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The Drosophila Extramacrochaetae Protein Antagonizes Sequence-Specific DNA Binding by Daughterless/Achaete-Scute Protein Complexes

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The Drosophila Extramacrochaetae Protein Antagonizes Sequence-Specific DNA Binding by Daughterless/Achaete-Scute Protein Complexes Development 113, 245-255 (1991) 245 Printed in Great Britain © The Company of Biologists Limited 1991 The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes MARK VAN DOREN, HILARY M. ELLIS* and JAMES W. POSAKONYf Department of Biology and Center for Molecular Genetics, University of California San Diego, LaJolla, CA 92093-0322, USA * Present address: Department of Biology, Emory University, 1510 Clifton Road, Atlanta, GA 30322, USA t Corresponding author Summary In Drosophila, a group of regulatory proteins of the of emc. Under the conditions of our experiments, the emc helix-loop-helix (HLH) class play an essential role in protein, but not the h protein, is able to antagonize conferring upon cells in the developing adult epidermis specifically the in vitro DNA-binding activity of da/AS-C the competence to give rise to sensory organs. Proteins and putative da/da protein complexes. We interpret encoded by the daughterless (da) gene and three genes of these results as follows: the heterodimerization capacity the achaete-scute complex (AS-C) act positively in the of the emc protein (conferred by its HLH domain) allows determination of the sensory organ precursor cell fate, it to act in vivo as a competitive inhibitor of the while the extramacrochaetae (emc) and hairy (h) gene formation of functional DNA-binding protein complexes products act as negative regulators. In the region by the da and AS-C proteins, thereby reducing the upstream of the achaete gene of the AS-C, we have effective level of their transcriptional regulatory activity identified three 'E box' consensus sequences that are within the cell. bound specifically in vitro by hetero-oligomeric com- plexes consisting of the da protein and an AS-C protein. Key words: helix-loop-helix, sensory organ development, We have used this DNA-binding activity to investigate peripheral nervous system, negative regulation, protein- the biochemical basis of the negative regulatory function protein interaction, DNA binding. Introduction lead to the loss of both larval and adult sensory organ precursors (Caudy et al. 1988a; Cline, 1989; Dambly- The adult peripheral nervous system (PNS) of Dros- Chaudiere and Ghysen, 1987; Garcia-Bellido, 1979; ophila is composed of several types of multicellular Garcia-Bellido and Santamaria, 1978; Ghysen and sensory organs, or sensilla, that appear on the body O'Kane, 1989; Romani et al. 1989; Ruiz-Gomez and surface in a relatively invariant spatial pattern. The Modolell, 1987). The activities of these genes are precursor cells that give rise to sensory organs develop negatively regulated by the extramacrochaetae (emc) during the late larval and early pupal stages within and hairy (h) genes; loss-of-function mutations in emc undifferentiated epithelial sheets (imaginal discs and and h cause the ectopic appearance of supernumerary histoblast nests) that also give rise to ordinary sensilla in the adult (Botas et al. 1982; Garcia Alonso epidermal cells (see Hartenstein and Posakony, 1989). and Garcfa-Bellido, 1988; Moscoso del Prado and Thus, a primary event in PNS development is the Garcia-Bellido, 1984). The genes of the ASIG interact position-dependent assignment of one of two alterna- in a dosage-sensitive manner, such that the phenotype tive fates, sensillum precursor or epidermal precursor, conferred by a mutation in one gene in the group is to the cells in the developing adult epidermis. A set of suppressed or enhanced by changes in the wild-type genes that we have referred to earlier as the 'achaete- dosage of another gene in the group (Botas etal. 1982; scute interacting group' (ASIG; Ellis etal. 1990) play an Dambly-Chaudiere et al. 1988; Moscoso del Prado and essential role in establishing the competence of cells to Garcia-Bellido, 1984). The discovery that the protein be sensory organ precursors. The daughterless (da) products of all of these genes contain the protein gene and three of the four genes of the achaete-scute dimerization domain characteristic of the helix-loop- complex (AS-C) [achaete (ac) or T5, scute (sc) or T4, helix (HLH) class of proteins (Murre et al. 1989a; and asense (ase) or T8 (Gonzalez et al. 1989)] act Alonso and Cabrera, 1988; Caudy et al. 19886; Ellis et positively to confer the sensory organ precursor cell al. 1990; Garrell and Modolell, 1990; Rushlow et al. fate: Loss-of-function mutations in da and the AS-C 1989; Villares and Cabrera, 1987) has led to the 246 M. Van Doren, H. M. Ellis andJ. W. Posakony suggestion that the dosage-sensitive interactions of the by the HLH proteins encoded by transcription units ASIG primarily reflect protein-protein interactions m5, ml, and m8 of the Enhancer of split [E(spl)] gene (Ellis et al. 1990; Garrell and Modolell, 1990). complex (Klambt etal. 1989). E(spl) is a member of the Most known members of the HLH family of proteins, so-called neurogenic class of genes; these are required including the da and AS-C proteins, have a region of for inhibitory cell-cell interaction events that restrict conserved basic residues adjacent to the HLH domain the expression of particular cell fates during both that is essential for sequence-specific DNA-binding central and peripheral neurogenesis in Drosophila (see activity (Davis etal. 1990; Lassar e/a/. 