Abstract
We have isolated two novel Krüppel-like zinc finger proteins containing the evolutionarily conserved Krüppel-associated box (KRAB), KRAZ1 and KRAZ2, and demonstrated that they repress transcription when heterologously targeted to DNA. Their repression activity appeared to be mediated by the putative corepressor KAP-1 (KRAB-associated protein-1), because KRAZ1/2 bind to KAP-1, but KRAB mutants of KRAZ1/2 that are unable to interact with KAP-1 lack repression activity, and KAP-1 has intrinsic repressor activity and potentiates KRAZ1/2-mediated repression. We dissected the KAP-1 protein into a KRAB-interacting domain and a region necessary for repression. Using a mammalian two-hybrid assay, we further demonstrated that KAP-1 deletions lacking repression activity fused to the VP16 transactivation domain strongly activated transcription when coexpressed with KRAZ1. In contrast, VP16-KAP-1 fusions retaining repression activity resulted in repression. These results provide the first evidence that KAP-1 functionally interacts with KRAB in mammalian cells and seems to exert repressor activity in the DNA-bound KRAB-KAP-1 complex, and they further support the hypothesis that KAP-1 functions as a corepressor for the large class of KRAB-containing zinc finger proteins.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s)AB024004 and AB024005 for KRAZ1 and KRAZ2, respectively
Our results present the first evidence that KRAB-associated protein-1 (KAP-1) functionally interacts with Kruppel-associated box (KRAB) in mammalian cells and that KAP-1 seems to function as a corepressor of KRAB-zinc finger proteins (ZFPs) in the complex with the DNA-bound KRAB domains
The KRAZ2 cDNA (2261 nt) contains an open reading frame for a polypeptide of 606 amino acids with a molecular mass of 71.2 kDa. Both KRAZ1 and KRAZ2 contain the KRAB-A domain at the N terminus and repeated zinc finger domains located in the C terminus (15 and 9 repeats) (Fig. 1A)
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s)AB024004 and AB024005 for KRAZ1 and KRAZ2, respectively. We examined the in vivo functional interaction of KRAB with KAP-1 using a mammalian two-hybrid assay in which GAL4-KRAB was coexpressed with the KAP-1 deletions fused to the VP16 transactivation domain (VP16AD) in NIH 3T3 cells.
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