Sequence-specific transcriptional repression by KS1, a multiple-zinc-finger-Kruppel-associated box protein

The vertebrate genome contains a large number of Kruppel-associated box-zin c finger genes that encode 10 or more C-2-H-2 zinc finger motifs. Members o f this gene family have been proposed to function as transcription factors by binding DNA through their zinc finger region and repressing gene express ion via the KRAB domain. To date, however, no Kruppel-associated box-zinc f inger protein (KRAB-ZFP) and few proteins with 10 or more zinc finger motif s have been shown to bind DNA in a sequence-specific manner. Our laboratory has recently identified KS1, a member of the KRAB-ZFP family that contains 10 different C-2-H-2 zinc finger motifs, 9 clustered at the C terminus wit h an additional zinc finger separated by a short linker region. In this stu dy, we used a random oligonucleotide binding assay to identify a 27-bp KS1 binding element (KBE). Reporter assays demonstrate that KS1 represses the e xpression of promoters containing this DNA sequence. Deletion and site-dire cted mutagenesis reveal that KS1 requires nine C-terminal zinc fingers and the KRAB domain for transcriptional repression through the KBE site, wherea s the isolated zinc finger and linker region are dispensable for this funct ion. Additional biochemical assays demonstrate that the KS1 KRAB domain int eracts with the KAP-1 corepressor, and mutations that abolish this interact ion alleviate KS1-mediated transcriptional repression. Thus, this study pro vides the first direct evidence that a KRAB-ZFP binds DNA to regulate gene expression and provides insight into the mechanisms used by multiple-zinc-f inger proteins to recognize DNA sequences.