1989; Murre e/a/. for review Campos-Ortega, 1990). Though the h and 1989a; Voronova and Baltimore, 1990). These are E(spl) HLH proteins share a set of basic residues referred to as bHLH proteins (Davis etal. 1990; Jones, adjacent to their HLH domains, the basic region 1990). They bind DNA with high affinity as dimeric or consensus sequence derived from them is quite differ- higher-order oligomeric complexes, the formation of ent from that derived from other bHLH proteins (see which requires the HLH dimerization domain (Davis et Discussion). Nevertheless, the existence of a conserved al. 1990; Lassar et al. 1989; Murre et al. 1989a,b; set of basic residues in the h and E(spl) proteins Voronova and Baltimore, 1990). The specific binding indicates that they may be capable of binding DNA, sites that have been identified for several bHLH and suggests that their mode of action may therefore be proteins include a common core sequence (CANNTG) different from that of emc and Id. called the 'E box' (see Blackwell and Weintraub, 1990; In this paper we report the identification, in the Murre and Baltimore, 1991; Murre et al. 1989a,b). region upstream of the ac gene, of specific 'E box' Detailed studies of certain bHLH proteins, such as the binding sites for hetero-oligomeric complexes com- myogenic regulator MyoD (Tapscott et al. 1988), have posed of the da protein and an AS-C protein. We also shown that they act as transcriptional activators, demonstrate that the emc protein can specifically controlling not only the expression of cell type-specific antagonize the DNA-binding activity of da/AS-C downstream genes (Davis etal. 1990; Lassar etal. 1989), complexes in vitro. Under the same conditions, the h but also their own expression (positive autoregulation) and E(spl) m8 proteins fail to exhibit this inhibitory (Thayer et al. 1989). By analogy to these results, the activity. Drosophila da and AS-C proteins are thought to exert their effects on neurogenesis as transcriptional acti- vators (Alonso and Cabrera, 1988; Murre etal. 1989ft). Materials and methods da and the AS-C T4 (sc) gene also play an essential role in sex determination by participating in the measure- General molecular biology methods ment of the X:autosome ratio in the preblastoderm General techniques not described in detail below were carried embryo (Cline, 1976; Cline, 1988; Erickson and Cline, out as described by Ausubel et al. (1987) and Sambrook et al. 1991; Parkhurst et al. 1990; Steinmann-Zwicky et al. (1989). 1990; Torres and Sanchez, 1989). Here again, hetero- dimeric complexes of the two proteins (da/T4) are Synthesis of capped mRNAs in vitro thought to act as transcriptional activators of the master cDNA clones representing mRNAs of the sc (AS-C T4), ac (AS-C T5), da, h, and E(spt) m8 genes were isolated froma regulatory gene Sex-lethal (Erickson and Cline, 1991; + Parkhurst et al. 1990). Consistent with these hypoth- library made from 4-8 h Drosophila embryo poly(A) eses, Murre et al. have shown that complexes of the da (Brown and Kafatos, 1988). These have been designated pNBT4, pNBT5, pNBda, pNBh, and pNBm8, respectively. and AS-C T3 {lethal of scute) proteins will bind in vitro Each clone contains a complete protein coding sequence, as to the KE2 site of the mouse immunoglobulin K light determined by comparing partial sequence data to published chain enhancer (Murre et al. 19896). sequences (Caudy etal. 19886; Cronmiller etal. 1988; Klambt etal. 1989; Rushlow etal. 1989; Villares and Cabrera, 1987); We and others have reported previously that the details are available upon request. An emc cDNA clone predicted emc protein, like its mouse homologue Id (pNB5b) has been described previously (Ellis etal. 1990). All (Benezra et al. 1990), shares the HLH dimerization cDNAs except that representing da were transcribed (see domain of the bHLH proteins but lacks the conserved below) directly from the library vector (pNB40). basic residues that constitute their DNA-binding For da, an Asel-Scal fragment of pNBda containing the domain (Ellis et al. 1990; Garrell and Modolell, 1990). protein coding region was ligated to Xbal linkers and On the basis of this finding, we proposed that the emc subcloned into the Xbal site of pBluescript KS(+) (Strata- protein negatively regulates sensory organ determi- gene) so that the 5' end is oriented towards the Sad site of the nation by forming heterodimers with the da and/or AS- vector (pKSda).
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