Secondary literature sources for the HOX domain

For each of the hand selected primary papers associated with the HOX domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

The yeast homeodomain protein MATalpha2 shows extended DNA binding specificity in complex with Mcm1.

Zhong H, Vershon AK
J Biol Chem. 1997; 272: 8402-9

The MATalpha2 (alpha2) repressor interacts with the Mcm1 protein to turn off a-cell type-specific genes in the yeast Saccharomyces cerevisiae. We compared five natural alpha2-Mcm1 sites with an alpha2-Mcm1 symmetric consensus site (AMSC) for their relative strength of repression and found that the AMSC functions slightly better than any of the natural sites. To further investigate the DNA binding specificity of alpha2 in complex with Mcm1, symmetric substitutions at each position in the alpha2 half-sites of AMSC were constructed and assayed for their effect on repression in vivo and DNA binding affinity in vitro. As expected, substitutions at positions in which there are base-specific contacts decrease the level of repression. Interestingly, substitutions at other positions, in which there are no apparent base-specific contacts made by the protein in the alpha2-DNA co-crystal structure, also significantly decrease repression. As an alternative method to examining the DNA binding specificity of alpha2, we performed in vitro alpha2 binding site selection experiments in the presence and absence of Mcm1. In the presence of Mcm1, the consensus sequences obtained were extended and more closely related to the natural alpha2 sites than the consensus sequence obtained in the absence of Mcm1. These results demonstrate that in the presence of Mcm1 the sequence specificity of alpha2 is extended to these positions.

Hox genes and the making of sphincters.

Zakany J, Duboule D
Nature. 1999; 401: 761-2

The nuclear hormone receptor Ftz-F1 is a cofactor for the Drosophila homeodomain protein Ftz.

Yu Y, Li W, Su K, Yussa M, Han W, Perrimon N, Pick L
Nature. 1997; 385: 552-5

Homeobox genes specify cell fate and positional identity in embryos throughout the animal kingdom. Paradoxically, although each has a specific function in vivo, the in vitro DNA-binding specificities of homeodomain proteins are overlapping and relatively weak. A current model is that homeodomain proteins interact with cofactors that increase specificity in vivo. Here we use a native binding site for the homeodomain protein Fushi tarazu (Ftz) to isolate Ftz-F1, a protein of the nuclear hormone-receptor superfamily and a new Ftz cofactor. Ftz and Ftz-F1 are present in a complex in Drosophila embryos. Ftz-F1 facilitates the binding of Ftz to DNA, allowing interactions with weak-affinity sites at concentrations of Ftz that alone bind only high-affinity sites. Embryos lacking Ftz-F1 display ftz-like pair-rule cuticular defects. This phenotype is a result of abnormal ftz function because it is expressed but fails to activate downstream target genes. Cooperative interaction between homeodomain proteins and cofactors of different classes may serve as a general mechanism to increase HOX protein specificity and to broaden the range of target sites they regulate.

Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS.

Yano M, Katayose Y, Ashikari M, Yamanouchi U, Monna L, Fuse T, Baba T, Yamamoto K, Umehara Y, Nagamura Y, Sasaki T
Plant Cell. 2000; 12: 2473-2484

A major quantitative trait locus (QTL) controlling response to photoperiod, Hd1, was identified by means of a map-based cloning strategy. High-resolution mapping using 1505 segregants enabled us to define a genomic region of approximately 12 kb as a candidate for Hd1. Further analysis revealed that the Hd1 QTL corresponds to a gene that is a homolog of CONSTANS in Arabidopsis. Sequencing analysis revealed a 43-bp deletion in the first exon of the photoperiod sensitivity 1 (se1) mutant HS66 and a 433-bp insertion in the intron in mutant HS110. Se1 is allelic to the Hd1 QTL, as determined by analysis of two se1 mutants, HS66 and HS110. Genetic complementation analysis proved the function of the candidate gene. The amount of Hd1 mRNA was not greatly affected by a change in length of the photoperiod. We suggest that Hd1 functions in the promotion of heading under short-day conditions and in inhibition under long-day conditions.

[Regulatory genes of flower development: members of the MADS box gene family]

Yamaguchi K, Kofuji R, Ueda K
Tanpakushitsu Kakusan Koso. 1994; 39: 2570-9

Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions.

Wolberger C, Vershon AK, Liu B, Johnson AD, Pabo CO
Cell. 1991; 67: 517-28

The MAT alpha 2 homeodomain regulates the expression of cell type-specific genes in yeast. We have determined the 2.7 A resolution crystal structure of the alpha 2 homeodomain bound to a biologically relevant DNA sequence. The DNA in this complex is contacted primarily by the third of three alpha-helices, with additional contacts coming from an N-terminal arm. Comparison of the yeast alpha 2 and the Drosophila engrailed homeodomain-DNA complexes shows that the protein fold is highly conserved, despite a 3-residue insertion in alpha 2 and only 27% sequence identity between the two homeodomains. Moreover, the orientation of the recognition helix on the DNA is also conserved. This docking arrangement is maintained by side chain contacts with the DNA--primarily the sugar-phosphate backbone--that are identical in alpha 2 and engrailed. Since these residues are conserved among all homeodomains, we propose that the contacts with the DNA are also conserved and suggest a general model for homeodomain-DNA interactions.

Structure and DNA binding of the yeast MAT alpha 2 homeodomain.

Wolberger C
Cold Spring Harb Symp Quant Biol. 1993; 58: 159-66

Conservation and diversification in homeodomain-DNA interactions: a comparative genetic analysis.

Wilson DS, Sheng G, Jun S, Desplan C
Proc Natl Acad Sci U S A. 1996; 93: 6886-91

Nearly all metazoan homeodomains (HDs) possess DNA binding targets that are related by the presence of a TAAT sequence. We use an in vitro genetic DNA binding site selection assay to refine our understanding of the amino acid determinants for the recognition of the TAAT site. Superimposed upon the conserved ability of metazoan HDs to recognize a TAAT core is a difference in their preference for the bases that lie immediately 3' to it. Amino acid position 50 of the HD has been shown to discriminate among these base pairs, and structural studies have suggested that water-mediated hydrogen bonds and van der Waals contacts underlie for this ability. Here, we show that each of six amino acids tested at position 50 can confer a distinct DNA binding specificity.

High resolution crystal structure of a paired (Pax) class cooperative homeodomain dimer on DNA.

Wilson DS, Guenther B, Desplan C, Kuriyan J
Cell. 1995; 82: 709-19

The crystal structure of the paired homeodomain bound to DNA as a cooperative dimer has been determined to 2.0 A resolution. Direct contacts between each homeodomain and the DNA are similar to those described previously. In addition, an extensive network of water molecules mediates contacts between the recognition helix and the DNA major groove. Several symmetrical contacts between the two homeodomains underlie the cooperative interaction, and deformations in the DNA structure are necessary for the establishment of these contacts. Comparison with structures of homeodomains bound monomerically to DNA suggests that the binding of a single paired homeodomain can introduce these DNA distortions, thus preparing a template for the cooperative interaction with a second homeodomain. This study shows how the paired (Pax) class homeodomains have achieved cooperativity in DNA binding without the assistance of other domains, thereby enabling the recognition of target sequences that are long enough to ensure specificity.

Structural basis of Hox specificity.

Wilson DS, Desplan C
Nat Struct Biol. 1999; 6: 297-300

DNA distortion and multimerization: novel functions of the glutamine-rich domain of GAGA factor.

Wilkins RC, Lis JT
J Mol Biol. 1999; 285: 515-25

GAGA factor (GAF) is an essential protein in Drosophila melanogaster, important for the transcriptional regulation of numerous genes. The effect of GAF on chromatin structure and promoter function has been the subject of much attention, yet little is known of the actual mechanism and the specific contributions of individual GAF domains to its function. The DNA-binding activity of GAF, as specified by the single zinc finger binding domain (Zn), has been examined in some detail; however, the functions of the POZ/BTB and glutamine domain (Q) remain poorly understood. Here, we report three separate activities of the Q domain of GAF; promoter distortion, single-strand binding, and multimerization. In vitro, GAF binding to the hsp70 promoter produces extended DNase I protection and KMnO4 hypersensitivity. These activities require both the Zn domain and Q domain of GAF, and appear independent of the POZ/BTB domain. GAF also has a single-stranded DNA binding affinity, as does the Q-rich region alone. GAF forms multimers both in vitro and in vivo, and the Q domain itself forms multimers. Protein-protein interactions mediated by the Q domain may, therefore, be at least partially responsible for the multimerization capabilities of GAF. We discuss these findings in the context of their possible function in GAF mediated transcriptional regulation.

Functional haploinsufficiency of the human homeobox gene MSX2 causes defects in skull ossification.

Wilkie AO, Tang Z, Elanko N, Walsh S, Twigg SR, Hurst JA, Wall SA, Chrzanowska KH, Maxson RE Jr
Nat Genet. 2000; 24: 387-90

The genetic analysis of congenital skull malformations provides insight into normal mechanisms of calvarial osteogenesis. Enlarged parietal foramina (PFM) are oval defects of the parietal bones caused by deficient ossification around the parietal notch, which is normally obliterated during the fifth fetal month. PFM are usually asymptomatic, but may be associated with headache, scalp defects and structural or vascular malformations of the brain. Inheritance is frequently autosomal dominant, but no causative mutations have been identified in non-syndromic cases. We describe here heterozygous mutations of the homeobox gene MSX2 (located on 5q34-q35) in three unrelated families with PFM. One is a deletion of approximately 206 kb including the entire gene and the others are intragenic mutations of the DNA-binding homeodomain (RK159-160del and R172H) that predict disruption of critical intramolecular and DNA contacts. Mouse Msx2 protein with either of the homeodomain mutations exhibited more than 85% reduction in binding to an optimal Msx2 DNA-binding site. Our findings contrast with the only described MSX2 homeodomain mutation (P148H), associated with craniosynostosis, that binds with enhanced affinity to the same target. This demonstrates that MSX2 dosage is critical for human skull development and suggests that PFM and craniosynostosis result, respectively, from loss and gain of activity in an MSX2-mediated pathway of calvarial osteogenic differentiation.

Crystal structure of Penicillium citrinum P1 nuclease at 2.8 A resolution.

Volbeda A, Lahm A, Sakiyama F, Suck D
EMBO J. 1991; 10: 1607-18

P1 nuclease from Penicillium citrinum is a zinc dependent glyco-enzyme consisting of 270 amino acid residues which cleaves single-stranded RNA and DNA into 5'-mononucleotides. The X-ray structure of a tetragonal crystal form of the enzyme with two molecules per asymmetric unit has been solved at 3.3 and refined at 2.8 A resolution to a crystallographic R-factor of 21.6%. The current model consists of 269 amino acid residues, three Zn ions and two N-acetyl glucosamines per subunit. The enzyme is folded very similarly to phospholipase C from Bacillus cereus, with 56% of the structure displaying an alpha-helical conformation. The three Zn ions are located at the bottom of a cleft and appear to be rather inaccessible for any phosphate group in double-stranded RNA or DNA substrates. A crystal soaking experiment with a dinucleotide gives clear evidence for two mononucleotide binding sites separated by approximately 20 A. One site shows binding of the phosphate group to one of the zinc ions. At both sites there is a hydrophobic binding pocket for the base, but no direct interaction between the protein and the deoxyribose. A cleavage mechanism is proposed involving nucleophilic attack by a Zn activated water molecule.

Structural study of homeodomain protein-DNA complexes using a homology modeling approach.

Tung CS
J Biomol Struct Dyn. 1999; 17: 347-54

The homeodomain is a conserved protein motif that binds to DNA and plays a central role in gene regulation. We use homeodomain as a model system to study the specific interactions between protein and DNA in a complex. Following the fundamental concept of homology modeling, we have developed an algorithm for predicting structures of both protein and DNA using the known structure of a similar complex as the template. The accuracies of the algorithm in predicting the complex structures are evaluated when two of the homeodomain protein-DNA complexes with known structures (antennapedia and MATalpha2) are selected as test systems. This algorithm allows structural studies of homeodomain binds to DNA with different sequences.

Modeling of the homoeo domain suggests similar structure to repressors.

Tsonis PA, Carperos V, Siahaan T, Shiahaan T
Biochem Biophys Res Commun. 1988; 157: 100-5

The sequences of the homoeo domain containing the three alpha-helices were modeled based on secondary structure prediction, sequence homologies and coordinates of known helix-turn-helix motif containing DNA-binding proteins. The model reveals very similar three dimensional structure to repressors and suggest binding to DNA with its helix 3.

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

Treisman J, Gonczy P, Vashishtha M, Harris E, Desplan C
Cell. 1989; 59: 553-62

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.

Several polymers enhance the sensitivity of the southwestern assay.

Tomaska L, Nosek J
Anal Biochem. 1995; 227: 387-9

A rapidly evolving homeobox at the site of a hybrid sterility gene.

Ting CT, Tsaur SC, Wu ML, Wu CI
Science. 1998; 282: 1501-4

The homeodomain is a DNA binding motif that is usually conserved among diverse taxa. Rapidly evolving homeodomains are thus of interest because their divergence may be associated with speciation. The exact site of the Odysseus (Ods) locus of hybrid male sterility in Drosophila contains such a homeobox gene. In the past half million years, this homeodomain has experienced more amino acid substitutions than it did in the preceding 700 million years; during this period, it has also evolved faster than other parts of the protein or even the introns. Such rapid sequence divergence is driven by positive selection and may contribute to reproductive isolation.

Crystal structure of the yeast MATalpha2/MCM1/DNA ternary complex.

Tan S, Richmond TJ
Nature. 1998; 391: 660-6

The structure of a complex containing the homeodomain repressor protein MATalpha2 and the MADS-box transcription factor MCM1 bound to DNA has been determined by X-ray crystallography at 2.25 A resolution. It reveals the protein-protein interactions responsible for cooperative binding of MATalpha2 and MCM1 to DNA. The otherwise flexible amino-terminal extension of the MATalpha2 homeodomain forms a beta-hairpin that grips the MCM1 surface through parallel beta-strand hydrogen bonds and close-packed, predominantly hydrophobic, side chains. DNA bending induced by MCM1 brings the two proteins closer together, facilitating their interaction. An unusual feature of the complex is that an eight-amino-acid sequence adopts an alpha-helical conformation in one of two copies of the MATalpha2 monomer and a beta-strand conformation in the other. This 'chameleon' sequence of MATalpha2 may be important for recognizing natural operator sites.

Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization.

Tan S, Hunziker Y, Pellegrini L, Richmond TJ
J Mol Biol. 2000; 297: 947-59

We describe our efforts to crystallize binary MCM1/DNA and ternary MATalpha2/MCM1/DNA complexes, including the unsuccessful attempts to crystallize MCM1/DNA complexes and the successful design of DNA crystal packing that resulted in high-resolution crystals of the MATalpha2/MCM1/DNA complex. We detail general procedures useful for preparing protein/DNA cocrystals, including improved methods for producing and purifying DNA-binding proteins and DNA fragments, for purifying protein/DNA complexes, and for controlling pH conditions during crystallization. We also describe the rational design of DNA for protein/DNA cocrystallization attempts, based on our analysis of how straight and bent DNA with single base-pair overhangs can pack end-to-end in a crystal.

The optimal binding sequence of the Hox11 protein contains a predicted recognition core motif.

Tang S, Breitman ML
Nucleic Acids Res. 1995; 23: 1928-35

HOX11 is a homeobox-containing oncogene of specific T-cell leukemias. We determined the DNA binding specificity of the Hox11 protein by using a novel technique of random oligonucleotide selection developed in this study. The optimal Hox11 binding sequence, GGCGGTAAGTGG, contained a core TAAGTG motif that is consistent with a prediction based on the residues at specific positions that potentially make DNA base contacts and models of homeodomain-DNA interaction proposed from studies with other homeodomains. The specific interaction between Hox11 and the selected optimal binding sequence was further confirmed by band-shift and DNA competition assays. Given that the Hox11 homeodomain shares low homology with other well studied homeodomains, the presence of a predictable recognition core motif in its optimal binding sequence supports the notion that different homeodomains interact with DNA in a similar manner, through highly conserved residues at specific positions that allow contact with DNA.

The structure of a pseudo intercalated complex between actinomycin and the DNA binding sequence d(GpC).

