Krüppel-associated boxes are potent transcriptional repression domains

Margolin et al. (1994). Proc Natl Acad Sci USA DOI: 10.1073/pnas.91.10.4509 Citations: 630

The KRAB domain occurs in a third of Kruppel zinc finger proteins but had no assigned function. This paper fused KRAB domains from four zinc finger proteins to GAL4 and tested repression. GAL4-KOX1 repressed transcription 17-fold, and a 45-residue KRAB A box sufficed alone.

Parts used

KRAB Domain

Key findings

  • Margolin et al. demonstrated that the KRAB domain of KOX1 functioned as a potent DNA-binding-site-dependent transcriptional repressor when fused to the GAL4 DNA-binding domain. The GAL4-KOX1-(1-161) fusion repressed a GAL-TK-CAT reporter 17-fold in NIH 3T3 cells, establishing that the KRAB domain transfers repression activity to heterologous DNA-binding contexts.

  • Truncation analysis defined a minimal 45-aa repression domain within the KRAB A box (residues 11–55 of KOX1). The KRAB B box was dispensable, and any deletion extending into the A box abolished repression. KRAB domains from four independent zinc finger proteins—KOX1, ZNF133, ZNF140, and ZNF141—each functioned as potent repressors when fused to GAL4.

  • Site-directed mutagenesis of conserved residues in the KRAB A box at DV, EEW, and MLE positions significantly inhibited transcriptional repression. The MLE sequence falls within the conserved VMLENY motif of a predicted amphipathic helix, suggesting that a protein-protein interaction surface on the KRAB A box mediates repression.

1 3

Abstract

The Krüppel-associated box (KRAB) is a highly conserved, 75-aa region containing two predicted amphipathic alpha-helices. The KRAB domain is present in the amino-terminal regions of more than one-third of all Krüppel-class Cys2His2 zinc finger proteins and is conserved from yeast to man; however, its function is unknown. Here it is shown that the KRAB domain functions as a DNA binding-dependent transcriptional repressor when fused to a heterologous DNA-binding domain from the yeast GAL4 protein. A 45-aa segment containing one of the predicted KRAB amphipathic helices was necessary and sufficient for repression. Amino acid substitutions in the predicted helix abolished the repression function. These results assign a function, transcriptional repression, to the highly conserved KRAB box and define a minimal repression domain which may aid in identifying mechanisms of repression.