A new bipartite DNA-binding domain: cooperative interaction between the cut repeat and homeo domain of the cut homeo proteins.
The recently cloned Clox (Cut-like homeo box) and CDP (CCAAT displacement protein), two mammalian counterparts of the Drosophila Cut homeo protein, correspond to alternatively spliced products of the same gene (mClox, for mammalian Cut-like homeo box). Although these proteins reportedly bind to apparently unrelated DNA sequences, we show by in vitro selection of optimal binding sites that both Clox and CDP have the same preferred DNA-binding specificity. The palindromic consensus target sequence, 5'-(t/a)(a/t)tATCGATTAt(t/c)(t/g)(t/a)-3', contains a bona fide homeo domain binding motif (ATTA). In addition, 37% of the in vitro-selected sequences have a CCAAT box, the canonical target for members of the family of CCAAT-binding factors. A characteristic feature of the cut homeo proteins is the presence of three evolutionarily conserved 73-amino-acid repeats of unknown function, the so-called cut repeats. We present evidence that the cut repeat II binds to mClox consensus targets independently of the DNA-binding activity of the homeo domain. In vitro selection of binding sites shows that the optimal targets for the cut repeat II contain one or more CCAAT boxes and, like the homeo domain, an ATTA core. These results indicate that the DNA-binding activity of the second cut repeat can account for the suggested role of CDP mClox as CCAAT displacement protein, a putative repressor of gene expression. We also report that the mClox homeo domain and cut repeat II interact in vitro in the absence of DNA. This interaction, which greatly enhances the DNA-binding activity of the binary complex, is specific to the cut homeo proteins. No cooperativity was observed between the cut repeat II and the homeo domains of Oct-1 and Gtx. Furthermore, the Drosophila cut repeat II, which does not appear to bind to DNA, also enhances the DNA-binding activity of the mClox homeo domain. Thus, the bifunctional cut repeat II, which defines a new family of bipartite DNA-binding proteins, is likely to play an important role in the function of the cut homeo proteins.
Andrés
V
V
Department of Cardiology, Children's Hospital, Boston, Massachusetts 02115.
Chiara
M D
MD
Mahdavi
V
V
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila; D005801:Genes, Homeobox; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D004247:DNA