Differential DNA binding properties of three human homeodomain proteins.

The products of three human homeobox containing (HOX) genes, 2C, 3C and 4B, were produced in insect cells using the Baculovirus expression system and purified to near homogeneity. In this system we observed that the DNA binding forms of the three proteins are not glycosylated. HOX 3C and 4B are phosphorylated in insect cells, while HOX 2C is not. The three HOX proteins bind to a DNA sequence known to be a target site for Antennapedia protein with a very similar affinity (Kd = 1-2 x 10(-9) M). We then measured their binding properties to four human sequences present in the HOX 3D, 4C, 1C and 4B promoters. Two of these sequences have been reported to be binding sites for HOX proteins. HOX 2C, 3C and 4B behaved quite differently, showing low affinity for promoters of genes located upstream from their own gene in the HOX clusters and a higher affinity for regulatory sequences of their own gene and downstream HOX genes.     

Isolation and sequence-specific DNA binding of the Antennapedia homeodomain.

The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was overproduced in a T7 expression vector in Escherichia coli. The corresponding polypeptide of 68 amino acids was purified to homogeneity. The homeodomain was analysed by ultracentrifugation and assayed for DNA binding. The secondary structure of the isolated homeodomain was determined by nuclear magnetic resonance spectroscopy. DNA-binding studies indicate that the isolated homeodomain binds to DNA in vitro. It selectively binds to the same sites as a longer Antp polypeptide and a full-length fushi tarazu (ftz) protein. Therefore, the homeodomain represents the DNA-binding domain of the homeotic proteins.     

RFX proteins, a novel family of DNA binding proteins conserved in the eukaryotic kingdom.

Until recently, the RFX family of DNA binding proteins consisted exclusively of four mammalian members (RFX1-RFX4) characterized by a novel highly conserved DNA binding domain. Strong conservation of this DNA binding domain precluded a precise definition of the motif required for DNA binding. In addition, the biological systems in which these RFX proteins are implicated remained obscure. The recent identification of four new RFX genes has now shed light on the evolutionary conservation of the RFX family, contributed greatly to a detailed characterization of the RFX DNA binding motif, and provided clear evidence for the function of some of the RFX proteins. RFX proteins have been conserved throughout evolution in a wide variety of species, including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, mouse and man. The characteristic RFX DNA binding motif has been recruited into otherwise very divergent regulatory factors functioning in a diverse spectrum of unrelated systems, including regulation of the mitotic cell cycle in fission yeast, the control of the immune response in mammals, and infection by human hepatitis B virus.     

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

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

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

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

Specific interactions between the K domains of AG and AGLs, members of the MADS domain family of DNA binding proteins

MADS domain (for M CM1, A G, D EFA and S RF) proteins are regulatory proteins found in all major eukaryotic kingdoms. Plant MADS domain regulatory proteins have a region of moderate sequence similarity that has been designated as the K domain, and its predicted coiled-coil structure suggests a role in establishing a protein—protein interaction. In vivo studies with the Arabidopsis AGAMOUS (AG) protein have indicated that the K domain is important for AG function. Using a bait fusion protein containing the K domain and the C-terminal region of AG in a yeast two-hybrid selection, 156 clones that encode potential AG-interacting proteins were identified. These clones each encode one of four highly related MADS domain proteins: AGL2, AGL4, AGL6 and AGL9. Additional analysis showed that the K domain of AG alone was able to bind the K domains of these AGLs. This binding was further confirmed by immunoprecipitation experiments using in vitro synthesized AG and AGL K domains. These results strongly suggest that AG interacts with AGL2, AGL4, AGL6 and AGL9 in vivo. Based on these results and previous observations, it is proposed that the AG function requires interaction with at least one of these AGL proteins, and such interactions contribute to the functional specificity of the AG protein.     

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

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.     

Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins.