Takusagawa F, Dabrow M, Neidle S, Berman HM
Nature. 1982; 296: 466-9

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

Stark MR, Johnson AD
Nature. 1994; 371: 429-32

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.

Homeodomain proteins Mox1 and Mox2 associate with Pax1 and Pax3 transcription factors.

Stamataki D, Kastrinaki M, Mankoo BS, Pachnis V, Karagogeos D
FEBS Lett. 2001; 499: 274-8

Mox1 and Mox2 homeobox genes have been shown to be critical in axial skeleton and in limb muscle development respectively. Pax1 and Pax3 gene products are also implicated in these processes. Mox and Pax expression patterns are highly overlapping both spatially and temporally during embryonic development. We show here for the first time that Mox proteins physically interact with Pax1 and Pax3 using the yeast two-hybrid protein interaction assay as well as in vitro biochemical assays. There is a strong preference of Mox1 to associate with Pax1 rather than Pax3 and of Mox2 to associate with Pax3 rather than Pax1. The observed interactions are mediated through the homeodomain of Mox.

Operator-constitutive mutations in a DNA sequence recognized by a yeast homeodomain.

Smith DL, Johnson AD
EMBO J. 1994; 13: 2378-87

Homeodomain proteins regulate transcription in organisms as diverse as yeasts, mammals and plants, often effecting key decisions in development. Although homeodomains can selectively recognize certain DNA sequences, a question has arisen as to how specific this interaction is and how much it contributes to the ability of these proteins to properly select target genes in the cell. This question is particularly an issue in cases where the homeodomain proteins recognize DNA cooperatively with other DNA-binding proteins. In this paper, we examine the issue of DNA binding specificity for the homeodomain of the yeast alpha 2 protein (which recognizes the a-specific gene operator cooperatively with the MCM1 protein) by examining both in vivo and in vitro the effects of point mutations in its recognition sequence. We found that most changes in the homeodomain recognition sequence produced only small effects on both homeodomain affinity as measured in vitro (with and without the helper protein MCM1) and operator function as determined in vivo. This tolerance for operator mutations illustrates in a systematic way the modest DNA-binding specificity of the alpha 2 homeodomain and contrasts with the behavior of many of the bacterial and phage repressors where single point mutations in the operator can have dramatic effects on affinity. This tolerance for different sequences may arise from the fact that most of the interactions made between the alpha 2 homeodomain and the DNA occur through long amino acid side chains; we suggest that these side chains can reconfigure in order to create surfaces complementary to many different DNA sequences. The relaxed DNA-binding specificity of homeodomain proteins such as alpha 2 may be an important feature that permits new regulatory circuits to evolve rapidly from existing components.

DNA bending by the a1 and alpha 2 homeodomain proteins from yeast.

Smith DL, Desai AB, Johnson AD
Nucleic Acids Res. 1995; 23: 1239-43

Structural and biochemical studies of monomer homeodomain-DNA complexes have not so far revealed any cases of pronounced DNA distortion. In this paper we show that multimeric complexes of the yeast homeodomain proteins a1 and alpha 2 induced significant bends in their operators upon binding. Based on a series of circular permutation experiments, we found that a dimer of alpha 2 bound to operator DNA produced a mild bend in the DNA, whereas the alpha 2-MCM1-DNA and the a1-alpha 2-DNA complexes exhibited much sharper bends. As these latter two complexes represent the in vivo form of DNA-bound a1 and alpha 2, we conclude that, in the cell, these homeodomain proteins are associated with pronounced bends in DNA. We discuss possible roles for these bends in transcriptional repression.

Design of a "minimAl" homeodomain: the N-terminal arm modulates DNA binding affinity and stabilizes homeodomain structure.

Shang Z, Isaac VE, Li H, Patel L, Catron KM, Curran T, Montelione GT, Abate C
Proc Natl Acad Sci U S A. 1994; 91: 8373-7

This report investigates the sequence specificity requirements for homeodomain structure and DNA binding activity by the design and synthesis of a "minimAl" homeodomain (for minimalist design and alanine scanning mutagenesis) which contains the consensus residues and in which all nonconsensus residues have been replaced with alanine. The murine homeodomain Msx served as the prototype for the minimAl homeodomain, Ala-Msx. We show that Ala-Msx binds to DNA specifically, albeit with lower affinity than Msx. A derivative of the minimAl homeodomain, Ala-Msx(NT), which contains a native rather than an alanine-substituted N-terminal arm, has similar DNA binding affinity as Msx. We show that the native N-terminal arm stabilizes the tertiary structure of the minimAl homeodomain. Although Ala-Msx resembles a molten-globule protein, the structure of Ala-Msx(NT) is similar to Msx. The requirement for an intact N-terminal arm is not unique to the minimAl homeodomain, since the N-terminal arm also promotes high-affinity binding activity and appropriate tertiary structure of Msx. Therefore, the homeodomain "scaffold" consists of consensus residues, which are sufficient for DNA recognition, and nonconsensus residues in the N-terminal arm, which are required for optimal DNA binding affinity and appropriate tertiary structure. MinimAl design provides a powerful strategy to probe homeodomain structure and function. This approach should be of general utility to study the sequence specificity requirements for structure and function of other DNA-binding domains.

DNA affinity cleaving analysis of homeodomain-DNA interaction: identification of homeodomain consensus sites in genomic DNA.

Shang Z, Ebright YW, Iler N, Pendergrast PS, Echelard Y, McMahon AP, Ebright RH, Abate C
Proc Natl Acad Sci U S A. 1994; 91: 118-22

We have incorporated the DNA-cleaving moiety o-phenanthroline-copper at amino acid 10 of the Msx-1 homeodomain, and we have analyzed site-specific DNA cleavage by the resulting Msx-1 derivative. We show that amino acid 10 of the Msx-1 homeodomain is close to the 5' end of the consensus DNA site 5'-(C/G)TAATTG-3' in the Msx-1-DNA complex. Our results indicate that the orientation of the Msx-1 homeodomain relative to DNA is analogous to the orientation of the engrailed and Antennapedia homeodomains. We show further that DNA affinity cleaving permits identification of consensus DNA sites for Msx-1 in kilobase DNA substrates. The specificity of the approach enabled us to identify an Msx-1 consensus DNA site within the transcriptional control region of the developmental regulatory gene Wnt-1. We propose that incorporation of o-phenanthroline-copper at amino acid 10 of a homeodomain may provide a generalizable strategy to determine the orientation of a homeodomain relative to DNA and to identify homeodomain consensus DNA sites in genomic DNA.

DNA binding and transcriptional properties of wild-type and mutant forms of the homeodomain protein Msx2.

Semenza GL, Wang GL, Kundu R
Biochem Biophys Res Commun. 1995; 209: 257-62

Msx2 is a mammalian homeodomain protein that is expressed during craniofacial development. A proline-to-histidine substitution at residue 148 of human Msx2 results in an autosomal dominant form of craniosynostosis. In this study, both wild-type and mutant Msx2 were shown to specifically bind to a DNA sequence previously identified as a high-affinity binding site for the related homeodomain protein Msx1. In co-transfection assays both wild-type and mutant Msx2 repressed reporter gene transcription in a dose-dependent but binding-site-independent manner. These results provide evidence that Msx2 is a transcriptional repressor and suggest that the mutant form of Msx2 may exert its pathophysiologic effects on craniofacial development by a gain-of-function mechanism.

A model for histone H5-DNA interaction: simultaneous minor and major groove binding.

Segers A, Wyns L, Lasters I
Biochem Biophys Res Commun. 1991; 174: 898-902

Using the tertiary structure of the globular domain of H5 (GH5) and based on an alternative sequence homology between GH5 and DNA-binding proteins containing the helix-turn-helix motif, a model for H5-DNA interaction is proposed. From molecular graphics it follows that helix II recognizes the major groove of the DNA, as does the second helix of the helix-turn-helix motif, while helix III makes minor groove contacts, in agreement with the hypothesis of Turnell et al. (FEBS letters 232, 263-268). In the resulting model GH5 makes contact with a full turn of DNA.

The structure and function of the homeodomain.

Scott MP, Tamkun JW, Hartzell GW 3rd
Biochim Biophys Acta. 1989; 989: 25-48

Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices.

Schumacher MA, Choi KY, Zalkin H, Brennan RG
Science. 1994; 266: 763-70

The three-dimensional structure of a ternary complex of the purine repressor, PurR, bound to both its corepressor, hypoxanthine, and the 16-base pair purF operator site has been solved at 2.7 A resolution by x-ray crystallography. The bipartite structure of PurR consists of an amino-terminal DNA-binding domain and a larger carboxyl-terminal corepressor binding and dimerization domain that is similar to that of the bacterial periplasmic binding proteins. The DNA-binding domain contains a helix-turn-helix motif that makes base-specific contacts in the major groove of the DNA. Base contacts are also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. Critical to hinge helix-minor groove binding is the intercalation of the side chains of Leu54 and its symmetry-related mate, Leu54', into the central CpG-base pair step. These residues thereby act as "leucine levers" to pry open the minor groove and kink the purF operator by 45 degrees.

Repression by HoxA7 is mediated by the homeodomain and the modulatory action of its N-terminal-arm residues.

Schnabel CA, Abate-Shen C
Mol Cell Biol. 1996; 16: 2678-88

Hox genes encode homeodomain-containing proteins that are presumed to control spatial patterning during murine embryogenesis through their actions as transcriptional regulatory proteins. In this study, we have investigated the transcriptional function of a prototypic member of this family, HoxA7. We demonstrate that HoxA7 function as a potent transcriptional repressor and that its action as such requires several domains, including both activator and repressor regions. The repressor regions are contained within the homeodomain and a C-terminal acidic region, both of which are well conserved among members of the Hox family. Accordingly, we show that two other members of this family also function as repressors, although they vary in their relative repressor potency. Finally, we explore the novel observation that the homeodomain of HoxA7 functions as a transcriptional repressor domain. We show that the homeodomain compared with two other DNA-binding domains, is unique in its ability to function as a repressor domain and that repression requires conserved residues, in helix III. We further show that residues in the N-terminal arm of the homeodomain contribute to the differential repressor actions of various Hox proteins. These findings demonstrate that the transcriptional function of HoxA7 and possibility of Hox proteins in general is determined by their unique combination of conserved and nonconserved regions as well as through the complex actions of their homeodomains.

The binding of Ku antigen to homeodomain proteins promotes their phosphorylation by DNA-dependent protein kinase.

Schild-Poulter C, Pope L, Giffin W, Kochan JC, Ngsee JK, Traykova-Andonova M, Hache RJ
J Biol Chem. 2001; 276: 16848-56

The Ku antigen (70- and 80-kDa subunits) is a regulatory subunit of DNA-dependent protein kinase (DNA-PK) that promotes the recruitment of the catalytic subunit of DNA-PK (DNA-PKcs) to DNA ends and to specific DNA sequences from which the kinase is activated. Ku and DNA-PKcs plays essential roles in double-stranded DNA break repair and V(D)J recombination and have been implicated in the regulation of specific gene transcription. In a yeast two-hybrid screen of a Jurkat T cell cDNA library, we have identified a specific interaction between the 70-kDa subunit of Ku heterodimer and the homeodomain of HOXC4, a homeodomain protein expressed in the hematopoietic system. Unexpectedly, a similar interaction with Ku was observed for several additional homeodomain proteins including octamer transcription factors 1 and 2 and Dlx2, suggesting that specific binding to Ku may be a property shared by many homeodomain proteins. Ku-homeodomain binding was mediated through the extreme C terminus of Ku70 and was abrogated by amino acid substitutions at Lys595/Lys596. Ku binding allowed the recruitment of the homeodomain to DNA ends and dramatically enhanced the phosphorylation of homeodomain-containing proteins by DNA-PK. These results suggest that Ku functions as a substrate docking protein for signaling by DNA-PK to homeodomain proteins from DNA ends.

Specific DNA binding of the two chicken Deformed family homeodomain proteins, Chox-1.4 and Chox-a.

Sasaki H, Yokoyama E, Kuroiwa A
Nucleic Acids Res. 1990; 18: 1739-47

The cDNA clones encoding two chicken Deformed (Dfd) family homeobox containing genes Chox-1.4 and Chox-a were isolated. Comparison of their amino acid sequences with another chicken Dfd family homeodomain protein and with those of mouse homologues revealed that strong homologies are located in the amino terminal regions and around the homeodomains. Although homologies in other regions were relatively low, some short conserved sequences were also identified. E. coli-made full length proteins were purified and used for the production of specific antibodies and for DNA binding studies. The binding profiles of these proteins to the 5'-leader and 5'-upstream sequences of Chox-1.4 and Chox-a coding regions were analyzed by immunoprecipitation and DNase I footprint assays. These two Chox proteins bound to the same sites in the 5'-flanking sequences of their coding regions with various affinities and their binding affinities to each site were nearly the same. The consensus sequences of the high and low affinity binding sites were TAATGA(C/G) and CTAATTTT, respectively. A clustered binding site was identified in the 5'-upstream of the Chox-a gene, suggesting that this clustered binding site works as a cis-regulatory element for auto- and/or cross-regulation of Chox-a gene expression.

Alx-4: cDNA cloning and characterization of a novel paired-type homeodomain protein.

Qu S, Li L, Wisdom R
Gene. 1997; 203: 217-23

Homeodomain containing transcription factors serve important functions in patterning the embryo during vertebrate development. We have isolated cDNA clones encoding a novel protein, named Alx-4, that contains a paired-type homeodomain. Analysis of the homeodomain sequence shows that Alx-4 belongs to a family of genes that are related to the Drosophila gene aristaless, and includes the mammalian genes Alx3, Cart-1, MHox, and S8. We have analyzed the expression of Alx-4 during development by Northern blot and whole mount in situ hybridization. In addition, we have generated antibodies to recombinant Alx-4 protein and identified Alx-4 protein in nuclear extracts prepared from mouse embryos. The expression pattern of Alx-4 suggests that it may play a role in the patterning of structures derived from craniofacial mesenchyme, the first branchial arch, and the limb bud. Our results provide a starting point for the analysis of a new member of the family of paired type homeodomain proteins.

Dissection of the bifunctional ARGRII protein involved in the regulation of arginine anabolic and catabolic pathways.

Qui HF, Dubois E, Messenguy F
Mol Cell Biol. 1991; 11: 2169-79

ARGRII is a regulatory protein which regulates the arginine anabolic and catabolic pathways in combination with ARGRI and ARGRIII. We have investigated, by deletion analysis and fusion to LexA protein, the different domains of ARGRII protein. In contrast to other yeast regulatory proteins, 92% of ARGRII is necessary for its anabolic repression function and 80% is necessary for its catabolic activator function. We can define three domains in this protein: a putative DNA-binding domain containing a zinc finger motif, a region more involved in the repression activity located around the RNase-like sequence, and a large activation domain.

Analysis of homeodomain function by structure-based design of a transcription factor.

Pomerantz JL, Pabo CO, Sharp PA
Proc Natl Acad Sci U S A. 1995; 92: 9752-6

The homeodomain is a 60-amino acid module which mediates critical protein-DNA and protein-protein interactions for a large family of regulatory proteins. We have used structure-based design to analyze the ability of the Oct-1 homeodomain to nucleate an enhancer complex. The Oct-1 protein regulates herpes simplex virus (HSV) gene expression by participating in the formation of a multiprotein complex (C1 complex) which regulates alpha (immediate early) genes. We recently described the design of ZFHD1, a chimeric transcription factor containing zinc fingers 1 and 2 of Zif268, a four-residue linker, and the Oct-1 homeodomain. In the presence of alpha-transinduction factor and C1 factor, ZFHD1 efficiently nucleates formation of the C1 complex in vitro and specifically activates gene expression in vivo. The sequence specificity of ZFHD1 recruits C1 complex formation to an enhancer element which is not efficiently recognized by Oct-1. ZFHD1 function depends on the recognition of the Oct-1 homeodomain surface. These results prove that the Oct-1 homeodomain mediates all the protein-protein interactions that are required to efficiently recruit alpha-transinduction factor and C1 factor into a C1 complex. The structure-based design of transcription factors should provide valuable tools for dissecting the interactions of DNA-bound domains in other regulatory circuits.

V(D)J recombination. Ragtime jumping.

