNA libraries containing 1 ϫ 106 phage from mouse E11.5 embryo and HeLa cells (CLONTECH). Antihuman RBaK Antisera—To generate mouse anti-human RBaK antiserum, we subcloned a PCR-generated cDNA fragment encoding the linker region of hRBaK (amino acids 80 –267, Fig. 1B) into the pGEX2T bacterial expression plasmid. Serum was obtained from mice for use in Western blotting
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