Murine homeobox genes play a fundamental role in directing embryogenesis by controlling gene expression during development. The homeobox encodes a DNA binding domain (the homeodomain) which presumably mediates interactions of homeodomain proteins with specific DNA sites in the control regions of target genes. However, the bases for these selective DNA-protein interactions are not well defined. In this report, we have characterized the DNA binding specificities of three murine homeodomain proteins, Hox 7.1, Hox 1.5, and En-1. We have identified optimal DNA binding sites for each of these proteins by using a random oligonucleotide selection strategy. Comparison of the sequences of the selected binding sites predicted a common consensus site that contained the motif (C/G)TAATTG. The TAAT core was essential for DNA binding activity, and the nucleotides flanking this core directed binding specificity. Whereas variations in the nucleotides flanking the 5' side of the TAAT core produced modest alterations in binding activity for all three proteins, perturbations of the nucleotides directly 3' of the core distinguished the binding specificity of Hox 1.5 from those of Hox 7.1 and En-1. These differences in binding activity reflected differences in the dissociation rates rather than the equilibrium constants of the protein-DNA complexes. Differences in DNA binding specificities observed in vitro may contribute to selective interactions of homeodomain proteins with potential binding sites in the control regions of target genes.     

DNA binding activity of the BarH1 homeodomain of Drosophila.

Using a series of deletion mutants of BarH1, a Drosophila homeobox gene required for eye morphogenesis, the DNA-binding region of the BarH1 protein was determined. Not only homeodomain but also its upstream sequence were found to be necessary for binding, whereas about a half of the conserved downstream sequence (Bar domain) was dispensable.     

Specific conjugation of DNA binding proteins to DNA templates through thiol-disulfide exchange

The double-stranded oligodeoxyribonucleotides with single internucleotide disulfide linkages were successfully used for covalent trapping of cysteine containing protein. In particular, an efficient conjugation of DNA methyltransferase SsoII to sequence-specific decoys was demonstrated. The obtained results assume that synthetic oligodeoxyribonucleotides bearing a new trapping site can be used as new tools to study and manipulate biological systems.     

Differential expression of two msh-related homeobox genes Chox-7 and Chox-8 during chick limb development.

We have isolated two closely related cDNAs, Chox-7 and Chox-8, encoding homeodomain-containing proteins homologous to Drosophila msh. The Chox-7 and Chox-8 genes are chicken cognates of mouse Hox-7.1 and Hox-8.1, respectively. In situ hybridization using 3' regions of the cDNAs as probes revealed that the Chox-7 gene is highly expressed in the mesenchyme subjacent to the apical ectodermal ridge whereas Chox-8 expression is localized in the anterodistal mesenchymal region at early stages of limb formation, suggesting different roles during limb development. At later stages, both genes are expressed in the anterior and posterior mesenchymes and in the interdigital mesenchyme where programmed cell death occurs.     

Two highly related homeodomain proteins, Nkx5-1 and Nkx5-2, display different DNA binding specificities.

The mouse Nkx5-1 and Nkx5-2 genes are related to NK genes in Drosophila and encode proteins with very similar homeodomains. In higher vertebrates Nkx5 genes are specifically expressed in the inner ear. Inactivation of the mouse Nkx5-1 gene by homologous recombination revealed a critical role for the formation of vestibular inner ear structures. Here, we investigated biochemical properties of the proteins encoded by the Nkx5 genes. A similar consensus binding sequence was isolated for both Nkx5 proteins using binding site selection. This sequence is related to target sequences previously identified for other Nkx proteins and contains the conserved homeodomain binding core TAAT. An additional, novel and unrelated high affinity binding sequence could be identified for the Nkx5-2 protein.     

ARID proteins: a diverse family of DNA binding proteins implicated in the control of cell growth, differentiation, and development.