Plasterk R
Nature. 1998; 394: 718-9

The beta-ribbon DNA recognition motif.

Phillips SE
Annu Rev Biophys Biomol Struct. 1994; 23: 671-701

The interaction with DNA of wild-type and mutant fushi tarazu homeodomains.

Percival-Smith A, Muller M, Affolter M, Gehring WJ
EMBO J. 1990; 9: 3967-74

The in vitro DNA binding properties of wild-type and mutant fushi tarazu homeodomains (ftz HD) have been analysed. The DNA binding properties of the ftz HD are very similar to those of the Antp HD. In interference experiments with mutant ftz HDs, close approaches between specific portions of the ftz HD peptide and specific regions of the binding site DNA were mapped. A methylation interference, G7 on the beta strand of BS2, is absent from the interference pattern with a mutant ftz HD [ftz (R43A) HD] in which the Arg43 at the second position of helix III (the recognition helix) is replaced by an Ala. This indicated that Arg43 of the ftz HD is in close proximity to the N7 of G7 of the beta strand of BS2 in the major groove. The methylation and ethylation interference patterns with the ftz (NTD) HD, in which the first six amino acids of the homeodomain were deleted, were extensively altered relative to the ftz HD patterns. Methylation of A11 and G12 of the alpha strand and ethylation of the phosphate of nucleotide A12 of the alpha strand no longer interfere with binding. This indicated that the first six amino acids of the homeodomain of ftz interact with A11 of the alpha strand in the minor groove, the phosphate of the nucleotide A13 on the alpha strand and G12 of the alpha strand in the adjacent major groove of BS2. In a binding study using a change of specificity mutation [ftz (Q50K) HD], in which the Gln50 at the ninth position of the third helix is exchanged for a Lys (as in the bicoid HD), and variant binding sites, we concluded that position 50 of the ftz HD and the ftz (Q50K) HD peptides interacts with base pairs at positions 6 and 7 of BS2. These three points of contact allowed us to propose a crude orientation of the ftz HD within the protein-DNA complex. We find that the ftz HD and the Antp HD peptides contact DNA in a similar way.

Functional interference between contacting amino acids of homeodomains.

Pellizzari L, Tell G, Fabbro D, Pucillo C, Damante G
FEBS Lett. 1997; 407: 320-4

In a protein, the function of an amino acid at some position depends on the amino acids at other positions. Here we demonstrate a functional interference between base-contacting amino acids (at positions 50 and 54) of homeodomains. When, in the context of Antennapedia or Goosecoid homeodomains, Lys50 is paired to Tyr54 or Ala54 and Gln50 is paired to Met54, the resulting proteins efficiently discriminate among different DNA sequences. In contrast, in the presence of the pair Lys50-Met54, both homeodomains show a reduced capability to discriminate among different DNA sequences. Sequence selection experiments performed in the context of the Goosecoid homeodomain suggest that the presence of Met54 precludes the base-discriminating function of Lys50. These results may explain why the pair Lys50-Met54 is never found in natural homeodomains.

Structure of a DNA-bound Ultrabithorax-Extradenticle homeodomain complex.

Passner JM, Ryoo HD, Shen L, Mann RS, Aggarwal AK
Nature. 1999; 397: 714-9

During the development of multicellular organisms, gene expression must be tightly regulated, both spatially and temporally. One set of transcription factors that are important in animal development is encoded by the homeotic (Hox) genes, which govern the choice between alternative developmental pathways along the anterior-posterior axis. Hox proteins, such as Drosophila Ultrabithorax, have low DNA-binding specificity by themselves but gain affinity and specificity when they bind together with the homeoprotein Extradenticle (or Pbxl in mammals). To understand the structural basis of Hox-Extradenticle pairing, we determine here the crystal structure of an Ultrabithorax-Extradenticle-DNA complex at 2.4 A resolution, using the minimal polypeptides that form a cooperative heterodimer. The Ultrabithorax and Extradenticle homeodomains bind opposite faces of the DNA, with their DNA-recognition helices almost touching each other. However, most of the cooperative interactions arise from the YPWM amino-acid motif of Ultrabithorax-located amino-terminally to its homeodomain-which forms a reverse turn and inserts into a hydrophobic pocket on the Extradenticle homeodomain surface. Together, these protein-DNA and protein-protein interactions define the general principles by which homeotic proteins interact with Extradenticle (or Pbx1) to affect development along the anterior-posterior axis of animals.

Positively charged residues at the N-terminal arm of the homeodomain are required for efficient DNA binding by homeodomain-leucine zipper proteins.

Palena CM, Tron AE, Bertoncini CW, Gonzalez DH, Chan RL
J Mol Biol. 2001; 308: 39-47

Plant homeodomain-leucine zipper proteins, unlike most animal homeodomains, bind DNA efficiently only as dimers. In the present work, we report that the deletion of the homeodomain N-terminal arm (first nine residues) of the homeodomain-leucine zipper protein Hahb-4 dramatically affects its DNA-binding affinity, causing a 70-fold increase in dissociation constant. The addition of the N-terminal arm of Drosophila Antennapedia to the truncated form restores the DNA-binding affinity of dimers to values similar to those of the native form. However, the Antennapedia N-terminal arm is not able to confer increased binding affinity to monomers of Hahb-4 lacking the leucine zipper motif, indicating that the inefficient binding of monomers must be due to structural differences in other parts of the molecule. The construction of proteins with modifications at residues 5 to 7 of the homeodomain suggests strongly that positively charged amino acids at these positions play essential roles in determining the DNA-binding affinity. However, the effect of mutations at positions 6 and 7 can be counteracted by introducing a stretch of positively charged residues at positions 1 to 3 of the homeodomain. Sequence comparisons indicate that all homeodomain-leucine zipper proteins might use contacts of the N-terminal arm with DNA for efficient binding. The occurrence of a homeodomain with a DNA-interacting N-terminal arm must then be an ancient acquisition in evolution, earlier than the separation of lines leading to metazoa, fungi and plants. Copyright 2001 Academic Press.

Protein--DNA contacts in the structure of a homeodomain--DNA complex determined by nuclear magnetic resonance spectroscopy in solution.

Otting G, Qian YQ, Billeter M, Muller M, Affolter M, Gehring WJ, Wuthrich K
EMBO J. 1990; 9: 3085-92

The 1:1 complex of the mutant Antp(C39----S) homeodomain with a 14 bp DNA fragment corresponding to the BS2 binding site was studied by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. The complex has a molecular weight of 17,800 and its lifetime is long compared with the NMR chemical shift time scale. Investigations of the three-dimensional structure were based on the use of the fully 15N-labelled protein, two-dimensional homonuclear proton NOESY with 15N(omega 2) half-filter, and heteronuclear three-dimensional NMR experiments. Based on nearly complete sequence-specific resonance assignments, both the protein and the DNA were found to have similar conformations in the free form and in the complex. A sufficient number of intermolecular 1H-1H Overhauser effects (NOE) could be identified to enable a unique docking of the protein on the DNA, which was achieved with the use of an ellipsoid algorithm. In the complex there are intermolecular NOEs between the elongated second helix in the helix-turn-helix motif of the homeodomain and the major groove of the DNA. Additional NOE contacts with the DNA involve the polypeptide loop immediately preceding the helix-turn-helix segment, and Arg5. This latter contact is of special interest, both because Arg5 reaches into the minor groove and because in the free Antp(C39----S) homeodomain no defined spatial structure could be found for the apparently flexible N-terminal segment comprising residues 0-6.

Comparison of the free and DNA-complexed forms of the DNA-binding domain from c-Myb.

Ogata K, Morikawa S, Nakamura H, Hojo H, Yoshimura S, Zhang R, Aimoto S, Ametani Y, Hirata Z, Sarai A, et al.
Nat Struct Biol. 1995; 2: 309-20

The DNA-binding domain of c-Myb consists of three imperfect tandem repeats (R1, R2 and R3). The three repeats have similar overall architectures, each containing a helix-turn-helix variation motif. The three conserved tryptophans in each repeat participate in forming a hydrophobic core. Comparison of the three repeat structures indicated that cavities are found in the hydrophobic core of R2, which is thermally unstable. On complexation with DNA, the orientations of R2 and R3 are fixed by tight binding and their conformations are slightly changed. No significant changes occur in the chemical shifts of R1 consistent with its loose interaction with DNA.

Sequence-specific DNA binding activity in the RAE28 protein, a mouse homologue of the Drosophila polyhomeotic protein.

Nomura M, Takihara Y, Abdul Motaleb M, Horie K, Higashinakagawa T, Shimada K
Biochem Mol Biol Int. 1998; 46: 905-12

The rae28 gene, a mouse homologue of the Drosophila polyhomeotic gene, is involved in the maintenance of the transcriptional repression states of Hox genes. In this study we synthesized the glutathione S transferase-RAE28 (GST-RAE28) fusion protein and examined sequence-specific DNA binding activity in the RAE28 protein by using the selected and amplified binding site method. After five rounds of enrichment, the eluted DNAs were amplified, cloned and sequenced. The sequences of individual oligonucleotides included the following consensus sequences; 5'-ACCA-3', 5'-ACCCA-3', 5'-CTATCA-3' and 5'-TGCC-3'. The oligonucleotides including these consensus sequences were show to have significant affinity with the GST-RAE28 fusion protein. The RAE28 protein was recently shown to form multimeric protein complexes with other members of mouse Pc-G proteins in the nucleus. These findings strongly suggest that the RAE28 protein constitutes a sequence-specific DNA binding domain in multimeric Pc-G protein complexes.

The hexapeptide LFPWMR in Hoxb-8 is required for cooperative DNA binding with Pbx1 and Pbx2 proteins.

Neuteboom ST, Peltenburg LT, van Dijk MA, Murre C
Proc Natl Acad Sci U S A. 1995; 92: 9166-70

The Hox gene products are DNA-binding proteins, containing a homeodomain, which function as a class of master control proteins establishing the body plan in organisms as diverse as Drosophila and vertebrates. Hox proteins have recently been shown to bind cooperatively to DNA with another class of homeodomain proteins that include extradenticle, Pbx1, and Pbx2. Hox gene products contain a highly conserved hexapeptide connected by a linker of variable length to the homeodomain. We show that the hexapeptide and the linker region are required for cooperativity with Pbx1 and Pbx2 proteins. Many of the conserved residues present in the Hoxb-8 hexapeptide are required to modulate the DNA binding of the Pbx proteins. Position of the hexapeptide relative to the homeodomain is important. Although deletions of two and four residues of the linker peptide still show cooperative DNA binding, removal of all six linker residues strongly reduces cooperativity. In addition, an insertion of 10 residues within the linker peptide significantly lowers cooperative DNA binding. These results show that the hexapeptide and the position of the hexapeptide relative to the homeodomain are important determinants to allow cooperative DNA binding involving Hox and Pbx gene products.

The DNA binding specificity of the Drosophila fushi tarazu protein: a possible role for DNA bending in homeodomain recognition.

Nelson HB, Laughon A
New Biol. 1990; 2: 171-8

Segmentation in Drosophila melanogaster is controlled by a network of interacting genes, many of which encode a homeodomain that confers sequence-specific binding to DNA. One of these, fushi tarazu (ftz), is a transcription factor that regulates a number of segmentation and homeotic genes, including Antennapedia (Antp). To determine the DNA binding specificity of the ftz homeodomain, we performed DNase I footprint analysis on ftz protein binding sites located near the two Antp promoters using a beta-galactosidase/ftz fusion protein synthesized in E. coli. A consensus sequence for the fusion protein's preferred binding site was derived from 19 sites. The consensus sequence contains an ATTA motif, as do the reported consensus sequences for the engrailed (en), even-skipped (eve), and bicoid (bcd) Drosophila homeodomain proteins. We propose DNA bending as an explanation for the presence of a shared motif between proteins with divergent recognition helices: according to this model, bases in ATTA would not directly contact amino acid side chains of the recognition helix but rather would be necessary for bending of the DNA around the homeodomain, perhaps facilitating important protein-DNA contacts.

A homology-based molecular model of the proline-rich homeodomain protein Prh, from haematopoietic cells.

Neidle S, Goodwin GH
FEBS Lett. 1994; 345: 93-8

A molecular structural model for the homeodomain of the haematopoietic protein Prh together with its DNA recognition sequence, has been built using the known crystal structure of the MAT alpha 2 homeodomain as a starting-point. The modelling procedure used main and side-chain optimisations by means of molecular mechanics/simulated annealing procedures to obtain stereochemically plausible geometries. The resulting structure has a number of specific interactions in both major and minor grooves of the DNA that serve to define the consensus binding sequence for Prh. In particular, the side-chain of glutamine 50 is postulated to be involved in hydrogen bonds to adjacent adenine and cytosine bases within the consensus sequence.

In vitro interactions between barley TALE homeodomain proteins suggest a role for protein-protein associations in the regulation of Knox gene function.

Muller J, Wang Y, Franzen R, Santi L, Salamini F, Rohde W
Plant J. 2001; 27: 13-23

This paper describes two-hybrid interactions amongst barley homeodomain proteins encoded by the Three Amino acid Loop Extension (TALE) superfamily. The class I KNOX protein BKN3 is shown to homodimerise and to associate with proteins encoded by the class I and II Knox genes BKn-1 and BKn-7. Furthermore, JUBEL1 and JUBEL2, two BELL1 homologous proteins, are identified and characterised as interacting partners of BKN3. Differences in the requirements of BKN3 derivatives for interactions with KNOX and JUBEL proteins imply the involvement of overlapping but slightly different domains. This set of results is an example for interactions amongst different classes of plant TALE homeodomain proteins, as previously described for related animal proteins. Apparently identical spatial and temporal expression patterns of BKn-1, BKn-3, BKn-7, JuBel1 and JuBel2, as determined by in situ hybridisation, are compatible with possible interactions of their protein products in planta. Contradictory to the common model, that the transcriptional down-regulation of certain class 1 Knox-genes is the prerequisite for organ differentiation, transcripts of all five genes were, similar to Tkn1 and Tkn2/LeT6 of tomato, detected in incipient and immature leaves as well as in meristematic tissues. A characteristic phenotype is induced by the overexpression of JuBel2 in transgenic tobacco plants.

DNA bending creates favored sites for retroviral integration: an explanation for preferred insertion sites in nucleosomes.

Muller HP, Varmus HE
EMBO J. 1994; 13: 4704-14

The choice of retroviral integration sites is strongly influenced by chromatin: integration in vitro occurs more efficiently into nucleosomal DNA than into naked DNA, and a characteristic pattern of preferred insertion sites with a 10 bp periodicity is observed at the outer face of the nucleosomal DNA. At least three features of nucleosomal DNA could be responsible for the creation of these favored sites: the presence of histones, attachment of the DNA to a protein surface, and DNA bending. To test each of these possibilities, we studied integration in vitro with human immunodeficiency virus and murine leukemia virus integrases into four model targets that mimic features of nucleosomal DNA: (i) catabolite activator protein-DNA complexes; (ii) lac repressor-operator complexes; (iii) lac repressor-induced loops; and (iv) intrinsically bent A-tract DNA. We found that bending of the target DNA can create favored integration sites at the outer face of the helix, irrespective of whether the bent DNA is attached to a protein surface. Our findings offer an explanation for the preferred usage of nucleosomes as integration targets. In addition, they suggest that bending of the target DNA might be an intrinsic feature of the integration reaction.

Groucho-mediated transcriptional repression establishes progenitor cell pattern and neuronal fate in the ventral neural tube.

Muhr J, Andersson E, Persson M, Jessell TM, Ericson J
Cell. 2001; 104: 861-73

The pattern of neuronal specification in the ventral neural tube is controlled by homeodomain transcription factors expressed by neural progenitor cells, but no general logic has emerged to explain how these proteins determine neuronal fate. We show that most of these homeodomain proteins possess a conserved eh1 motif that mediates the recruitment of Gro/TLE corepressors. The eh1 motif underlies the function of these proteins as repressors during neural patterning in vivo. Inhibition of Gro/TLE-mediated repression in vivo results in a deregulation of cell pattern in the neural tube. These results imply that the pattern of neurogenesis in the neural tube is achieved through the spatially controlled repression of transcriptional repressors-a derepression strategy of neuronal fate specification.

Transformation properties of the E2a-Pbx1 chimeric oncoprotein: fusion with E2a is essential, but the Pbx1 homeodomain is dispensable.

