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

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.

[1]  G. Sarkar,et al.  The "megaprimer" method of site-directed mutagenesis. , 1990, BioTechniques.

[2]  Claude Desplan,et al.  Crystal structure of a paired domain-DNA complex at 2.5 å resolution reveals structural basis for pax developmental mutations , 1995, Cell.

[3]  C. Desplan,et al.  Cooperative interactions between paired domain and homeodomain. , 1996, Development.

[4]  V. Sheffield,et al.  Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[5]  M. Weir,et al.  Functional dissection of the paired segmentation gene in Drosophila embryos. , 1991, Genes & development.

[6]  R. Balling,et al.  undulated, a mutation affecting the development of the mouse skeleton, has a point mutation in the paired box of Pax 1 , 1988, Cell.

[7]  M. Fujioka,et al.  Early even-skipped stripes act as morphogenetic gradients at the single cell level to establish engrailed expression. , 1995, Development.

[8]  M P Weir,et al.  Patterns of engrailed protein in early Drosophila embryos. , 1989, Development.

[9]  C. Desplan,et al.  In vivo requirement for the paired domain and homeodomain of the paired segmentation gene product. , 1996, Development.

[10]  K. Schughart,et al.  Mouse Hox-2.2 specifies thoracic segmental identity in Drosophila embryos and larvae , 1990, Cell.

[11]  Further elucidation of the genomic structure of PAX3, and identification of two different point mutations within the PAX3 homeobox that cause Waardenburg syndrome type 1 in two families. , 1995, American journal of human genetics.

[12]  S. Brain,et al.  Substance P regulates the vasodilator activity of calcitonin gene-related peptide , 1988, Nature.

[13]  J. Yon,et al.  Precise gene fusion by PCR. , 1989, Nucleic acids research.

[14]  J. Kuriyan,et al.  High resolution crystal structure of a paired (Pax) class cooperative homeodomain dimer on DNA , 1995, Cell.

[15]  M. Fujioka,et al.  Drosophila Paired regulates late even-skipped expression through a composite binding site for the paired domain and the homeodomain. , 1996, Development.

[16]  W. Gehring,et al.  Functional redundancy: the respective roles of the two sloppy paired genes in Drosophila segmentation. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M. Noll,et al.  Isolation of two tissue‐specific Drosophila paired box genes, Pox meso and Pox neuro. , 1989, The EMBO journal.

[18]  H. Krause,et al.  Control of segmental asymmetry in Drosophila embryos. , 1993, Development.

[19]  P. O’Farrell,et al.  Establishment and refinement of segmental pattern in the Drosophila embryo: spatial control of engrailed expression by pair-rule genes. , 1987, Genes & development.

[20]  C. Nüsslein-Volhard,et al.  Mutations affecting segment number and polarity in Drosophila , 1980, Nature.

[21]  J. Mohler,et al.  Molecular organization and embryonic expression of the hedgehog gene involved in cell-cell communication in segmental patterning of Drosophila. , 1992, Development.

[22]  References , 1971 .

[23]  G. Schaffner,et al.  DNA sequence recognition by Pax proteins: bipartite structure of the paired domain and its binding site. , 1993, Genes & development.

[24]  P. Ingham.  The molecular genetics of embryonic pattern formation in Drosophila , 1988, Nature.

[25]  C. Baldwin,et al.  Mutations in the paired domain of the human PAX3 gene cause Klein-Waardenburg syndrome (WS-III) as well as Waardenburg syndrome type I (WS-I). , 1993, American journal of human genetics.

[26]  M. Levine,et al.  Cross-regulatory interactions among pair-rule genes in Drosophila. , 1986, Science.

[27]  M. Noll,et al.  Structure of two genes at the gooseberry locus related to the paired gene and their spatial expression during Drosophila embryogenesis. , 1987, Genes & development.

[28]  M. Noll,et al.  Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network , 1986, Cell.

[29]  M. Levine,et al.  Synergistic activation and repression of transcription by Drosophila homeobox proteins , 1989, Cell.

[30]  W. Gehring,et al.  The white gene as a marker in a new P-element vector for gene transfer in Drosophila. , 1987, Nucleic acids research.

[31]  M. Noll,et al.  Isolation of the paired gene of Drosophila and its spatial expression during early embryogenesis , 1986, Nature.

[32]  G. Eichele,et al.  9-cis-retinoic acid, a potent inducer of digit pattern duplications in the chick wing bud. , 1993, Development.

[33]  M. Noll,et al.  Conservation of a large protein domain in the segmentation gene paired and in functionally related genes of Drosophila , 1986, Cell.

