A single amino acid can determine the DNA binding specificity of homeodomain proteins

Many Drosophila developmental genes contain a DNA binding domain encoded by the homeobox. This homeodomain contains a region distantly homologous to the helix-turn-helix motif present in several prokaryotic DNA binding proteins. We investigated the nature of homeodomain-DNA interactions by making a series of mutations in the helix-turn-helix motif of the Drosophila homeodomain protein Paired (Prd). This protein does not recognize sequences bound by the homeodomain proteins Fushi tarazu (Ftz) or Bicoid (Bcd). We show that changing a single amino acid at the C-terminus of the recognition helix is both necessary and sufficient to confer the DNA binding specificity of either Ftz or Bcd on Prd. This simple rule indicates that the amino acids that determine the specificity of homeodomains are different from those mediating protein-DNA contacts in prokaryotic proteins. We further show that Prd contains two DNA binding activities. The Prd homeodomain is responsible for one of them while the other is not dependent on the recognition helix.     

Interaction of the BELL-like protein ATH1 with DNA: role of homeodomain residue 54 in specifying the different binding properties of BELL and KNOX proteins

We have studied the interaction of the BELL-like Arabidopsis homeodomain protein ATH1 with DNA. Analysis of oligonucleotides selected by the ATH1 homeodomain from a random mixture suggests that ATH1 preferentially binds the sequence TGACAGGT. Single nucleotide replacements at positions 2 or 3 of this sequence abolish binding, while changes at position 4 are more tolerated. Changes outside this core differentially affect binding, depending on the position. Hydroxyl radical footprinting and missing nucleoside experiments showed that ATH1 interacts with a 7-bp region of the strand carrying the GAC core. On the other strand, protection was observed over a 7-bp region, comprising one additional nucleotide complementary to T in position 1. A comparative analysis of the binding preferences of the homeodomains of ATH1 and STM (a KNOX homeodomain protein) indicated that they bind similar sequences, but with differences in affinity and specificity. The decreased affinity displayed by the ATH1 homeodomain correlates with the presence of valine (instead of lysine as in STM) at position 54. This difference also explains the decreased and increased selectivities, respectively, at positions 4 and 5. Our results point to an essential role of residue 54 in determining the different binding properties of BELL and KNOX homeodomains.     

Interaction of the PHD-finger homeodomain protein HAT3.1 from Arabidopsis thaliana with DNA. Specific DNA binding by a homeodomain with histidine at position 51

HAT3.1 is a member of the PHD-finger homeodomain protein family. The HAT3.1 homeodomain is highly divergent in sequence even at positions that are almost invariable among homeodomains. In this work, we have applied the random oligonucleotide selection technique to investigate if the HAT3.1 homeodomain is able to recognize specific DNA sequences. Analysis of the selected molecules followed by hydroxyl radical footprinting experiments and yeast one-hybrid assays indicated that HAT3.1 shows a preference for the sequence T(A/G)(A/C)ACCA, different from those bound by other homeodomains. Binding was dependent on homeodomain residues located at positions 47, 50, 51, and 54, the same positions that usually participate in DNA binding in most homeodomains. The study of the interaction of mutants at these positions with DNA carrying nucleotide changes at specific sites suggested that H51 and K50 most likely interact with nucleotides 2 to 4 and 5 to 6, respectively, while W54 would establish contacts with position 4. The presence of H51 and W54 represents an innovation among homeodomain structures. The fact that the HAT3.1 homeodomain is able to interact with specific DNA sequences is evidence of the inherent plasticity of the homeodomain as a DNA binding unit.     

DNA-binding specificity of the S8 homeodomain.

The murine S8 homeobox gene is expressed in a mesenchyme-specific pattern in embryos, as well as in mesodermal cell lines. The S8 homeodomain is overall similar to paired type homeodomains, but at position 50, which is crucial for specific DNA recognition, it contains a Gln, as is found in Antennapedia (Antp)-type homeodomains. We determined the DNA-binding specificity of the purified S8 homeodomain by in vitro selection of random oligonucleotides. The resulting 11-bp consensus binding site, ANC/TC/TAATTAA/GC resembles, but subtly differs from, the recognition sequences of Antp-type homeodomains. Equilibrium binding constants of down to 6.0 x 10(-10) M were found for binding of the S8 homeodomain to selected oligonucleotides. Using specific antibodies and an oligonucleotide containing an S8-site, we detected by band-shift two abundant DNA binding activities in mesodermal cell lines that correspond to S8 and two more that correspond to its close relative MHox. These S8 protein forms are differentially expressed in retinoic acid-treated P19 EC cells.     

Analysis of homeodomain specificities allows the family-wide prediction of preferred recognition sites

We describe the comprehensive characterization of homeodomain DNA-binding specificities from a metazoan genome. The analysis of all 84 independent homeodomains from D. melanogaster reveals the breadth of DNA sequences that can be specified by this recognition motif. The majority of these factors can be organized into 11 different specificity groups, where the preferred recognition sequence between these groups can differ at up to four of the six core recognition positions. Analysis of the recognition motifs within these groups led to a catalog of common specificity determinants that may cooperate or compete to define the binding site preference. With these recognition principles, a homeodomain can be reengineered to create factors where its specificity is altered at the majority of recognition positions. This resource also allows prediction of homeodomain specificities from other organisms, which is demonstrated by the prediction and analysis of human homeodomain specificities.     

The two homeodomains of the ZmHox2a gene from maize originated as an internal gene duplication and have evolved different target site specificities.

The maize ZmHox2a gene encodes two homeodomains which originated by a 699 bp duplication within an ancestral precursor. The sequences of the two ZmHox2a homeodomains are highly diverged in the N-terminal arm, while residues in the helical part have mostly been conserved. We show here that both ZmHox2a homeodomains are functional DNA-binding motifs but exhibit different target site specificities. CASTing experiments reveal a TCCT motif recognized by HD1 but a GATC tetranucleotide as the recognition sequence of HD2. Mutation of the central nucleotides in both tetranucleotide core motifs abolishes DNA binding. A domain swap experiment indicates that target site specificity is achieved in a combinatorial manner by the contributions of the diverged N-terminal arms together with the slightly different recognition helices. Computer modelling suggests that K47 and H54 in the recognition helices preferentially contact the bases at the 3'-terminus of the tetranucleotide target sequences.     

A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an alpha 2 dimer.

DNA recognition sequences for dimeric proteins typically contain two types of information. The first is the DNA sequence of each half-site, and the second is the arrangement of these half-sites. We show that dimers of the yeast homeodomain protein alpha 2, although able to read the first type of information, lack the ability to assess the second type. Rather, alpha 2 dimers bind with equal affinity to artificial operators in which the two half-sites are arrayed as inverted repeats, as direct repeats, or as everted (inside-out) repeats. We show that a second protein-MCM1-sets the exact spacing and orientation of the homeodomains in the alpha 2 dimer so that they accommodate only the geometry of the naturally occurring operators. These experiments show directly how the target specificity of a homeodomain protein is raised by an auxiliary protein, allowing it to distinguish the biologically correct operators from closely related sequences in the cell.