Monica K, LeBrun DP, Dedera DA, Brown R, Cleary ML
Mol Cell Biol. 1994; 14: 8304-14

The t(1;19) chromosomal translocation in acute lymphoblastic leukemias creates chimeric E2a-Pbx1 oncoproteins that can act as DNA-binding activators of transcription. A structural analysis of the functional domains of E2a-Pbx1 showed that portions of both E2a and Pbx1 were essential for transformation of NIH 3T3 cells and transcriptional activation of synthetic reporter genes containing PBX1 consensus binding sites. Hyperexpression of wild-type or experimentally truncated Pbx1 proteins was insufficient for transformation, consistent with their inability to activate transcription. When fused with E2a, the Pbx-related proteins Pbx2 and Pbx3 were also transformation competent, demonstrating that all known members of this highly similar subfamily of homeodomain proteins have latent oncogenic potential. The oncogenic contributions of E2a to the chimeras were localized to transactivation motifs AD1 and AD2, as their mutation significantly impaired transformation. Either the homeodomain or Pbx1 amino acids flanking this region could mediate transformation when fused to E2a. However, the homeodomain was not essential for transformation, since a mutant E2a-Pbx1 protein (E2a-Pbx delta HD) lacking the homeodomain efficiently transformed fibroblasts and induced malignant lymphomas in transgenic mice. Thus, transformation mediated by the chimeric oncoprotein E2a-Pbx1 is absolutely dependent on motifs acquired from E2a but the Pbx1 homeodomain is optional. The latter finding suggests that E2a-Pbx1 may interact with cellular proteins that assist or mediate alterations in gene expression responsible for oncogenesis even in the absence of homeodomain-DNA interactions.

The phage 434 Cro/OR1 complex at 2.5 A resolution.

Mondragon A, Harrison SC
J Mol Biol. 1991; 219: 321-34

The crystal structure of phage 434 Cro protein in complex with a 20 base-pair DNA fragment has been determined to 2.5 A resolution. The DNA fragment contains the sequence of the OR1 operator site. The structure shows a bent conformation for the DNA, straighter at the center and more bent at the ends. The central base-pairs adopt conformations with significant deviations from coplanarity. The two molecules interact extensively along their common interface, both through hydrogen bonds and van der Waals interactions. The significance of these interactions for operator binding and recognition is discussed.

Both the paired domain and homeodomain are required for in vivo function of Drosophila Paired.

Miskiewicz P, Morrissey D, Lan Y, Raj L, Kessler S, Fujioka M, Goto T, Weir M
Development. 1996; 122: 2709-18

Drosophila paired, a homolog of mammalian Pax-3, is key to the coordinated regulation of segment-polarity genes during embryogenesis. The paired gene and its homologs are unusual in encoding proteins with two DNA-binding domains, a paired domain and a homeodomain. We are using an in vivo assay to dissect the functions of the domains of this type of molecule. In particular, we are interested in determining whether one or both DNA-binding activities are required for individual in vivo functions of Paired. We constructed point mutants in each domain designed to disrupt DNA binding and tested the mutants with ectopic expression assays in Drosophila embryos. Mutations in either domain abolished the normal regulation of the target genes engrailed, hedgehog, gooseberry and even-skipped, suggesting that these in vivo functions of Paired require DNA binding through both domains rather than either domain alone. However, when the two mutant proteins were placed in the same embryo, Paired function was restored, indicating that the two DNA-binding activities need not be present in the same molecule. Quantitation of this effect shows that the paired domain mutant has a dominant-negative effect consistent with the observations that Paired protein can bind DNA as a dimer.

Homeobox genes and the zootype.

Miller DJ, Miles A
Nature. 1993; 365: 215-6

The yeast alpha2 and Mcm1 proteins interact through a region similar to a motif found in homeodomain proteins of higher eukaryotes.

Mead J, Zhong H, Acton TB, Vershon AK
Mol Cell Biol. 1996; 16: 2135-43

Homeodomain proteins are transcriptional regulatory factors that, in general, bind DNA with relatively low sequence specificity and affinity. One mechanism homeodomain proteins use to increase their biological specificity is through interactions with other DNA-binding proteins. We have examined how the yeast (Saccharomyces cerevisiae) homeodomain protein alpha2 specifically interacts with Mcm1, a MADS box protein, to bind DNA specifically and repress transcription. A patch of predominantly hydrophobic residues within a region preceding the homeodomain of alpha2 has been identified that specifies direct interaction with Mcm1 in the absence of DNA. This hydrophobic patch is required for cooperative DNA binding with Mcm1 in vitro and for transcriptional repression in vivo. We have also found that a conserved motif, termed YPWM, frequently found in homeodomain proteins of insects and mammals, partially functions in place of the patch in alpha2 to interact with Mcm1. These findings suggest that homeodomain proteins from diverse organisms may use analogous interaction motifs to associate with other proteins to achieve high levels of DNA binding affinity and specificity.

Isolation and characterization of target sequences of the chicken CdxA homeobox gene.

Margalit Y, Yarus S, Shapira E, Gruenbaum Y, Fainsod A
Nucleic Acids Res. 1993; 21: 4915-22

The DNA binding specificity of the chicken homeodomain protein CDXA was studied. Using a CDXA-glutathione-S-transferase fusion protein, DNA fragments containing the binding site for this protein were isolated. The sources of DNA were oligonucleotides with random sequence and chicken genomic DNA. The DNA fragments isolated were sequenced and tested in DNA binding assays. Sequencing revealed that most DNA fragments are AT rich which is a common feature of homeodomain binding sites. By electrophoretic mobility shift assays it was shown that the different target sequences isolated bind to the CDXA protein with different affinities. The specific sequences bound by the CDXA protein in the genomic fragments isolated, were determined by DNase I footprinting. From the footprinted sequences, the CDXA consensus binding site was determined. The CDXA protein binds the consensus sequence A, A/T, T, A/T, A, T, A/G. The CAUDAL binding site in the ftz promoter is also included in this consensus sequence. When tested, some of the genomic target sequences were capable of enhancing the transcriptional activity of reporter plasmids when introduced into CDXA expressing cells. This study determined the DNA sequence specificity of the CDXA protein and it also shows that this protein can further activate transcription in cells in culture.

The specificity of homeotic gene function.

Mann RS
Bioessays. 1995; 17: 855-63

How transcription factors achieve their in vivo specificities is a fundamental question in biology. For the Homeotic Complex (HOM/Hox) family of homeoproteins, specificity in vivo is likely to be in part determined by subtle differences in the DNA binding properties inherent in these proteins. Some of these differences in DNA binding are due to sequence differences in the N-terminal arms of HOM/Hox homeodomains. Evidence also exists to suggest that cofactors can modify HOM/Hox function by cooperative DNA binding interactions. The Drosophila homeoprotein extradenticle (exd) is likely to be one such cofactor. In HOM/Hox proteins, both the conserved 'YPWM' peptide motif and the homeodomain are important for interacting with exd. Although exd provides part of the answer as to how specificity is achieved, there may be additional cofactors and mechanisms that have yet to be identified.

Target genes of homeodomain proteins.

Mannervik M
Bioessays. 1999; 21: 267-70

Homeodomain proteins are transcription factors that share a related DNA binding domain, the homeodomain. This class of proteins was first recognized in the fruitfly Drosophila melanogaster where they cause homeotic transformations such as a fly with four wings instead of two (Lewis EB. A gene complex controlling segmentation in Drosophila. Nature 1978;276:565-570 [Ref. 18]). They are now known to exist in all eukaryotes where they perform important functions during development. Given that homeodomain proteins are transcription factors, they control the expression of downstream genes to regulate development. Which genes are controlled by homeodomain proteins and how many of them are there? This review focuses on a recent paper by Liang and Biggin (Liang Z, Biggin MD. Eve and Ftz regulate a wide array of genes in blastoderm embryos: the selector homeoproteins directly or indirectly regulate most genes in Drosophila. Development 1998; 125:4471-4482 [Ref. 1]), which proposes that the Drosophila homeodomain proteins Even-skipped and Fushi-tarazu directly control the expression of the majority of genes in the Drosophila genome. An alternative view, that most genes are only indirectly affected by homeodomain proteins is also discussed.

The human cut homeodomain protein can repress gene expression by two distinct mechanisms: active repression and competition for binding site occupancy.

Mailly F, Berube G, Harada R, Mao PL, Phillips S, Nepveu A
Mol Cell Biol. 1996; 16: 5346-57

By analogy with other homeodomain proteins conserved in evolution, mammalian Cut proteins are believed, as in Drosophila melanogaster, to play an important role in determining cell type specificity in several tissues. At the molecular level, Cut proteins appear to serve as transcriptional repressors. In this study, we have examined the mechanism by which the human Cut (hCut) protein down-regulates gene expression. The homeodomain and the three regions called Cut repeats are evolutionarily conserved and were previously shown to function as DNA binding domains. The carboxy-terminal region, although it does not show amino acid sequence homology per se, in all cases is enriched in alanine and proline residues, a distinctive feature of some transcriptional repression domains. Our results reveal two distinct modes of repression: competition for binding site occupancy and active repression. On one hand, the composite DNA binding domain formed by Cut repeat 3 and the Cut homeodomain was shown to bind to CCAAT and Sp1 sites within the tk gene promoter and to reduce gene expression, presumably by preventing activation by the corresponding transcription factors. On the other hand, the carboxy-terminal region of mammalian Cut proteins was found to function as an active repression domain in a distance-independent manner. We have further narrowed this activity to two subdomains that can independently repress activated transcription. Finally, we present a model to illustrate the two mechanisms by which Cut proteins repress gene expression.

Interaction of the A alpha Y and Z mating-type homeodomain proteins of Schizophyllum commune detected by the two-hybrid system.

Magae Y, Novotny C, Ullrich R
Biochem Biophys Res Commun. 1995; 211: 1071-6

The A alpha locus is one of four mating-type loci that control sexual development in Schizophyllum commune. A alpha has nine alternative mating types (A alpha 1-A alpha 9) which encode specific alleles of two homeodomain-related proteins, Y and Z. For example, proteins Y4 and Z4 are encoded by A alpha 4, and Y5 and Z5 are encoded by A alpha 5. Our previous studies showed that A alpha-regulated development is activated in fusion cells between haploid strains by the presence of Y and Z proteins derived from different A alpha mating types (e.g., Y4 and Z5), but not by proteins from the same mating type (e.g., Y4 and Z4). In this study we analyzed protein-protein interactions of Y4 with the Z4 and Z5 isoforms in a two-hybrid system. Protein interactions were detected in filter and liquid assays for beta-galactosidase with Y4 and Z5, a non-self, developmentally active combination, but not with Y4 and Z4, a self, developmentally inactive combination. These results support the hypothesis that non-self combinations of Y and Z form a heteromultimer that activates development, but that self combinations do not. Although developmental target genes directly regulated by the A alpha locus have yet to be identified, it is assumed on the basis of motifs encoded that the Y-Z heteromultimer acts as a transcription factor.

Crystal structure of the MATa1/MAT alpha 2 homeodomain heterodimer bound to DNA.

Li T, Stark MR, Johnson AD, Wolberger C
Science. 1995; 270: 262-9

The Saccharomyces cerevisiae MATa1 and MAT alpha 2 homeodomain proteins, which play a role in determining yeast cell type, form a heterodimer that binds DNA and represses transcription in a cell type-specific manner. Whereas the alpha 2 and a1 proteins on their own have only modest affinity for DNA, the a1/alpha 2 heterodimer binds DNA with high specificity and affinity. The three-dimensional crystal structure of the a1/alpha 2 homeodomain heterodimer bound to DNA was determined at a resolution of 2.5 A. The a1 and alpha 2 homeodomains bind in a head-to-tail orientation, with heterodimer contacts mediated by a 16-residue tail located carboxyl-terminal to the alpha 2 homeodomain. This tail becomes ordered in the presence of a1, part of it forming a short amphipathic helix that packs against the a1 homeodomain between helices 1 and 2. A pronounced 60 degree bend is induced in the DNA, which makes possible protein-protein and protein-DNA contacts that could not take place in a straight DNA fragment. Complex formation mediated by flexible protein-recognition peptides attached to stably folded DNA binding domains may prove to be a general feature of the architecture of other classes of eukaryotic transcriptional regulators.

Crystal structure of the MATa1/MATalpha2 homeodomain heterodimer in complex with DNA containing an A-tract.

Li T, Jin Y, Vershon AK, Wolberger C
Nucleic Acids Res. 1998; 26: 5707-18

The crystal structure of the heterodimer formed by the DNA binding domains of the yeast mating type transcription factors, MATa1 and MATalpha2, bound to a 21 bp DNA fragment has been determined at 2.5 A resolution. The DNA fragment in the present study differs at four central base pairs from the DNA sequence used in the previously studied ternary complex. These base pair changes give rise to a (dA5).(dT5) tract without changing the overall base composition of the DNA. The resulting A-tract occurs near the center of the overall 60 degrees bend in the DNA. Comparison of the two structures shows that the structural details of the DNA bend are maintained despite the DNA sequence changes. Analysis of the A5-tract DNA subfragment shows that it contains a bend toward the minor groove centered at one end of the A-tract. The observed bend is larger than that observed in the crystal structures of A-tracts embedded in uncomplexed DNA, which are straight and have been presumed to be quite rigid. Variation of the central DNA base sequence reverses the two AT base pairs contacted in the minor groove by Arg7 of the alpha2 N-terminal arm without significantly altering the DNA binding affinity of the a1/alpha2 heterodimer. The Arg7 side chain accommodates the sequence change by forming alternate H bond interactions, in agreement with the proposal that minor groove base pair recognition is insensitive to base pair reversal. Furthermore, the minor groove spine of hydration, which stabilizes the narrowed minor groove caused by DNA bending, is conserved in both structures. We also find that many of the water-mediated hydrogen bonds between the a1 and alpha2 homeodomains and the DNA are highly conserved, indicating an important role for water in stabilization of the a1/alpha2-DNA complex.

pH-dependent enhancement of DNA binding by the ultrabithorax homeodomain.

Li L, von Kessler D, Beachy PA, Matthews KS
Biochemistry. 1996; 35: 9832-9

Ultrabithorax (Ubx) and Deformed (Dfd) proteins of Drosophila melanogaster contain homeodomains (HD) that are structurally similar and recognize similar DNA sequences, despite functionally distinct genetic regulatory roles for Ubx and Dfd. We report in the present study that Ubx-HD binding to a single optimal target site displayed significantly increased affinity and higher salt concentration dependence at lower pH, while Dfd-HD binding to DNA was unaffected by pH. Results from studies of chimeric Ubx-Dfd homeodomains showed that the N- and C-terminal regions of the Ubx-HD are required for this pH dependence. The increase in binding affinity at lower pH was greater for the Ubx optimal binding site than for other DNA binding sites, indicating that subtle sequence alterations in DNA binding sites may influence pH-dependent behavior. These data demonstrate enhanced DNA binding affinity at lower pH for the Ubx-HD in vitro and suggest the potential for significant discrimination of DNA binding sites in vivo.

The structure of the four-way junction in DNA.

Lilley DM, Clegg RM
Annu Rev Biophys Biomol Struct. 1993; 22: 299-328

Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis.

Lemaire P, Garrett N, Gurdon JB
Cell. 1995; 81: 85-94

Using an expression cloning strategy that relies on a functional assay, we have cloned a novel Xenopus homeobox-containing gene, Siamois. Embryos injected in a ventral-vegetal blastomere with as little as 5 pg of Siamois mRNA develop a complete secondary axis, but the progeny of the injected cells do not participate in the secondary axis formation. In normal development, Siamois mRNA is first detected shortly after the midblastula transition, which is earlier than mRNAs for goosecoid or Xbrachyury, and is present most abundantly in the dorsal endoderm of early gastrulae. The activation of this gene can be obtained cell autonomously in dispersed embryo cells. These results indicate that Siamois may play an important role in the formation of the Nieuwkoop center.

Induced structural changes in protein-DNA complexes.

Kyogoku Y, Kojima C, Lee SJ, Tochio H, Suzuki N, Matsuo H, Shirakawa M
Methods Enzymol. 1995; 261: 524-41

A gamete-specific, sex-limited homeodomain protein in Chlamydomonas.