The ARID family of DNA binding proteins was first recognized approximately 5 years ago. The founding members, murine Bright and Drosophila dead ringer (Dri), were independently cloned on the basis of their ability to bind to AT-rich DNA sequences, although neither cDNA encoded a recognizable DNA binding domain. Mapping of the respective binding activities revealed a shared but previously unrecognized DNA binding domain, the consensus sequence of which extends across approximately 100 amino acids. This novel DNA binding domain was designated AT-rich interactive domain (ARID), based on the behavior of Bright and Dri. The consensus sequence occurs in 13 distinct human proteins and in proteins from all sequenced eukaryotic organisms. The majority of ARID-containing proteins were not cloned in the context of DNA binding activity, however, and their features as DNA binding proteins are only beginning to be investigated. The ARID region itself shows more diversity in structure and function than the highly conserved consensus sequence suggests. The basic structure appears to be a series of six alpha-helices separated by beta-strands, loops, or turns, but the structured region may extend to an additional helix at either or both ends of the basic six. It has also become apparent that the DNA binding activity of ARID-containing proteins is not necessarily sequence specific. What is consistent is the evidence that family members play vital roles in the regulation of development and/or tissue-specific gene expression. Inappropriate expression of ARID proteins is also increasingly implicated in human tumorigenesis. This review summarizes current knowledge about the structure and function of ARID family members, with a particular focus on the human proteins.     

Hox homeodomain proteins exhibit selective complex stabilities with Pbx and DNA.

Eight of the nine homeobox genes of the Hoxb locus encode proteins which contain a conserved hexapeptide motif upstream from the homeodomain. All eight proteins (Hoxb-1-Hoxb-8) bind to a target oligonucleotide in the presence of Pbx1a under conditions where minimal or no binding is detected for the Hox or Pbx1a proteins alone. The stabilities of the Hox-Pbx1a-DNA complexes vary >100-fold, with the proteins from the middle of the locus (Hoxb-5 and Hoxb-6) forming very stable complexes, while Hoxb-4, Hoxb-7 and Hoxb-8 form complexes of intermediate stability and proteins at the 3'-side of the locus (Hoxb-1-Hoxb-3) form complexes which are very unstable. Although Hox-b proteins containing longer linker sequences between the hexapeptide and homeodomains formed unstable complexes, shortening the linker did not confer complex stability. Homeodomain swapping experiments revealed that this motif does not independently determine complex stability. Naturally occurring variations within the hexapeptides of specific Hox proteins also do not explain complex stability differences. However, two core amino acids (tryptophan and methionine) which are absolutely conserved within the hexapeptide domains appear to be required for complex formation. Removal of N- and C-terminal flanking regions did not influence complex stability and the members of paralog group 4 (Hoxa-4, b-4, c-4 and d-4), which share highly conserved hexapeptides, linkers and homeodomains but different flanking regions, form complexes of similar stability. These data suggest that the structural features of Hox proteins which determine Hox-Pbx1a-DNA complex stability reside within the precise structural relationships between the homeodomain, hexapeptide and linker regions.     

DNA binding properties of two Arabidopsis MADS domain proteins: binding consensus and dimer formation.

MADS domain proteins are members of a highly conserved family found in all eukaryotes. Genetic studies clearly indicate that many plant MADS domain proteins have different regulatory functions in flower development, yet they share a highly conserved DNA binding domain and can bind to very similar sequences. How, then, can these MADS box genes confer their specific functions? Here, we describe results from DNA binding studies of AGL1 and AGL2 (for AGAMOUS-like), two Arabidopsis MADS domain proteins that are preferentially expressed in flowers. We demonstrate that both proteins are sequence-specific DNA binding proteins and show that each binding consensus has distinct features, suggestion a mechanism for specificity. In addition, we show that the proteins with more similar amino acid sequences have more similar binding sequences. We also found that AGL2 binds to DNA in vitro as a dimer and determined the region of AGL2 that is sufficient for DNA binding and dimerization. Finally, we show that several plant MADS domain proteins can bind to DNA either as homodimers or as heterodimers, suggesting that the number of different regulators could be much greater than the number of MADS box genes.