Specific residues in the Pbx homeodomain differentially modulate the DNA-binding activity of Hox and Engrailed proteins

Two classes of homeodomain proteins, Hox and Engrailed, have been shown to act in concert with the atypical homeodomain proteins Pbx and extradenticle. We now show that specific residues located within the Pbx homeodomain are essential for cooperative DNA binding with Hox and Engrailed gene products. Within the N-terminal region of the Pbx homeodomain, we have identified a residue that is required for cooperative DNA binding with three Hox gene products but not for cooperativity with Engrailed-2 (En-2). Furthermore, there are similarities between heterodimeric interactions involving the yeast mating type proteins MATa1 and MATalpha2 and those that allow the formation of Pbx/Hox and Pbx/En-2 heterodimers. Specifically, residues located in the a1 homeodomain that were previously shown to form a hydrophobic pocket allowing the alpha2 C-terminal tail to bind, are also required for Pbx/Hox and Pbx/En-2 cooperativity. Furthermore, we show that three residues located in the turn between helix 1 and helix 2, characteristic of many atypical homeodomain proteins, are required for cooperative DNA binding involving both Hox and En-2. Replacement of the three residues located in the turn between helix 1 and helix 2 of the Pbx homeodomain with those of the atypical homeodomain proteins controlling cell fate in the basidiomycete Ustilago maydis, bE5 and bE6, allows cooperative DNA binding with three Hox members but abolishes interactions with En-2. The data suggest that the molecular mechanism of homeodomain protein interactions that control cell fate in Saccharomyces cerevisiae and in the basidiomycetes may well be conserved in part in multicellular organisms.     

Bright light: a countermeasure for jet lag?

We report the cDNA cloning, partial genomic organization, and expression pattern of Stra10, a novel retinoic acid-inducible gene in P19 embryonal carcinoma cells. Four murine cDNA isoforms have been isolated, which are likely to result from alternative splicing. The predicted protein sequences exhibit approximately 85% identity with the Pbx-related Meis1 homeobox gene products, which are involved in myeloid leukemia in BXH-2 mice, and one of the Stra10 isoforms corresponds to the recently published Meis2 sequence (Nakamura et al. [1996] Oncogene 13:2235-2242). The Meis2 homeodomain is identical to that of Meis1, and is most closely related to those of the Pbx/TGIF homeobox gene products. By in situ hybridization analysis, we show that the Meis2 gene displays spatially restricted expression patterns in the developing nervous system, limbs, face, and in various viscera. In adult mice, Meis2 is mainly expressed in the brain and female genital tract, with a different distribution of the alternative splice forms in these organs.     

Specification of anteroposterior cell fates in Caenorhabditis elegans by Drosophila Hox proteins

Antennapedia class homeobox (Hox) genes specify cell fates in successive anteroposterior body domains in vertebrates, insects and nematodes. The DNA-binding homeodomain sequences are very similar between vertebrate and Drosophila Hox proteins, and this similarity allows vertebrate Hox proteins to function in Drosophila. In contrast, the Caenorhabditis elegans homeodomains are substantially divergent. Further, C. elegans differs from both insects and vertebrates in having a non-segmented body as well as a distinctive mode of development that involves asymmetric early cleavages and invariant cell lineages. Here we report that, despite these differences, Drosophila Hox proteins expressed in C. elegans can substitute for C. elegans Hox proteins in the control of three different cell-fate decisions: the regulation of cell migration, the specification of serotonergic neurons, and the specification of a sensory structure. We also show that the specificity of one C. elegans Hox protein is partly determined by two amino acids that have been implicated in sequence-specific DNA binding. Together these findings suggest that factors important for target recognition by specific Hox proteins have been conserved throughout much of the animal kingdom.     

Pax-3-DNA interaction: flexibility in the DNA binding and induction of DNA conformational changes by paired domains

The mouse Pax-3 gene encodes a protein that is a member of the Pax family of DNA binding proteins. Pax-3 contains two DNA binding domains: a paired domain (PD) and a paired type homeodomain (HD). Both domains are separated by 53 amino acids and interact synergistically with a sequence harboring an ATTA motif (binding to the HD) and a GTTCC site (binding to the PD) separated by 5 base pairs. Here we show that the interaction of Pax-3 with these two binding sites is independent of their angular orientation. In addition, the protein spacer region between the HD and the PD can be shortened without changing the spatial flexibility of the two DNA binding domains which interact with DNA. Furthermore, by using circular permutation analysis we determined that binding of Pax-3 to a DNA fragment containing a specific binding site causes conformational changes in the DNA, as indicated by the different mobilities of the Pax-3-DNA complexes. The ability to change the conformation of the DNA was found to be an intrinsic property of the Pax-3 PD and of all Pax proteins that we tested so far. These in vitro studies suggest that interaction of Pax proteins with their specific sequences in vivo may result in an altered DNA conformation.     