Kurvari V, Grishin NV, Snell WJ
J Cell Biol. 1998; 143: 1971-80

During fertilization in Chlamydomonas, gametes of opposite mating types interact with each other through sex-specific adhesion molecules on their flagellar surfaces. Flagellar adhesion brings the cell bodies of the gametes into close contact and initiates a signal transduction pathway in preparation for cell-cell fusion. We have identified a cDNA, gsp1, whose transcript levels are upregulated during flagellar adhesion. The GSP1 polypeptide is a novel, gamete-specific homeodomain protein, the first to be identified in an alga. Its homeodomain shows significant identity with several higher plant homeodomain proteins. Although encoded by a single copy gene present in cells of both mating types, immunoblot analysis showed that GSP1 was expressed in mating type (mt)+ gametes, but was not detectable in mt- gametes or in vegetative cells of either mating type. Moreover, GSP1 appeared late during gametogenesis, suggesting that it may function during adhesion with mt- gametes or after zygote formation. GSP1 is expressed in imp11, mt- mutant gametes, which have a lesion in the mid gene involved in sex determination and exhibit many phenotypic characteristics of mt+ gametes. Thus, gsp1 is negatively regulated by mid and is the first molecule to be identified in Chlamydomonas that shows sex-limited expression.

A mating-type factors of Coprinus cinereus have variable numbers of specificity genes encoding two classes of homeodomain proteins.

Kues U, Tymon AM, Richardson WV, May G, Gieser PT, Casselton LA
Mol Gen Genet. 1994; 245: 45-52

We have identified the seven genes that constitute the A43 mating-type factor of Coprinus cinereus and compare the organisation of A43 with the previously characterised A42 factor. In both, the genes that trigger clamp cell development, the so-called specificity genes, are separated into alpha and beta loci by 7 kb of noncoding sequence and are flanked by homologous genes alpha-fg and beta-fg. The specificity genes are known to encode two classes of dissimilar homeodomain (HD1 and HD2) proteins and have different allelic forms which show little or no cross-hybridisation. By partial sequencing we identified a divergently transcribed HD1 (a1-2) and HD2 (a2-2) gene in the A43 alpha locus. a2-2 failed to elicit clamp cell development in three different hosts, suggesting that it is non-functional. a1-2 elicited clamp cells in an A42 host that has only an HD2 gene (a2-1) in its alpha locus, thus demonstrating that the compatible A alpha mating interaction is between an HD1 and an HD2 protein. The A43 beta locus contains three specificity genes, the divergently transcribed HD1 and HD2 genes b1-2 and b2-2 and a third HD1 gene (d1-1) that was shown by hybridisation and transformation analyses to be functionally equivalent to d1-1 in A42. An untranscribed footprint of a third A42 HD1 gene, c1-1, was detected between the A43 b2-2 and d1-1 genes by Southern hybridisation.

Homeodomains and regulation of sexual development in basidiomycetes.

Kues U, Casselton LA
Trends Genet. 1992; 8: 154-5

Residues in the WD repeats of Tup1 required for interaction with alpha2.

Komachi K, Johnson AD
Mol Cell Biol. 1997; 17: 6023-8

The yeast transcriptional repressor Tup1 contains seven WD repeats which interact with the DNA-binding protein alpha2. We have identified mutations in Tup1 that disrupt this interaction. The positions of the amino acids changed by these mutations are consistent with Tup1 being folded into a seven-bladed propeller like that formed by another WD repeat-containing protein, the beta subunit of the heterotrimeric G protein used in signal transduction. Our results also indicate that the interaction between Tup1 and alpha2 resembles the interaction between Gbeta and G alpha, suggesting that a similar structural interface is formed by WD repeat proteins that are used in both transcriptional regulation and signal transduction.

The pentapeptide motif of Hox proteins is required for cooperative DNA binding with Pbx1, physically contacts Pbx1, and enhances DNA binding by Pbx1.

Knoepfler PS, Kamps MP
Mol Cell Biol. 1995; 15: 5811-9

The vertebrate Hox genes, which represent a subset of all homeobox genes, encode proteins that regulate anterior-posterior positional identity during embryogenesis and are cognates of the Drosophila homeodomain proteins encoded by genes composing the homeotic complex (HOM-C). Recently, we demonstrated that multiple Hox proteins bind DNA cooperatively with both Pbx1 and its oncogenic derivative, E2A-Pbx1. Here, we show that the highly conserved pentapeptide motif F/Y-P-W-M-R/K, which occurs in numerous Hox proteins and is positioned 8 to 50 amino acids N terminal to the homeodomain, is essential for cooperative DNA binding with Pbx1 and E2A-Pbx1. Point mutational analysis demonstrated that the tryptophan and methionine residues within the core of this motif were critical for cooperative DNA binding. A peptide containing the wild-type pentapeptide sequence, but not one in which phenylalanine was substituted for tryptophan, blocked the ability of Hox proteins to bind cooperatively with Pbx1 or E2A-Pbx1, suggesting that the pentapeptide itself provides at least one surface through which Hox proteins bind Pbx1. Furthermore, the same peptide, but not the mutant peptide, stimulated DNA binding by Pbx1, suggesting that interaction of Hox proteins with Pbx1 through the pentapeptide motif raises the DNA-binding ability of Pbx1.

Oligonucleotide X-ray structures in the study of conformation and interactions of nucleic acids.

Kennard O, Salisbury SA
J Biol Chem. 1993; 268: 10701-4

Oligonucleotide structure: a decade of results from single crystal X-ray diffraction studies.

Kennard O, Hunter WN
Q Rev Biophys. 1989; 22: 327-79

Ordered water structure in an A-DNA octamer at 1.7 A resolution.

Kennard O, Cruse WB, Nachman J, Prange T, Shakked Z, Rabinovich D
J Biomol Struct Dyn. 1986; 3: 623-47

The crystal structure of the deoxyoctamer d(G-G-Br U-A-BrU-A-C-C) was refined to a resolution of 1.7 A using combined diffractometer and synchrotron data. The analysis was carried out independently in two laboratories using different procedures. Although the final results are identical the comparison of the two approaches highlights potential problems in the refinement of oligonucleotides when only limited data are available. As part of the analysis the positions of 84 solvent molecules in the asymmetric unit were established. The DNA molecule is highly solvated, particularly the phosphate-sugar back-bone and the functional groups of the bases. The major groove contains, in the central BrU-A-BrU-A region, a ribbon of water molecules forming closed pentagons with shared edges. These water molecules are linked to the base O and N atoms and to the solvent chains connecting the O-1 phosphate oxygen atoms on each strand. The minor groove is also extensively hydrated with a continuous network in the central region and other networks at each end. The pattern of hydration is briefly compared with that observed in the structure of a B-dodecamer.

Multiallelic recognition: nonself-dependent dimerization of the bE and bW homeodomain proteins in Ustilago maydis.

Kamper J, Reichmann M, Romeis T, Bolker M, Kahmann R
Cell. 1995; 81: 73-83

In the plant pathogenic fungus Ustilago maydis, sexual and pathogenic development are controlled by the multiallelic b mating-type locus. The b locus encodes a pair of unrelated homeodomain proteins termed bE and bW, with allelic differences clustering in the N-terminal domains of both polypeptides. Only combinations of bE and bW of different allelic origin are active. We have investigated the underlying molecular mechanism for this intracellular self/nonself recognition phenomenon. By using the two-hybrid system, we were able to show that bE and bW dimerize only if they are derived from different alleles. Dimerization involves the N-terminal variable domains. Different point mutants of bE2 were isolated that function in combination with bW2. The majority of such bE2 mutant polypeptides were also able to form heterodimers with bW2 in the two-hybrid system. Nonself-dependent dimerization of bE and bW was supported with a biochemical interaction assay with immobilized proteins. Our results suggest a model for self/nonself recognition in which variable cohesive contacts direct dimerization.

[Electrostatic interaction and differences in specific and nonspecific interactions of the Lac-repressor with DNA]

Kamashev DE, Ebralidze KK, Esipova NG, Mirzabekov AL
Biofizika. 1994; 39: 1101-3

To investigate the complexes of lac-repressor with specific and non-specific DNA sequences method of covalent cross-linking of proteins to partially depurinated DNA was used. We have identified the amino acid resides which form cross-links and hence are proximate to DNA. Only lysine 33 is cross-linkable in specific complex. In the case of non-specific complex (at the presents of inducer molecules) lysine 33 remains cross-linkable. Some consequences of these phenomena in aspects of the physical backgrounds of protein-DNA interactions have been discussed.

A cAMP-regulatory sequence (CRS1) of CYP17 is a cellular target for the homeodomain protein Pbx1.

Kagawa N, Ogo A, Takahashi Y, Iwamatsu A, Waterman MR
J Biol Chem. 1994; 269: 18716-9

Cytochrome P450c17 encoded by CYP17, whose expression is regulated by peptide hormones via cAMP, is required for cortisol and sex hormone biosynthesis thereby playing a key role in biological processes including sexual differentiation. Utilizing the cAMP-regulatory sequence CRS1 of the bovine CYP17 gene as an affinity ligand, four CRS1-binding proteins have been purified from nuclear extracts of mouse adrenocortical Y1 cells and shown to enhance the in vitro transcription of a reporter gene promoted by CRS1. Microsequencing of these four proteins established two of them to be the homeodomain proteins Pbx1a and Pbx1b, originally discovered by their involvement in the t(1;19) chromosomal translocation in pre-B-cell acute lymphoblastic leukemias. Overexpression of Pbx1 in Y1 cells enhances cAMP-dependent transcription of the CRS1-dependent reporter gene. These results identify the CRS1 of bovine CYP17 as a cellular target for Pbx1 and suggest that one role of this homeodomain protein is in the regulation of steroidogenesis and subsequently sexual development.

The yeast a1 and alpha2 homeodomain proteins do not contribute equally to heterodimeric DNA binding.

Jin Y, Zhong H, Vershon AK
Mol Cell Biol. 1999; 19: 585-93

In diploid cells of the yeast Saccharomyces cerevisiae, the alpha2 and a1 homeodomain proteins bind cooperatively to sites in the promoters of haploid cell-type-specific genes (hsg) to repress their expression. Although both proteins bind to the DNA, in the alpha2 homeodomain substitutions of residues that are involved in contacting the DNA have little or no effect on repression in vivo or cooperative DNA binding with a1 protein in vitro. This result brings up the question of the contribution of each protein in the heterodimer complex to the DNA-binding affinity and specificity. To determine the requirements for the a1-alpha2 homeodomain DNA recognition, we systematically introduced single base-pair substitutions in an a1-alpha2 DNA-binding site and examined their effects on repression in vivo and DNA binding in vitro. Our results show that nearly all substitutions that significantly decrease repression and DNA-binding affinity are at positions which are specifically contacted by either the alpha2 or a1 protein. Interestingly, an alpha2 mutant lacking side chains that make base-specific contacts in the major groove is able to discriminate between the wild-type and mutant DNA sites with the same sequence specificity as the wild-type protein. These results suggest that the specificity of alpha2 DNA binding in complex with a1 does not rely solely on the residues that make base-specific contacts. We have also examined the contribution of the a1 homeodomain to the binding affinity and specificity of the complex. In contrast to the lack of a defective phenotype produced by mutations in the alpha2 homeodomain, many of the alanine substitutions of residues in the a1 homeodomain have large effects on a1-alpha2-mediated repression and DNA binding. This result shows that the two proteins do not make equal contributions to the DNA-binding affinity of the complex.

Altered DNA recognition and bending by insertions in the alpha 2 tail of the yeast a1/alpha 2 homeodomain heterodimer.

Jin Y, Mead J, Li T, Wolberger C, Vershon AK
Science. 1995; 270: 290-3

The yeast MAT alpha 2 and MATa1 homeodomain proteins bind cooperatively as a heterodimer to sites upstream of haploid-specific genes, repressing their transcription. In the crystal structure of alpha 2 and a1 bound to DNA, each homeodomain makes independent base-specific contacts with the DNA and the two proteins contact each other through an extended tail region of alpha 2 that tethers the two homeodomains to one another. Because this extended region may be flexible, the ability of the heterodimer to discriminate among DNA sites with altered spacing between alpha 2 and a1 binding sites was examined. Spacing between the half sites was critical for specific DNA binding and transcriptional repression by the complex. However, amino acid insertions in the tail region of alpha 2 suppressed the effect of altering an a1/alpha 2 site by increasing the spacing between the half sites. Insertions in the tail also decreased DNA bending by a1/alpha 2. Thus tethering the two homeodomains contributes to DNA bending by a1/alpha 2, but the precise nature of the resulting bend is not essential for repression.

Structure of Pit-1 POU domain bound to DNA as a dimer: unexpected arrangement and flexibility.

Jacobson EM, Li P, Leon-del-Rio A, Rosenfeld MG, Aggarwal AK
Genes Dev. 1997; 11: 198-212

Pit-1, a member of the POU domain family of transcription factors, characterized by a bipartite DNA-binding domain, serves critical developmental functions based on binding to diverse DNA elements in its target genes. Here we report a high resolution X-ray analysis of the Pit-1 POU domain bound to a DNA element as a homodimer. This analysis reveals that Pit-1 subdomains bind to perpendicular faces of the DNA, rather than opposite faces of the DNA as in Oct-1. This is accomplished by different spacing and orientation of the POU-specific domain. Contrary to previous predictions, the dimerization interface involves the carboxyl terminus of the DNA recognition helix of the homeodomain, which in an extended conformation interacts with specific residues at the amino terminus of helix alpha1 and in the loop between helices alpha3 and alpha4 of the POU-specific domain of the symmetry related monomer. These features suggest the molecular basis of disease-causing mutations in Pit-1 and provide potential basis for the flexible allostery between protein domains and DNA sites in the activation of target genes.

Developmental genes and the origin and evolution of Metazoa.

Jacobs DK
EXS. 1994; 69: 537-49

Multiple amino acids determine the DNA binding specificity of the Msx-1 homeodomain.

Isaac VE, Sciavolino P, Abate C
Biochemistry. 1995; 34: 7127-34

This study investigates the sequence features that contribute to the differential DNA binding properties of two divergent homeodomains, Msx-1 and HoxA3. We show that these homeodomains have overlapping, but nonidentical, DNA binding site preferences. We defined the amino acid residues that contribute to the observed differences in DNA binding specificity by producing a series of mutated polypeptides in which selected residues in Msx-1 were replaced with the corresponding ones in HoxA3. These analyses show that the DNA binding specificity of Msx-1 versus HoxA3 results from the cumulative action of multiple residues in all segments of the homeodomain (i.e., the N-terminal arm and helices I, II, and III). Therefore, substitutions of residues in both helix III and the N-terminal arm (but not in either segment alone) produced an Msx-1 polypeptide whose binding site preference was indistinguishable from that of HoxA3. Residues in helices I and II also influence DNA binding activity; these oppositely charged residues (e.g., lysine 19 and glutamate 30) may mediate ionic interactions between helices I and II which stabilize DNA binding by Msx-1. These findings demonstrate a critical interplay between residues in each homeodomain segment for appropriate conformation of the protein-DNA complex.

Detection and isolation of DNA-binding proteins using single-pulse ultraviolet laser crosslinking.

Ho DT, Sauve DM, Roberge M
Anal Biochem. 1994; 218: 248-54

Ultraviolet laser crosslinking of proteins to DNA is a potentially powerful tool for studying protein-nucleic acid interactions in vitro and in vivo. We describe a simple, rapid, and reliable procedure to detect protein-DNA complexes using crosslinking with a single 5-ns pulse of 266-nm light from a uv laser. The method provides an estimate of the molecular mass of DNA-binding proteins in crude extracts or in purified preparations. It is also well suited for kinetic analysis, and can detect transient protein-DNA interactions as well as interactions that are labile in band-shift gels. We show that the method is generally applicable to DNA-binding proteins. In addition, we describe a technique to isolate crosslinked protein-DNA complexes from crude extracts in one rapid step, using biotinylated DNA probes. Ultraviolet laser crosslinking is a useful alternative or complement to commonly used techniques for the detection and characterization of DNA-binding proteins.

Structure of the even-skipped homeodomain complexed to AT-rich DNA: new perspectives on homeodomain specificity.

