Interaction between two homeodomain proteins is specified by a short C-terminal tail

Abstract

Two yeast homeodomain proteins, a1 and alpha 2, interact and cooperatively bind the haploid-specific gene (hsg) operator, resulting in the repression of a set of genes involved in the determination of cell type. The cooperative binding of a1 and alpha 2 to DNA can be reconstituted in vitro using purified fragments of a1 and alpha 2. Only the homeodomain is needed for a1, but for alpha 2 a C-terminal 22-amino-acid tail is required as well. As most of the specificity of DNA binding appears to derive from a1, we proposed that alpha 2 functions in the a1/alpha 2 heterodimer to contact a1 with its tail. By construction and analysis of several chimaeric proteins, we investigate how two DNA-binding proteins, one with low intrinsic specificity (alpha 2) and one with no apparent intrinsic DNA-binding ability (a1), can together create a highly specific DNA-binding activity. We show that the 22-amino-acid region of alpha 2 immediately C-terminal to the homeodomain, when grafted onto the a1 homeodomain, converts a1 to a strong DNA-binding protein. This alpha 2 tail can also be attached to the Drosophila engrailed homeodomain, and the chimaeric protein now binds cooperatively to DNA with a1, showing how a simple change can create a new homeodomain combination that specifically recognizes a new DNA operator.

Structural Determinants within Pbx1 That Mediate Cooperative DNA Binding with Pentapeptide-Containing Hox Proteins: Proposal for a Model of a Pbx1-Hox-DNA Complex

“…At this time, we cannot distinguish between the possibilities that residues 296 to 313 contribute directly to cooperativity by providing a protein-protein interaction surface or that they alter the conformation of the adjacent HD, maximizing its interaction with the Hox protein. In a somewhat similar case, fusion of the C-terminal tail of yeast ␣2 to a1 also enhanced the DNA binding by a1 HD, suggesting that in the a1-␣2 complex, binding of the C-terminal tail of ␣2 to a1 enhances DNA binding by a1 (51). Unlike the C-terminal tail of ␣2, which mediates cooperative DNA binding between ␣2 and a1 (51), the C-terminal conserved sequence of Pbx1 is not absolutely essential for Pbx1/Hox heterodimerization.…”

“…In yeasts, interaction of the HD protein ␣2 with either a second HD protein, a1, or with MCM1 (a homolog of the serum response factor [SRF]) specifies different DNA recognition motifs, impacts transcription of different target genes, and specifies distinct yeast cell types (22). A short protein sequence of ␣2 immediately N terminal of the HD mediates ␣2-MCM1 interaction (56), while the C-terminal tail of ␣2 is involved in the ␣2-a1 interaction (51). Cooperative interactions involving HD proteins are also documented for Phox1 (17), POU HD proteins (21,52,53,57,62), and Paired class HD proteins (60).…”

Differential Control of Transcription by Homologous Homeodomain Coregulators†

“…homeodomain, whereas a1 and MCM1 recognize very different regions of ␣2 that lie outside and on opposite sides of the homeodomain (30,39). Thus, the association of the Oct-1 homeodomain with the VP16 homologs demonstrates that more closely related coregulators can provide different specificities for transcriptional regulation through differential recognition of even closely related DNA-binding sites.…”

Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox proteins.

“…Most of the DNA-binding specificity derives from al which alone has no intrinsic DNA-binding activity. Contact of al, with the carboxy-terminal tail of a2, a low specificity DNA-binding protein by itself, creates a highly specific DNA-binding complex (Stark and Johnson 1994). Similar to al, full-length Pbxl has little or no intrinsic DNA binding ability even at high concentrations of 200 nM (C-P. Chang and M.L.…”