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

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.     

A conserved C-terminal domain in PBX increases DNA binding by the PBX homeodomain and is not a primary site of contact for the YPWM motif of HOXA1

HOX proteins are dependent upon cofactors of the PBX family for specificity of DNA binding. Two regions that have been implicated in HOX/PBX cooperative interactions are the YPWM motif, found N-terminal to the HOX homeodomain, and the GKFQ domain (also known as the Hox cooperativity motif) immediately C-terminal to the PBX homeodomain. Using derivatives of the E2A-PBX oncoprotein, we find that the GKFQ domain is not essential for cooperative interaction with HOXA1 but contributes to the stability of the complex. By contrast, the YPWM motif is strictly required for cooperative interactions in vitro and in vivo, even with mutants of E2A-PBX lacking the GKFQ domain. Using truncated PBX proteins, we show that the YPWM motif contacts the PBX homeodomain. The presence of the GKFQ domain increases monomer binding by the PBX homeodomain 5-fold, and the stability of the HOXA1.E2A-PBX complex 2-fold. These data suggest that the GKFQ domain acts mainly to increase DNA binding by PBX, rather than providing a primary contact site for the YPWM motif of HOXA1. We have identified 2 residues, Glu-301 and Tyr-305, required for GKFQ function and suggest that this is dependent on alpha-helical character.     

Mapping the RNA binding sites for human immunodeficiency virus type-1 gag and NC proteins within the complete HIV-1 and -2 untranslated leader regions

Encapsidation of HIV-1 genomic RNA is mediated by specific interactions between the RNA packaging signal and the Gag protein. During maturation of the virion, the Gag protein is processed into smaller fragments, including the nucleocapsid (NC) domain which remains associated with the viral genomic RNA. We have investigated the binding of glutathione- S -transferase (GST) Gag and NC fusion proteins from HIV-1, to the entire HIV-1 and -2 leader RNAencompassing the packaging signal. We have mapped the binding sites at conditions where only about two complexes are formed and find that GST-Gag and GST-NC fusion proteins bind specifically to discrete sites within the leader. Analysis of the HIV-1 leader indicated that GST-Gag strongly associates with the PSI stem-loop and to a lesser extent with regions near the primer binding site. GST-NC binds the same regions but with reversed preferences. The HIV-1 proteins also interact specifically with the 5'-leader of HIV-2 and the major site of interaction mapped to a stem-loop, with homology to the HIV-1 PSI stem-loop structure. The different specificities of Gag and NC may reflect functionally distinct roles in the viral replication, and suggest that the RNA binding specificity of NC is modulated by its structural context.     

Mapping viral DNA specificity to the central region of integrase by using functional human immunodeficiency virus type 1/visna virus chimeric proteins

We previously described the construction and analysis of the first set of functional chimeric lentivirus integrases, involving exchange of the N-terminal, central, and C-terminal regions of the human immunodeficiency virus type 1 (HIV-1) and visna virus integrase (IN) proteins. Based on those results, additional HIV-1/visna virus chimeric integrases were designed and purified. Each of the chimeric enzymes was functional in at least one oligonucleotide-based IN assay. Of a total of 12 chimeric IN proteins, 3 exhibit specific viral DNA processing, 9 catalyze insertion of viral DNA ends, 12 can reverse that reaction, and 11 are active for nonspecific alcoholysis. Functional data obtained with the processing assay indicate that the central region of the protein is responsible for viral DNA specificity. Target site selection for nonspecific alcoholysis again mapped to the central domain of IN, confirming our previous data indicating that this region can position nonviral DNA for nucleophilic attack. However, the chimeric proteins created patterns of viral DNA insertion distinct from that of either wild-type IN, suggesting that interactions between regions of IN influence target site selection for viral DNA integration. The results support a new model for the functional organization of IN in which viral DNA initially binds nonspecifically to the C-terminal portion of IN but the catalytic central region of the enzyme has a prominent role both in specific recognition of viral DNA ends and in positioning the host DNA for viral DNA integration.     