Hirsch JA, Aggarwal AK
EMBO J. 1995; 14: 6280-91

even-skipped is a homeobox gene important in controlling segment patterning in the embryonic fruit fly. Its homeobox encodes a DNA binding domain which binds with similar affinities to two DNA consensus sequences, one AT-rich, the other GC-rich. We describe a crystallographic analysis of the Even-skipped homeodomain complexed to an AT-rich oligonucleotide at 2.0 A resolution. The structure reveals a novel arrangement of two homeodomains bound to one 10 bp DNA sequence in a tandem fashion. This arrangement suggests a mechanism for the homeoproteins' regulatory specificity. In addition, the functionally important residue Gln50 is observed in multiple conformations making direct and water-mediated hydrogen bonds with the DNA bases.

Template-directed interference footprinting of cytosine contacts in a protein-DNA complex: potent interference by 5-aza-2'-deoxycytidine.

Hayashibara KC, Verdine GL
Biochemistry. 1992; 31: 11265-73

In the template-directed interference (TDI) footprinting method (Hayashibara & Verdine, 1990), analogs of the naturally occurring DNA bases are incorporated into DNA enzymatically and assayed for interference of sequence-specific binding by a protein. Here we extend this method to include analysis of contacts of amino acid residues to the major groove surface of cytosine residues (TDI-C footprinting). The base analog 5-aza-2'-deoxycytidine, in which the hydrophobic 5-CH of cytosine is replaced by a hydrophilic aza nitrogen, was incorporated into DNA via the corresponding 5'-triphosphate. The analog was found to base pair with guanine during polymerization, resulting in substitution of 2'-deoxycytidine residues. TDI-C footprints of the lambda repressor-OL1 operator complex revealed apparent contacts to the cytosines at operator positions 7 and 8. Inspection of the high-resolution X-ray crystal structure of the lambda-OL1 complex (Clarke et al., 1992; Beamer & Pabo, 1992) revealed that C8 makes a hydrogen binding contact with the Lys3; C7, on the other hand, makes a previously unnoticed hydrophobic contact with the alkane side chain of Lys3. In only the consensus operator half-site was cytosine interference observed, suggesting that the nonconsensus arm binds DNA very differently if at all. The N-terminal arm represents the archetypal case of a sequence-specific peptide-DNA complex characterized at high resolution; thus, the present studies suggest strategies for design and screening of DNA binding peptides. The finding that 5-aza-2'-deoxycytidine inhibits sequence-specific DNA binding proteins may suggest an alternative rationale for the biological activities of this and related azapyrimidine nucleosides.

DNA-binding specificity of the cut repeats from the human cut-like protein.

Harada R, Berube G, Tamplin OJ, Denis-Larose C, Nepveu A
Mol Cell Biol. 1995; 15: 129-40

The Drosophila Cut and mammalian Cut-like proteins contain, in addition to the homeodomain, three other DNA-binding regions called Cut repeats. Cut-like proteins, therefore, belong to a distinct class of homeodomain proteins with multiple DNA-binding domains. In this study, we assessed the DNA-binding specificity of the human Cut repeats by performing PCR-mediated random oligonucleotide selection with glutathione S-transferase fusion proteins. Cut repeat 1, Cut repeat 3, and Cut repeat 3 plus the homeodomain selected related yet distinct sequences. Therefore, sequences selected by one of the fusion proteins were often, but not always, recognized by the other proteins. Consensus binding sites were derived for each fusion protein. In each case, however, some selected sequences diverged from the consensus but were confirmed to be high-affinity recognition sites by electrophoretic mobility shift assay. We conclude that Cut DNA-binding domains have broad, overlapping DNA-binding specificities. Determination of dissociation constants indicated that in addition to the core consensus, flanking sequences have a moderate but significant effect on sequence recognition. Evidence from electrophoretic mobility shift assay, DNase footprinting, and dissociation constant analyses strongly suggested that glutathione S-transferase/Cut fusion proteins bind to DNA as dimers. The implications of these findings are discussed in relation to the DNA-binding capabilities of Cut repeats. In contrast to other studies, we found that the human Cut-like protein does not preferably bind to a site that includes an ATTA homeodomain-binding motif. Here we demonstrate that the native human Cut-like protein recognizes more efficiently a site containing an ATCGAT core consensus flanked with G/C-rich sequences.

A genetic model for interaction of the homeodomain recognition helix with DNA.

Hanes SD, Brent R
Science. 1991; 251: 426-30

The Bicoid homeodomain protein controls anterior development in the Drosophila embryo by binding to DNA and regulating gene expression. With the use of genetic assays in yeast, the interaction between the Bicoid homeodomain and a series of mutated DNA sites was studied. These experiments defined important features of homeodomain binding sites, identified specific amino acid-base pair contacts, and suggested a model for interaction of the recognition alpha-helices of Bicoid and Antennapedia-class homeodomain proteins with DNA. The model is in general agreement with results of crystallographic and magnetic resonance studies, but differs in important details. It is likely that genetic studies of protein-DNA interaction will continue to complement conventional structural approaches.

[A code governing specific binding of regulatory proteins to DNA and structure of stereospecific sites of regulatory proteins]

Gurskii GV, Tumanian VG, Zasedatelev AS, Zhuze AL, Grokhovskii SL, Gottikh BP
Mol Biol (Mosk). 1975; 9: 635-51

A model is proposed for the structure of stereospecific sites in regulatory proteins. On its basis a possible code is suggested that governs the binding of regulatory proteins at specific control sites on DNA. Stereospecific sites of regulatory proteins are assumed to contain pairs of antiparallel polypeptide chain segments which form a right-hand twisted antiparallel beta-sheet, with single-stranded regions at the ends of the beta-structure. The model predicts that binding reaction between a regulatory protein and double-helical DNA is a cooperative phenomenon and is accompanied by significant structural alteration at the stereospecific site of the protein. Half of hydrogen bonds normally existing in beta-structure are broken upon complex formation with DNA and a new set of hydrogen bonds is formed between polypeptide amide groups and DNA base pairs. In a stereospecific site, one chain (t-chain) is attached through hydrogen bonds to the carbonyl oxygens of pyramides and N3 adenines lying in one DNA strand, while the second polypeptide chain (g chain) is hydrogen bonded to the 2-amino groups of guanine residues lying in the opposite DNA strand. The amide groups serve as specific reaction sites being hydrogen bond acceptors in g-chain and hydrogen bond donors in t-chain. The single-stranded portions of t- and g-chains lying in neighbouring subunits of regulatory protein interact with each other forming deformed beta-sheets. The recognition of regulatory sequences by proteins is based on the structural complementarity between stereospecific sites of regulatory proteins and base pairs sequences at the control sites. An essential feature of these sequences is the asymmetrical distribution of guanine residues between the two DNA strands. The code predicts that there are six fundamental amino acid residues (serine, threonine, asparagine, histidine, glutamine and cysteine) whose sequence in stereospecific site determines the base pair sequence to which a given regulatory protein would bind preferentially. The code states a correspondence between four amino acid residues at the stereospecific site of regulatory protein with the two residues being in t- and g-segments, respectively, and AT(GC) base pair at the control site. It is thus possible to determine which amino acid residues in the repressor and which base pairs in the operator DNA are involved in specific interactions with each other, as exemplified by lac repressor binding to lac operator.

Parameters that influence the extent of site occupancy by a candidate telomere end-binding protein.

Gualberto A, Lowry J, Santoro IM, Walsh K
J Biol Chem. 1995; 270: 4509-17

The MF3 protein specifically recognizes telomeric and non-telomeric DNA probes that can form G.G base-paired structures (Gualberto, A., Patrick, R. M., and Walsh, K. (1992) Genes & Dev. 6, 815-824). Here we further characterize the nucleic acid recognition properties of MF3 and present a mathematical analysis that evaluates the potential extent of telomere site occupancy by this factor. The substitution of dI at dG positions in telomeric DNA probes revealed that a single dG at any position within the internal repeat was sufficient for high affinity binding to MF3. The RNA analogs of high affinity DNA sites were not bound specifically by MF3, but the substitution of dU for dT in a DNA probe had little or no effect on binding. These data demonstrate that ribose ring structure is a critical feature of nucleoprotein complex formation, and this ribose specificity may enable MF3 to occupy sites of unusual DNA structure while minimizing interactions with cellular RNAs. Collectively, the nucleic acid binding properties of MF3 suggest that it may occupy a significant fraction of sites at telomere ends or other G-rich regions of altered DNA structure in vivo.

Interactions of the vnd/NK-2 homeodomain with DNA by nuclear magnetic resonance spectroscopy: basis of binding specificity.

Gruschus JM, Tsao DH, Wang LH, Nirenberg M, Ferretti JA
Biochemistry. 1997; 36: 5372-80

The interactions responsible for the nucleotide sequence-specific binding of the vnd/NK-2 homeodomain of Drosophila melanogaster to its consensus DNA binding site have been identified. A three-dimensional structure of the vnd/NK-2 homeodomain-DNA complex is presented, with emphasis on the structure of regions of observed protein-DNA contacts. This structure is based on protein-DNA distance restraints derived from NMR data, along with homology modeling, solvated molecular dynamics, and results from methylation and ethylation interference experiments. Helix III of the homeodomain binds in the major groove of the DNA and the N-terminal arm binds in the minor groove, in analogy with other homeodomain-DNA complexes whose structures have been reported. The vnd/NK-2 homeodomain recognizes the unusual DNA consensus sequence 5'-CAAGTG-3'. The roles in sequence specificity and strength of binding of individual amino acid residues that make contact with the DNA are described. We show, based primarily on the observed protein-DNA contacts, that the interaction of Y54 with the DNA is the major determinant of this uncommon nucleotide binding specificity in the vnd/NK-2 homeodomain-DNA complex.

Human and Drosophila homeodomain proteins that enhance the DNA-binding activity of serum response factor.

Grueneberg DA, Natesan S, Alexandre C, Gilman MZ
Science. 1992; 257: 1089-95

Cells with distinct developmental histories can respond differentially to identical signals, suggesting that signals are interpreted in a fashion that reflects a cell's identity. How this might occur is suggested by the observation that proteins of the homeodomain family, including a newly identified human protein, enhance the DNA-binding activity of serum response factor, a protein required for the induction of genes by growth and differentiation factors. Interaction with proteins of the serum response factor family may allow homeodomain proteins to specify the transcriptional response to inductive signals. Moreover, because the ability to enhance the binding of serum response factor to DNA residues within the homeodomain but is independent of homeodomain DNA-binding activity, this additional activity of the homeodomain may account for some of specificity of action of homeodomain proteins in development.

Complementarity, specificity and the nature of epitopes and paratopes in multivalent interactions.

Greenspan NS, Cooper LJ
Immunol Today. 1995; 16: 226-30

Structural elements of an antibody (Ab) or antigen (Ag) distant from the actual sites mediating contact between Ab and Ag can exert substantial influence on binding to, and discrimination among, multivalent targets. Consequently, multivalent molecules that express the same number of identical binding sites, but that differ in other structural features, can exhibit differences in their ability to discriminate between multivalent ligands. Here, Neil Greenspan and Laurence Cooper review evidence for these effects, and explore implications of the conclusion that effective specificity in multivalent interactions is not completely determined by the degree of complementarity between epitopes and paratopes.

Yeast a1 and alpha 2 homeodomain proteins form a DNA-binding activity with properties distinct from those of either protein.

Goutte C, Johnson AD
J Mol Biol. 1993; 233: 359-71

The yeast a1 and alpha 2 proteins are examples of homeodomain proteins that display cell-type-specific expression. They are co-expressed in only one type of cell, the a/alpha cell, where they repress the expression of a group of target genes. Using purified proteins, we demonstrate that a1 and alpha 2 form a highly specific DNA-binding activity, which recognizes an operator found upstream of each target gene. These proteins interact with DNA to form a ternary complex in which both a1 and alpha 2 contact the DNA through their respective homeodomains. An alpha 2 homodimer can recognize the same operator as the a1/alpha 2 heterodimer, but the affinity and specificity of the alpha 2 homodimer for DNA are much weaker than those of the a1/alpha 2 heterodimer. This difference results in part from the fact that the heterodimer is formed using a set of protein-protein contacts that is distinct from those that form the alpha 2/alpha 2 homodimer. Although a1 contacts DNA in the presence of alpha 2, it does not on its own bind DNA specifically, even at very high concentrations. These results suggest that the dimerization of heterologous partners can produce a DNA-binding activity that is not a simple hybrid of the known properties of each constituent.

Recognition of a DNA operator by a dimer composed of two different homeodomain proteins.

Goutte C, Johnson AD
EMBO J. 1994; 13: 1434-42

The yeast homeodomain proteins a1 and alpha 2 interact to form a heterodimer that binds DNA with high specificity. The DNA recognition element consists of two similar half sites, arranged with dyad symmetry and separated by a fixed number of base pairs. We demonstrate that in the a1 alpha 2-DNA complex, one of these half-sites is bound by a1 while the other is bound by alpha 2. These assignments allow a comparison of the chemical and nuclease protection patterns produced by both proteins when bound together to the hsg operator. Contrary to simple expectations, we propose that the a1 and alpha 2 homeodomains are arranged on the DNA in tandem, despite the fact that the recognition sequence is dyad symmetric.

The specificities of Sex combs reduced and Antennapedia are defined by a distinct portion of each protein that includes the homeodomain.

Gibson G, Schier A, LeMotte P, Gehring WJ
Cell. 1990; 62: 1087-103

The sequence requirements for distinguishing the functional specificities of two homeodomain proteins, Antennapedia and Sex combs reduced, involved in the specification of segmental identities in Drosophila, have been determined. A series of deletions and hybrid proteins was generated and assayed for their function in vivo after heat shock-induced ectopic expression during development. A distinct portion of each protein, including the residues within and adjacent to both ends of the homeodomain, has been found to almost entirely determine its functional specificity as measured by diagnostic cuticular transformations of embryonic and adult head structures. The remaining sequences contribute to the potency with which the proteins act in different cells and are to a limited extent functionally transferable from one protein to the other.

Barnacle duplicate engrailed genes: divergent expression patterns and evidence for a vestigial abdomen.

Gibert JM, Mouchel-Vielh E, Queinnec E, Deutsch JS
Evol Dev. 2000; 2: 194-202

Cirripedes (barnacles) are crustaceans that are characterized by a very peculiar body plan, in particular by the lack of an abdomen. To study their body plan, we searched for their engrailed gene. We found two engrailed (en.a/en.b) genes in cirripedes. The two engrailed genes of the rhizocephalan barnacle Sacculina carcini are expressed in the posterior compartment of developing segments and appendages. When the neuroectoderm differentiates into epidermis and neuroderm the expression patterns of en.a and en.b diverge dramatically. en.a expression fades in segment epidermis whereas it is subsequently detected ventrally in reiterated putative neural cells. At the same time, en.b expression increases in the epidermis, which makes it a very good segmentation marker. Five tiny en.b stripes are observed between the sixth thoracic segment and the telson. We interpret these stripes as the molecular definition of vestigial abdominal segments, being the remnant of an ancestral state in keeping with the bodyplan of maxillopod crustaceans. engrailedexpression is the first molecular evidence for a segmented abdomen in barnacles.

The structure of the homeodomain and its functional implications.

Gehring WJ, Muller M, Affolter M, Percival-Smith A, Billeter M, Qian YQ, Otting G, Wuthrich K
Trends Genet. 1990; 6: 323-9

The three-dimensional structure of the homeodomain, as determined by nuclear magnetic resonance spectroscopy, reveals the presence of a helix-turn-helix motif, similar to the one found in prokaryotic gene regulatory proteins. Isolated homeodomains bind with high affinity to specific DNA sequences. Thus, the structure-function relationship is highly conserved in evolution.

The homeobox in perspective.

Gehring WJ
Trends Biochem Sci. 1992; 17: 277-80

The discovery of the homeobox marks the beginning of a new era in developmental biology in which a class of master control genes, which determine the body plan, have been identified. Their mechanism of action can now be studied at the molecular level and their occurrence seems to be much more universal than originally anticipated.

Engrailed homeodomain-DNA complex at 2.2 A resolution: a detailed view of the interface and comparison with other engrailed structures.