[34]  C. Desplan,et al.  Cooperative dimerization of paired class homeo domains on DNA. , 1993, Genes & development.

[35]  P. Gros,et al.  splotch (Sp2H ), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homeodomain of Pax-3 , 1991, Cell.

[36]  C. Desplan,et al.  The paired box encodes a second DNA-binding domain in the paired homeo domain protein. , 1991, Genes & development.

[37]  Juan Botas,et al.  The DNA binding specificity of ultrabithorax is modulated by cooperative interactions with extradenticle, another homeoprotein , 1994, Cell.

[38]  P. Ingham,et al.  Isolation, structure, and expression of even-skipped: A second pair-rule gene of Drosophila containing a homeo box , 1986, Cell.

[39]  Pierre Gönczy,et al.  A single amino acid can determine the DNA binding specificity of homeodomain proteins , 1989, Cell.

[40]  Dana L. Smith,et al.  A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an α2 dimer , 1992, Cell.

[41]  M. Levine,et al.  Divergent homeo box proteins recognize similar DNA sequences in Drosophila , 1988, Nature.

[42]  M. Benedyk,et al.  odd-paired: a zinc finger pair-rule protein required for the timely activation of engrailed and wingless in Drosophila embryos. , 1994, Genes & development.

[43]  P. Ingham,et al.  Regulatory interactions between the segmentation genes fushi tarazu, hairy, and engrailed in the Drosophila blastoderm , 1986, Cell.

[44]  P. Beachy,et al.  Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog , 1992, Cell.

[45]  C. Baldwin,et al.  An exonic mutation in the HuP2 paired domain gene causes Waardenburg's syndrome , 1992, Nature.

[46]  R. Baler,et al.  Synergistic activation of transcription is mediated by the N-terminal domain of Drosophila fushi tarazu homeoprotein and can occur without DNA binding by the protein , 1993, Molecular and cellular biology.

[47]  T. Tabata,et al.  The Drosophila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation. , 1992, Genes & development.

[48]  H. Krause,et al.  Concentration-dependent activities of the even-skipped protein in Drosophila embryos. , 1992, Genes & development.

[49]  M. Noll,et al.  Complex regulation of early paired expression: initial activation by gap genes and pattern modulation by pair-rule genes. , 1993, Development.

[50]  M. Noll,et al.  Evolution of distinct developmental functions of three Drosophila genes by acquisition of different cis-regulatory regions , 1994, Nature.

[51]  D. Grueneberg,et al.  Human and Drosophila Homeodomain Proteins That Enhance the DNA-Binding Activity of Serum Response Factor , 1992, Science.

[52]  M. Akam,et al.  The molecular basis for metameric pattern in the Drosophila embryo. , 1987, Development.

[53]  Wolfgang Driever,et al.  Determination of spatial domains of zygotic gene expression in the Drosophila embryo by the affinity of binding sites for the bicoid morphogen , 1989, Nature.

[54]  L. Kauvar,et al.  The engrailed locus of drosophila: Structural analysis of an embryonic transcript , 1985, Cell.

[55]  M. Weir,et al.  Dissection of the Drosophila paired protein: Functional requirements for conserved motifs , 1994, Mechanisms of Development.

[56]  S. Ho,et al.  Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. , 1989, Gene.

[57]  M. Goulding,et al.  Molecular basis of splotch and Waardenburg Pax-3 mutations. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[58]  P. Ingham,et al.  Regulation of segment polarity genes in the Drosophila blastoderm by fushi tarazu and even skipped , 1988, Nature.

[59]  C. Goutte,et al.  a1 Protein alters the dna binding specificity of α2 repressor , 1988, Cell.

[60]  M. Goulding,et al.  The molecular basis of the undulated/Pax-1 mutation , 1991, Cell.

[61]  J. Gall.  Gametogenesis and the early embryo , 1986 .

[62]  R. Brent,et al.  A genetic model for interaction of the homeodomain recognition helix with DNA. , 1991, Science.

[63]  M. Noll,et al.  Conservation of the paired domain in metazoans and its structure in three isolated human genes. , 1989, The EMBO journal.

[64]  M. Scott,et al.  The structure and function of the homeodomain. , 1989, Biochimica et biophysica acta.

[65]  B. Edgar,et al.  Spatial regulation of engrailed expression in the Drosophila embryo. , 1988, Genes & development.

[66]  R. Balling,et al.  Waardenburg's syndrome patients have mutations in the human homologue of the Pax-3 paired box gene , 1992, Nature.