Cooperative assembly of the bovine papilloma virus E1 and E2 proteins on the replication origin requires an intact E2 binding site

Using quantitative gel retardation assays the properties of the bovine papilloma virus (BPV) origin recognition protein E1 and the effect of the viral E2 protein on the binding of E1 to BPV origin DNA were examined. As reported previously (Seo, Y.S., Mueller, F., Lusky, M., Gibbs, E., Kim, H.-Y., Phillips, B. and J. Hurwitz (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2865-2869), the E1 protein binds specifically to DNA sequences within the BPV origin (ori+) of replication. We also show that the presence of MgCl2 and ATP could stabilize the E1 ori+ DNA complex. At low levels of E1, ori+ DNA binding was greatly stimulated by the viral E2 protein when the intact E2 binding site 12 was present on the DNA. In addition DNA-protein complexes formed in the presence of both E1 and E2 were more stable than those formed with E1 alone. In the absence of an E2 binding site the E2 protein inhibited the binding of E1 to the BPV origin. Spacing of 0 or 9 base pairs between the E1 binding site and the E2 binding site 12 abolished the stimulation of E1-DNA binding by E2, whereas spacing of 6 base pairs between the two binding sites allowed for efficient stimulation. The data presented account for a direct role of E2 in BPV DNA replication. We propose that the cooperative binding of both the E1 and E2 proteins to BPV ori+ DNA is mediated by protein-protein interactions and by protein-DNA interactions, which include the formation of specific contacts of E2 with DNA.     

Pesticide avoidance behavior in Anopheles albimanus, a malaria vector in the Americas

We show that homothorax (hth) is required for the Hox genes to pattern the body of the fruit fly, Drosophila melanogaster. hth is necessary for the nuclear localization of an essential HOX cofactor, Extradenticle (EXD), and encodes a homeodomain protein that shares extensive identity with the product of Meis1, a murine proto-oncogene. MEIS1 is able to rescue hth mutant phenotypes and can induce the cytoplasmic-to-nuclear translocation of EXD in cell culture and Drosophila embryos. Thus, Meis1 is a murine homolog of hth. MEIS1/HTH also specifically binds to EXD with high affinity in vitro. These data suggest a novel and evolutionarily conserved mechanism for regulating HOX activity in which a direct protein-protein interaction between EXD and HTH results in EXD's nuclear translocation.     

Association with capsid proteins promotes nuclear targeting of simian virus 40 DNA

All animal DNA viruses except pox virus utilize the cell nucleus as the site for virus reproduction. Yet, a critical viral infection process, nuclear targeting of the viral genome, is poorly understood. The role of capsid proteins in nuclear targeting of simian virus 40 (SV40) DNA, which is assessed by the nuclear accumulation of large tumor (T) antigen, the initial sign of the infectious process, was tested by two independent approaches: antibody interception experiments and reconstitution experiments. When antibody against viral capsid protein Vp1 or Vp3 was introduced into the cytoplasm, the nuclear accumulation of T antigen was not observed in cells either infected or cytoplasmically injected with virion. Nuclearly introduced anti-Vp3 IgG also showed the inhibitory effect. In the reconstitution experiments, SV40 DNA was allowed to interact with protein components of the virus, either empty particles or histones, and the resulting complexes were tested for the capability of protein components to target the DNA to the nucleus from cytoplasm as effectively as the targeting of DNA in the mature virion. In cells injected with empty particle-DNA, but not in minichromosome-injected cells, T antigen was observed as effectively as in SV40-injected cells. These results demonstrate that SV40 capsid proteins can facilitate transport of SV40 DNA into the nucleus and indicate that Vp3, one of the capsid proteins, accompanies SV40 DNA as it enters the nucleus during virus infection.     

Sequence specificity of viral end DNA binding by HIV-1 integrase reveals critical regions for protein-DNA interaction

HIV-1 integrase specifically recognizes and cleaves viral end DNA during the initial step of retroviral integration. The protein and DNA determinants of the specificity of viral end DNA binding have not been clearly identified. We have used mutational analysis of the viral end LTR sequence, in vitro selection of optimal viral end sequences, and specific photocrosslinking to identify regions of integrase that interact with specific bases in the LTR termini. The results highlight the involvement of the disordered loop of the integrase core domain, specifically residues Q148 and Y143, in binding to the terminal portion of the viral DNA ends. Additionally, we have identified positions upstream in the LTR termini which interact with the C-terminal domain of integrase, providing evidence for the role of that domain in stabilization of viral DNA binding. Finally, we have located a region centered 12 bases from the viral DNA terminus which appears essential for viral end DNA binding in the presence of magnesium, but not in the presence of manganese, suggesting a differential effect of divalent cations on sequence-specific binding. These results help to define important regions of contact between integrase and viral DNA, and assist in the formulation of a molecular model of this vital interaction.