Fraenkel E, Rould MA, Chambers KA, Pabo CO
J Mol Biol. 1998; 284: 351-61

We report the 2.2 A resolution structure of the Drosophila engrailed homeodomain bound to its optimal DNA site. The original 2.8 A resolution structure of this complex provided the first detailed three-dimensional view of how homeodomains recognize DNA, and has served as the basis for biochemical studies, structural studies and molecular modeling. Our refined structure confirms the principal conclusions of the original structure, but provides important new details about the recognition interface. Biochemical and NMR studies of other homeodomains had led to the notion that Gln50 was an especially important determinant of specificity. However, our refined structure shows that this side-chain makes no direct hydrogen bonds to the DNA. The structure does reveal an extensive network of ordered water molecules which mediate contacts to several bases and phosphates (including contacts from Gln50), and our model provides a basis for detailed comparison with the structure of an engrailed Q50K altered-specificity variant. Comparing our structure with the crystal structure of the free protein confirms that the N and C termini of the homeodomain become ordered upon DNA-binding. However, we also find that several key DNA contact residues in the recognition helix have the same conformation in the free and bound protein, and that several water molecules also are "preorganized" to contact the DNA. Our structure helps provide a more complete basis for the detailed analysis of homeodomain-DNA interactions.

Electrostatic effects in homeodomain-DNA interactions.

Fogolari F, Elcock AH, Esposito G, Viglino P, Briggs JM, McCammon JA
J Mol Biol. 1997; 267: 368-81

We report here an investigation of the role of electrostatics in homeodomain-DNA interactions using techniques based around the use of the Poisson-Boltzmann equation. In the present case such a study is of particular interest, since in contrast to other proteins previously studied with this method, the homeodomain is a small, highly charged protein that forms extensive ion pairs upon binding DNA. We have investigated the salt dependence of the binding constant for specific association and for a variety of models for non-specific association. The results indicate that, in line with the models proposed by Manning and Record, the entropy of counterion release accounts for a significant fraction of the salt dependence of the binding free energy, though this is perhaps due to fortuitous cancellation of other contributing terms. The thermodynamic effects of a number of specific homeodomain mutants were also investigated, and partly rationalized in terms of favorable electrostatic interactions in the major goove of DNA. Investigation of the temperature-dependence of the free energy of association indicates that the electrostatic contributions become increasingly favorable as the temperature rises. For this particular system, however, there appears to be no significant electrostatic contribution to the delta(delta C(p)) of association. Finally, an analysis of the free energy of interaction when the homeodomain is moved ca one Debye length from the DNA suggests that pure electrostatic forces are able to steer the homeodomain into a partially correct orientation for binding to the DNA.

Molecular dynamics simulations of the glucocorticoid receptor DNA-binding domain in complex with DNA and free in solution.

Eriksson MA, Hard T, Nilsson L
Biophys J. 1995; 68: 402-26

Molecular dynamics simulations have been performed on the glucocorticoid receptor DNA binding domain (GR DBD) in aqueous solution as a dimer in complex with DNA and as a free monomer. In the simulated complex, we find a slightly increased bending of the DNA helix axis compared with the crystal structure in the spacer region of DNA between the two half-sites that are recognized by GR DBD. The bend is mainly caused by an increased number of interactions between DNA and the N-terminal extended region of the sequence specifically bound monomer. The recognition helices of GR DBD are pulled further into the DNA major groove leading to a weakening of the intrahelical hydrogen bonds in the middle of the helices. Many ordered water molecules with long residence times are found at the intermolecular interfaces of the complex. The hydrogen-bonding networks (including water bridges) on either side of the DNA major groove involve residues that are highly conserved within the family of nuclear receptors. Very similar hydrogen-bonding networks are found in the estrogen receptor (ER) DBD in complex with DNA, which suggests that this is a common feature for proper positioning of the recognition helix in ER DBD and GR DBD.

Targeting of nucleic acids by iron complexes.

Draganescu A, Tullius TD
Met Ions Biol Syst. 1996; 33: 453-84

The DNA binding specificity of engrailed homeodomain.

Draganescu A, Tullius TD
J Mol Biol. 1998; 276: 529-36

The engrailed gene of Drosophila melanogaster is an integral member of the highly complex cascade which results in a fully developed fruitfly. The gene product of engrailed contains a homeodomain which is responsible for DNA binding via a helix-turn-helix motif. The crystal structure of this 60 amino acid residue domain complexed to DNA is analogous to structures of other homeodomain-DNA complexes, consistent with the high degree of sequence conservation within both protein and DNA. Despite the high degree of homology, homeodomains do exhibit distinct preferences for certain DNA sequences. Such specificity may be at least partly responsible for the interactions necessary for normal development. Using the hydroxyl radical as a chemical probe, we have examined complexes of Engrailed homeodomain with several DNA sequences to determine the protein's binding specificity in solution. We find that Engrailed forms a single, specific complex with a unique DNA binding site which is analogous to the complex seen in the co-crystal structure. In contrast, our chemical probe experiments show that the binding site of Engrailed that was determined by in vitro selection and that also was present in the co-crystal structure contains two possible binding sites. Modification of the sequence of this site to yield single binding sites removes the ambiguity, and results in two different, well-behaved Engrailed-DNA complexes. Our results underscore the utility of chemical probe experiments for defining the variety of modes of interaction of proteins with DNA that can occur in solution, but that might not be apparent in a crystal structure.

Homeodomain proteins: what governs their ability to recognize specific DNA sequences?

Draganescu A, Levin JR, Tullius TD
J Mol Biol. 1995; 250: 595-608

Deformed (Dfd) and Ultrabithorax (Ubx) are homeodomain proteins from Drosophila melanogaster that exert regulatory effects on gene expression by binding to specific target sites in the fly genome using a helix-turn-helix (HTH) motif. The recognition helices of these two proteins are almost identical and the DNA sequences they recognize are similar, containing a conserved TAAT core sequence flanked by a somewhat variable sequence. Yet the in vivo functions of the two proteins are quite different. We have used the homeodomains of these two proteins and in vitro selected DNA binding sites to characterize the structural details of homeodomain binding to DNA and to understand the basis for the differences in sequence specificity between homeodomains with similar recognition helices. We have employed hydroxyl radical cleavage of DNA to study the positioning of the proteins on the binding sites and have analyzed the effects of missing nucleosides and purine methylation on homeodomain binding. Our results indicate that the positioning of the Ubx and Dfd homeodomains on their binding sites is consistent with reported structures of other homeodomain/DNA complexes. Dfd and Ubx bind to DNA with the recognition helix in the major groove 3' to the TAAT core sequence and the N-terminal arm in the adjacent minor groove. However, we observe striking differences between the two homeodomains in their specific interactions with DNA. Missing nucleosides within the selected binding sites have differential effects on protein binding, which are dependent on the identity of the homeodomain. Differences at the 3' end of the binding site on the top strand indicate that the N-terminal arm of a homeodomain is capable of distinguishing an A.T base-pair from T.A in the minor groove. Specific orientation of the N-terminal arm within the binding site appears to vary between the homeodomains and influences the interaction of the recognition helix with the major groove.

Distamycin-induced inhibition of homeodomain-DNA complexes.

Dorn A, Affolter M, Muller M, Gehring WJ, Leupin W
EMBO J. 1992; 11: 279-86

The mobility shift assay was used to study the competition of the minor groove binder distamycin A with either an Antennapedia homeodomain (Antp HD) peptide or derivatives of a fushi tarazu homeodomain (ftz HD) peptide for their AT-rich DNA binding site. The results show that distamycin and the homeodomain peptides compete under the conditions: (i) preincubation of DNA with distamycin and subsequent addition of HD peptide; (ii) simultaneous incubation of DNA with distamycin and HD peptide; and (iii) preincubation of DNA with HD peptide and subsequent addition of distamycin. There is also competition when using a peptide which lacks the N-terminal arm of ftz HD that is involved in contacts in the minor groove. It is proposed that the protein's binding affinity is diminished by distamycin-induced conformational changes of the DNA. The feasibility of the propagation of conformational changes upon binding in the minor groove is also shown for the inhibition of restriction endonucleases differing in the AT content of their recognition site and of their flanking DNA sequences. Thus, it is demonstrated that minor groove binders can compete with the binding of proteins in the major groove, providing an experimental indication for the influence of biological activities exerted by DNA ligands binding in the minor groove.

Homeodomain proteins in development and therapy.

Dorn A, Affolter M, Gehring WJ, Leupin W
Pharmacol Ther. 1994; 61: 155-84

Homeobox genes encode transcriptional regulators found in all organisms ranging from yeast to humans. In Drosophila, a specific class of homeobox genes, the homeotic genes, specifies the identity of certain spatial units of development. Their genomic organization, in Drosophila, as well as in vertebrates, is uniquely connected with their expression which follows a 5'-posterior-3'-anterior rule along the longitudinal body axis. The 180-bp homeobox is part of the coding sequence of these genes, and the sequence of 60 amino acids it encodes is referred to as the homeodomain. Structural analyses have shown that homeodomains consist of a helix-turn-helix motif that binds the DNA by inserting the recognition helix into the major groove of the DNA and its amino-terminal arm into the adjacent minor groove. Developmental as well as gene regulatory functions of homeobox genes are discussed, with special emphasis on one group, the Antennapedia (Antp) class homeobox genes and a representative 60-amino acid Antennapedia peptide (pAntp). In cultured neuronal cells, pAntp translocates through the membrane specifically and efficiently and accumulates in the nucleus. The internalization process is followed by a strong induction of neuronal morphological differentiation, which raises the possibility that motoneuron growth is controlled by homeodomain proteins. It has been demonstrated that chimeric peptide molecules encompassing pAntp are also captured by cultured neurons and conveyed to their nuclei. This may be of enormous interest for the internalization of drugs.

Determination of axial polarity in the vertebrate embryo: homeodomain proteins and homeogenetic induction.

De Robertis EM, Oliver G, Wright CV
Cell. 1989; 57: 189-91

Characterization of a telomere-binding protein from Physarum polycephalum.

Coren JS, Epstein EM, Vogt VM
Mol Cell Biol. 1991; 11: 2282-90

We have partially purified a nuclear protein (PPT) from Physarum polycephalum that binds to the extrachromosomal ribosomal DNA telomeres of this acellular slime mold. Binding is specific for the (T2AG3)n telomere repeats, as evidenced by nitrocellulose filter binding assays, by gel mobility shift assays with both DNA fragments and double-stranded oligonucleotides, and by DNase I footprinting. PPT is remarkably heat stable, showing undiminished binding activity after incubation at 90 degrees C. It sediments at 1.2S, corresponding to a molecular weight of about 10,000 (for a globular protein), and its binding activity is undiminished by incubation with RNase, suggesting that it is not a ribonucleoprotein. We hypothesize that PPT plays a structural role in telomeres, perhaps preventing nucleolytic degradation or promoting telomere extension by a telomere-specific terminal transferase.

Mutational analysis of the engrailed homeodomain recognition helix by phage display.

Connolly JP, Augustine JG, Francklyn C
Nucleic Acids Res. 1999; 27: 1182-9

The homeodomain (HD) is a ubiquitous protein fold that confers DNA binding function on a superfamily of eukaryotic gene regulatory proteins. Here, the DNA binding of recognition helix variants of the HD from the engrailed gene of Drosophila melanogaster was investigated by phage display. Nineteen different combinations of pairwise mutations at positions 50 and 54 were screened against a panel of four DNA sequences consisting of the engrailed consensus, a non-specific DNA control based on the lambda repressor operator OR1 and two model sequence targets con-taining imperfect versions of the 5'-TAAT-3' consensus. The resulting mutant proteins could be divided into four groups that varied with respect to their affinity for DNA and specificity for the engrailed consensus. The altered specificity phenotypes of several mutant proteins were confirmed by DNA mobility shift analysis. Lys50/Ala54 was the only mutant protein that exhibited preferential binding to a sequence other than the engrailed consensus. Arginine was also demonstrated to be a functional replacement for Ala54. The functional combinations at 50 and 54 identified by these experiments recapitulate the distribution of naturally occurring HD sequences and illustrate how the engrailed HD can be used as a framework to explore covariation among DNA binding residues.

Covariation of residues in the homeodomain sequence family.

Clarke ND
Protein Sci. 1995; 4: 2269-78

Homeodomains are 60 amino acid DNA binding domains found in numerous eukaryotic transcription factors. The homeodomain family is a useful system for studying sequence-structure relationships because several hundred sequences are known and the structures of several homeodomains have been determined. Covariation of amino acid residues in the homeodomain family has been investigated to see whether strongly covariant residue pairs can be understood in terms of the structure and function of these domains. Among 16 strongly covariant pairs examined, 2 are explained by the ability to form salt bridges, and 9 appear related to the DNA binding function of the proteins. For the remaining 5 pairs, the rationale for covariance remains unclear and the likelihood of artifactual correlations is discussed in the context of experimental and evolutionary biases in the selection of sequences. No significant correlation was found between covariance and structural proximity in the hydrophobic core.

The primary structure of the murine multifinger gene mKr2 and its specific expression in developing and adult neurons.

Chowdhury K, Dressler G, Breier G, Deutsch U, Gruss P
EMBO J. 1988; 7: 1345-53

The complete amino acid sequence of the murine finger-containing gene mKr2 was determined. On the basis of sequence similarities in the repeated finger domain, mKr2 belongs to the same class of developmentally expressed genes as Drosophila Kruppel and hunchback. The presence of metal ion and DNA-binding finger domains similar to those identified in TFIIIA supports the hypothesis that these genes regulate transcription. mKr2 transcripts are restricted to neurons in the central and peripheral nervous system of adult animals. Furthermore, mKr2 transcripts can be detected in all the major structures of the developing nervous system during embryogenesis. The data are consistent with the hypothesis that mKr2 is a regulatory factor required for the differentiation and/or phenotypic maintenance of neurons.

Identification of novel DNA binding targets and regulatory domains of a murine tinman homeodomain factor, nkx-2.5.

Chen CY, Schwartz RJ
J Biol Chem. 1995; 270: 15628-33

A murine cardiac-specific homeodomain gene named csx (Komuro, I., and Izumo. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8145-8149) and nkx-2.5 (Lints, T. J., Parsons, L. M., Hartley, L., Lyons, I., and Harvey, R. P. (1993) Development 119, 419-431) was identified as a potential vertebrate homologue of Drosophila tinman, a mesoderm determination factor required for insect heart formation (Bodmer, R. (1993) Development 118, 719-729). Bacterial expression of the nkx-2.5 homeodomain allowed us to identify downstream DNA targets from a library of randomly generated oligonucleotides. High affinity nkx-2.5 DNA binding sites, 5'-TNNAGTG-3', represented novel binding sequences, whereas intermediate and weaker affinity sites, 5'-C(A/T)TTAATTN-3', contained the typical 5'-TAAT-3' core required by most homeodomain factors for DNA binding. We also observed that nkx-2.5 served as a modest transcription activator in transfection assays done in 10T1/2 fibroblasts with multimerized binding sites linked to a luciferase reporter gene. Functional dissection of nkx-2.5 revealed a COOH-terminal inhibitory domain composed mainly of clusters of alanines and prolines, which appeared to mask a potent activation domain composed of hydrophobic and highly charged amino acids.

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

Chang CP, Shen WF, Rozenfeld S, Lawrence HJ, Largman C, Cleary ML
Genes Dev. 1995; 9: 663-74

The human proto-oncogene PBX1 codes for a homolog of Drosophila extradenticle, a divergent homeo domain protein that modulates the developmental and DNA-binding specificity of select HOM proteins. We demonstrate that wild-type Pbx proteins and chimeric E2a-Pbx1 oncoproteins cooperatively bind a consensus DNA probe with HoxB4, B6, and B7 of the Antennapedia class of Hox/HOM proteins. Specificity of Hox-Pbx interactions was suggested by the inability of Pbx proteins to cooperatively bind the synthetic DNA target with HoxA10 or Drosophila even-skipped. Site-directed mutagenesis showed that the hexapeptide motif (IYPWMK) upstream of the Hox homeo domain was essential for HoxB6 and B7 to cooperatively bind DNA with Pbx proteins. Engraftment of the HoxB7 hexapeptide onto HoxA10 endowed it with robust cooperative properties, demonstrating a functional role for the highly conserved hexapeptide element as one of the molecular determinants delimiting Hox-Pbx cooperativity. The Pbx homeo domain was necessary but not sufficient for cooperativity, which required conserved amino acids carboxy-terminal of the homeo domain. These findings demonstrate that interactions between Hox and Pbx proteins modulate their DNA-binding properties, suggesting that Pbx and Hox proteins act in parallel as heterotypic complexes to regulate expression of specific subordinate genes.

Energetics of folding and DNA binding of the MAT alpha 2 homeodomain.

Carra JH, Privalov PL
Biochemistry. 1997; 36: 526-35

Homeodomains are a class of DNA-binding protein domains which play an important role in genetic regulation in eukaryotes. We have characterized the thermodynamics of folding and sequence-specific association with DNA of the MAT alpha 2 homeodomain of yeast. Using differential scanning and isothermal titration calorimetry, we measured the enthalpy, heat capacity, and Gibbs free energy changes of these processes. The protein-DNA interaction is enthalpically driven at physiological temperatures. DSC data on the process of melting the protein-DNA complex at different salt concentrations were dissected into its endothermic components, yielding the enthalpy change and dissociation constant of binding. A comparison of the circular dichroism spectra of the free and DNA-bound protein species revealed the formation of additional alpha-helical structure upon binding to DNA. We propose that the latter half of helix 3, the recognition helix, is substantially unfolded in the free protein under the conditions used, as has been observed with other homeodomains [Tsao, D. H. H., et al. (1994) Biochemistry 33, 15053-15060: Cox, M., et al. (1995) J. Biomol. NMR 5, 23-32]. Formation of protein structure is induced by DNA binding, and the energies measured for association therefore include a component due to folding.

Crystallography in the study of protein-DNA interactions.

Brown DG, Freemont PS
Methods Mol Biol. 1996; 56: 293-318

The helix-turn-helix DNA binding motif.

Brennan RG, Matthews BW
J Biol Chem. 1989; 264: 1903-6

Formation of a monomeric DNA binding domain by Skn-1 bZIP and homeodomain elements.

Blackwell TK, Bowerman B, Priess JR, Weintraub H
Science. 1994; 266: 621-8

Maternally expressed Skn-1 protein is required for the correct specification of certain blastomere fates in early Caenorhabditis elegans embryos. Skn-1 contains a basic region similar to those of basic leucine zipper (bZIP) proteins but, paradoxically, it lacks a leucine zipper dimerization segment. Random sequence selection methods were used to show that Skn-1 binds to specific DNA sequences as a monomer. The Skn-1 basic region lies at the carboxyl terminus of an 85-amino acid domain that binds preferentially to a bZIP half-site and also recognizes adjacent 5' AT-rich sequences in the minor groove, apparently with an amino (NH2)-terminal "arm" related to those of homeodomain proteins. The intervening residues appear to stabilize interactions of these two subdomains with DNA. The Skn-1 DNA binding domain thus represents an alternative strategy for promoting binding of a basic region segment recognition helix to its cognate half-site. The results point to an underlying modularity in subdomains within established DNA binding domains.

Determination of the nuclear magnetic resonance solution structure of an Antennapedia homeodomain-DNA complex.

Billeter M, Qian YQ, Otting G, Muller M, Gehring W, Wuthrich K
J Mol Biol. 1993; 234: 1084-93

The nuclear magnetic resonance (NMR) solution structure of a complex formed by the mutant Antennapedia homeodomain with Cys39 replaced by Ser, Antp(C39S), and a 14 base-pair DNA duplex containing the BS2 operator sequence was determined using uniform 13C and 15N-labeling of the protein. Two-dimensional nuclear Overhauser enhancement spectroscopy ([1H,1H]NOESY) with 15N(omega 2)-half-filter and 13C(omega 1, omega 2)-double-half-filter, and three-dimensional heteronuclear-correlated [1H,1H]NOESY yielded a total of 855 intramolecular NOE upper distance constraints in the homeodomain, 151 upper distance constraints within the DNA duplex, and 39 intermolecular protein-DNA upper distance constraints. These data were used as the input for the structure calculation with simulated annealing followed by molecular dynamics in a water bath and energy refinement. A group of 16 conformers was thus generated which represent the solution structure of the Antp(C39S) homeodomain-DNA complex. The new structure determination confirms the salient features reported previously from a preliminary investigation of the same complex, in particular the location of the recognition helix in the major groove with the turn of the helix-turn-helix motif outside the contact area with the DNA, and the N-terminal arm of the homeodomain contacting the minor groove of the DNA. In addition, distinct amino acid side-chain-DNA contacts could be identified, and evidence was found that the invariant residue Asn51 (and possibly also Gln50) is in a slow dynamic equilibrium between two or several different DNA contact sites. The molecular dynamics calculations in a water bath yielded structures with hydration water molecules in the protein-DNA interface, which coincides with direct NMR observations of hydration waters. In the Appendix the experimental data obtained with the Antp(C39S) homeodomain-DNA complex and the techniques used for the structure calculation are evaluated using a simulated input data set derived from the X-ray crystal structure of a DNA complex with a homologous homeodomain. This study indicates that a nearly complete set of NOE upper distance constraints for the Antp(C39S) homeodomain and the protein-DNA interface was presently obtained. It further shows that the structure calculation used here yields a precise reproduction of the crystal structure from the simulated input data, and also results in hydration of the protein-DNA interface in the recalculated complex.

Homeodomain-type DNA recognition.

Billeter M
Prog Biophys Mol Biol. 1996; 66: 211-25

Homeobox genes and heart development.

Biben C, Palmer S, Elliott DA, Harvey RP
Cold Spring Harb Symp Quant Biol. 1997; 62: 395-403

Hairpin coding end opening is mediated by RAG1 and RAG2 proteins.

Besmer E, Mansilla-Soto J, Cassard S, Sawchuk DJ, Brown G, Sadofsky M, Lewis SM, Nussenzweig MC, Cortes P
Mol Cell. 1998; 2: 817-28

Despite the importance of hairpin opening in antigen receptor gene assembly, the molecular machinery that mediates this reaction has not been defined. Here, we show that RAG1 plus RAG2 can open DNA hairpins. Hairpin opening by RAGs is not sequence specific, but in Mg2+, hairpin opening occurs only in the context of a regulated cleavage complex. The chemical mechanism of hairpin opening by RAGs resembles RSS cleavage and 3' end processing by HIV integrase and Mu transposase in that these reactions can proceed through alcoholysis. Mutations in either RAG1 or RAG2 that interfere with RSS cleavage also interfere with hairpin opening, suggesting that RAGs have a single active site that catalyzes several distinct DNA cleavage reactions.

Ustilago maydis, the delightful blight.

Banuett F
Trends Genet. 1992; 8: 174-80

Recent studies of the corn smut fungus life cycle and its regulation by two mating type loci and other genes provide a cornucopia of challenges in cell biology, genetics and protein structure. The fungus can exist in two states: nonpathogenic and pathogenic. The change from one state to the other is accompanied by a change in morphology (yeast-like to filamentous) and growth properties (saprophytic to parasitic).

The Homeodomain Resource: sequences, structures, DNA binding sites and genomic information.

Banerjee-Basu S, Sink DW, Baxevanis AD
Nucleic Acids Res. 2001; 29: 291-3

The Homeodomain Resource is an annotated collection of non-redundant protein sequences, three-dimensional structures and genomic information for the homeodomain protein family. Release 3.0 contains 795 full-length homeodomain-containing sequences, 32 experimentally-derived structures and 143 homeo-box loci implicated in human genetic disorders. Entries are fully hyperlinked to facilitate easy retrieval of the original records from source databases. A simple search engine with a graphical user interface is provided to query the component databases and assemble customized data sets. A new feature for this release is the addition of DNA recognition sites for all human homeodomain proteins described in the literature. The Homeodomain Resource is freely available through the World Wide Web at http://genome.nhgri.nih.gov/homeodomain.

Threading analysis of prospero-type homeodomains.

Banerjee-Basu S, Landsman D, Baxevanis AD
In Silico Biol. 1999; 1: 163-73

The homeodomain is a common structural motif found in many transcription factors involved in cell fate determination during development. We have used threading analysis techniques to predict whether the atypical homeodomain of prospero (pros) family members could form the three-helical homeodomain structural motif, even though these proteins are not statistically similar to canonical homeodomains as assessed by BLAST searches. Amino acid sequences of these divergent homeodomain proteins were threaded through the X-ray coordinates of the Drosophila engrailed homeodomain protein [23]. The analysis confirms that the prospero class of homeodomain proteins is indeed capable of forming the homeodomain structure despite its low degree of sequence identity to the canonical homeodomain. Energy calculations indicate that the homeodomain structure is stabilized primarily by hydrophobic interactions between residues at the helical interfaces. Although the atypical prospero-type homeodomain shows very little sequence similarity when compared to other homeodomain proteins, the critical amino acids responsible for maintaining the three-dimensional structure are highly conserved. A number of other homeodomain proteins, such as PHO2p from Saccharomyces and Pax6 from human, were also included in the threading analysis and were shown to be able to form the engrailed structure, indicating that there are no rigid overall sequence requirements for the formation of the homeodomain structural motif. Based on the threading experiments and the subsequent structural alignment, a new amino acid signature that unambiguously identifies the prospero-type proteins was deduced.

The Homeodomain Resource: sequences, structures and genomic information.

Banerjee-Basu S, Ferlanti ES, Ryan JF, Baxevanis AD
Nucleic Acids Res. 1999; 27: 336-7

The Homeodomain Resource is a comprehensive collection of sequence, structure and genomic information on the homeodomain protein family. Available through the Resource are both full-length and domain-only sequence data, as well as X-ray and NMR structural data for proteins and protein-DNA complexes. Also available is information on human genetic diseases and disorders in which proteins from the homeodomain family play an important role; genomic information includes relevant gene symbols, cytogenetic map locations, and specific mutation data. Search engines are provided to allow users to easily query the component databases and assemble specialized data sets. The Homeodomain Resource is available through the World Wide Web at http://genome.nhgri.nih.gov/homeodomain

A tale of two synthetases.

Artymiuk PJ, Rice DW, Poirrette AR, Willet P
Nat Struct Biol. 1994; 1: 758-60

The DNA binding activity and the dimerization ability of the thyroid transcription factor I are redox regulated.

Arnone MI, Zannini M, Di Lauro R
J Biol Chem. 1995; 270: 12048-55

The DNA binding activity of the thyroid transcription factor-1 (TTF-1), a homeodomain-containing protein implicated in the control of thyroid- and lung-specific transcription, is controlled, in vitro, by the redox potential. Oxidation decreases TTF-1 DNA binding activity, which is fully restored upon exposure to reducing agents. The decrease in DNA binding activity is due to the formation of disulfide bond(s), formed between two specific cysteine residues located outside the TTF-1 homeodomain; hence, oxidation does not appear to directly hinder TTF-1/DNA contacts. Disulfide bond formation seems to stabilize preexisting, loosely associated, TTF-1 dimers, which, upon oxidation, proceed in the formation of specific, higher order oligomers.

A heterodimeric transcriptional repressor becomes crystal clear.

Andrews BJ, Donoviel MS
Science. 1995; 270: 251-3

A new bipartite DNA-binding domain: cooperative interaction between the cut repeat and homeo domain of the cut homeo proteins.

Andres V, Chiara MD, Mahdavi V
Genes Dev. 1994; 8: 245-57

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.

Proposed alpha-helical super-secondary structure associated with protein-dna recognition.

Anderson WF
J Mol Biol. 1982; 159: 745-51

Cooperative ordering in homeodomain-DNA recognition: solution structure and dynamics of the MATa1 homeodomain.

Anderson JS, Forman MD, Modleski S, Dahlquist FW, Baxter SM
Biochemistry. 2000; 39: 10045-54

The mating type homeodomain proteins, MATa1 and MATalpha2, combine to form a heterodimer to bind DNA in diploid yeast cells. The a1-alpha2 heterodimer tightly and specifically binds haploid-specific gene operators to repress transcription. On its own, however, the a1 homeodomain does not bind DNA in a sequence-specific manner. To help understand this interaction, we describe the solution structure and backbone dynamics of the free a1 homeodomain. Free a1 in solution is an ensemble of structures having flexible hinges at the two turns in the small protein fold. Conformational changes in the a1 homeodomain upon ternary complex formation are located in the loop between helix 1 and helix 2, where the C-terminal tail of alpha2 binds to form the heterodimer, and at the C-terminus of helix 3, the DNA recognition helix. The observed differences, comparing the free and bound a1 structures, suggest a mechanism linking van der Waals stacking changes to the ordering of a final turn in the DNA-binding helix of a1. The tail of alpha2 induces changes in loop 1 of a1 that push it toward a properly folded DNA binding conformation.

A triploblast origin for Myxozoa?

Anderson CL, Canning EU, Okamura B
Nature. 1998; 392: 346-7

Hox genes: from master genes to micromanagers.

Akam M
Curr Biol. 1998; 8: 6768-6768

We still have little idea how the differential expression of one 'master' gene can control the morphology of complex structures, but recent studies suggest that the Drosophila Hox gene Ultrabithorax micromanages segment development by manipulating a large number of different targets at many developmental stages.

Evolution of distinct expression patterns for engrailed paralogues in higher crustaceans (Malacostraca).

Ahzhanov A, Kaufman TC
Dev Genes Evol. 2000; 210: 493-506

The segment-polarity gene engrailed of Drosophila melanogaster and its homologues in other arthropods possess a highly conserved expression domain in the posterior portion of each segment. We report here that the two pan-specific antibodies, Mab4D9 and Mab4F11, reveal strikingly different accumulation patterns in both of the malacostracan crustaceans Porcellio scaber (Isopoda) and Procambarus clarkii (Decapoda), compared with insects. The signal detected with Mab4D9 resides in the posterior part of each segment, including the appendages, the ventral and lateral sides of the trunk and the CNS. However, Mab4F11 reveals a signal only in small groups of neurons in the CNS and PNS, primarily localized in the pereon. We observe similar Mab4D9 and Mab4F11 patterns in the crayfish P. clarkii, except that no Mab4F11 signal is detected in the pleon. To address the possibility of multiple engrailed paralogues, we cloned partial cDNAs of two engrailed genes, Ps-en1 and Ps-en2, from P. scaber, and studied their expression patterns using whole-mount in situ hybridization. Although the Ps-en1 and Ps-en2 patterns are comparable in early development, they become distinct in late embryogenesis. Ps-en1 is expressed in the CNS, where Mab4F11 stains, but also accumulates in the epidermis. In contrast, Ps-en2 is expressed in the lateral aspect and limbs of all segments. Phylogenetic analysis of en sequences from crustaceans and insects suggests that the two en genes from the apterygote insect Thermobia domestica (Thysanura) may be related to en1 and en2 of higher crustaceans.

Homeodomain proteins and the regulation of gene expression.

Affolter M, Schier A, Gehring WJ
Curr Opin Cell Biol. 1990; 2: 485-95

Balancing import and export in development.

Affolter M, Marty T, Vigano MA
Genes Dev. 1999; 13: 913-5

Specificity of minor-groove and major-groove interactions in a homeodomain-DNA complex.

Ades SE, Sauer RT
Biochemistry. 1995; 34: 14601-8

To assess the importance of minor-groove and major-groove interactions in homeodomain-DNA recognition, the binding properties of variants of the altered-specificity engrailed homeodomain, containing Lys50, and its DNA site TAATCC were determined. This homeodomain contacts bases in the minor groove of the DNA using Arg3 and Arg5 from its N-terminal arm and contacts bases in the major groove of the DNA using Ile47, Lys50, and Asn51 from its third alpha-helix. Mutation of Arg3 or Ile47 to alanine reduces binding affinity 10-20-fold while mutation of Arg5, Asn51, or Lys50 to alanine reduces binding affinity > 100-fold, indicating that both minor-groove and major-groove interactions contribute to the overall binding energy. Binding site selections and affinity measurements show that the homeodomain can also discriminate among different base pairs in the minor groove and the major groove. However, the interactions between Lys50 of the recognition helix and the major-groove edges of base pairs 5 and 6 are more specific than interactions mediated by Arg3 and Arg5 in the N-terminal arm and the minor-groove edges of base pairs 1 and 2.

Human HOX homeobox genes.

Acampora D, Simeone A, Boncinelli E
Oxf Surv Eukaryot Genes. 1991; 7: 1-28