The spineless-aristapedia and tango bHLH-PAS proteins interact to control antennal and tarsal development in Drosophila

Development, Sep 1999

R.B. Emmons, D. Duncan, P.A. Estes, P. Kiefel, J.T. Mosher, M. Sonnenfeld, M.P. Ward, I. Duncan, S.T. Crews

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The spineless-aristapedia and tango bHLH-PAS proteins interact to control antennal and tarsal development in Drosophila

Richard B. Emmons 1 Dianne Duncan 1 Patricia A. Estes 0 Paula Kiefel 1 Jack T. Mosher 0 Margaret Sonnenfeld 0 Mary P. Ward 0 Ian Duncan 1 Stephen T. Crews ) 0 0 Department of Biochemistry and Biophysics, the University of North Carolina at Chapel Hill , Chapel Hill, NC 27599-7260 , USA 1 Department of Biology, Washington University , St. Louis, MO , USA SUMMARY The Drosophila spineless (ss) gene encodes a basic-helixloop-helix-PAS transcription factor that is required for proper specification of distal antennal identity, establishment of the tarsal regions of the legs, and normal bristle growth. ss is the closest known homolog of the mammalian aryl hydrocarbon receptor (Ahr), also known as the dioxin receptor. Dioxin and other aryl hydrocarbons bind to the PAS domain of Ahr, causing Ahr to translocate to the nucleus, where it dimerizes with another bHLHPAS protein, the aryl hydrocarbon receptor nuclear translocator (Arnt). Ahr:Arnt heterodimers then activate transcription of target genes that encode enzymes involved in metabolizing aryl hydrocarbons. In this report, we present evidence that Ss functions as a heterodimer with the Drosophila ortholog of Arnt, Tango (Tgo). We show that the ss and tgo genes have a close functional relationship: loss-of-function alleles of tgo were recovered as dominant enhancers of a ss mutation, and tgo-mutant somatic clones show antennal, leg, and bristle defects almost identical to those caused by ss- mutations. The results of yeast twohybrid assays indicate that the Ss and Tgo proteins interact directly, presumably by forming heterodimers. Loss-of-function mutations in the Drosophila spineless (ss) gene cause three distinct phenotypes: transformation of the distal antenna to distal second leg, deletion of most of the tarsal region in each leg, and reduction in the size of almost all bristles (Struhl, 1982; Lindsley and Zimm, 1992; Duncan et al., 1998). Consistent with these phenotypes, ss is expressed in the distal portion of the antennal imaginal disc, in the tarsal regions of the leg discs, and in bristle precursor cells (Duncan et al., 1998). ss is also expressed in the antennal segment, gnathal segments, limb primordia, and peripheral nervous system of the embryo. ss appears to be a primary determinant of distal antennal identity, as ectopic expression causes transformation of the distal leg, maxillary palp, and rostral membrane to antenna. In the legs, ss appears to function in the establishment of the tarsal primordia, as it is expressed only Coexpression of Ss and Tgo in Drosophila SL2 cells causes transcriptional activation of reporters containing mammalian Ahr:Arnt response elements, indicating that Ss:Tgo heterodimers are very similar to Ahr:Arnt heterodimers in DNA-binding specificity and transcriptional activation ability. During embryogenesis, Tgo is localized to the nucleus at sites of ss expression. This localization is lost in a ss null mutant, suggesting that Tgo requires heterodimerization for translocation to the nucleus. Ectopic expression of ss causes coincident ectopic nuclear localization of Tgo, independent of cell type or developmental stage. This suggests that the interaction of Ss and Tgo does not require additional signals, unlike the ligand-dependent interaction of Ahr and Arnt. Despite the very different biological roles of Ahr and Arnt in insects and mammals, the molecular mechanisms by which these proteins function appear to be largely conserved. early in the tarsal region, and is required for later expression of the tarsal gene bric brac (Duncan et al., 1998). The protein encoded by ss is a member of the basic-helixloop-helix-PAS domain (bHLH-PAS) family of transcription factors. PAS is named for the founding members of the family, Period, Aryl hydrocarbon receptor (Ahr) and Single-minded (Sim) (for review see Crews, 1998). PAS domains can function in dimerization (Huang et al., 1993), dimerization specificity (Pongratz et al., 1998), binding of aryl hydrocarbons (Dolwick et al., 1993) and interaction with non-PAS proteins (Coumailleau et al., 1995; Gekakis et al., 1995). Recently, it has become clear that several bHLH-PAS proteins share a common bHLH-PAS dimerization partner. In mammals, this is the so-called Aryl hydrocarbon receptor nuclear translocator, or Arnt. Arnt serves as the dimerization partner for Ahr and probably at least five other mammalian bHLH-PAS proteins (Crews 1998). These include the small family of proteins related to Hypoxia-inducible factor-1a (HIF-1a ) that function in the physiological and developmental aspects of oxygen homeostasis (Semenza, 1998), and Sim1 and Sim2, the mammalian orthologs of Drosophila Sim. The Sim1 gene controls the formation of several hypothalamic nuclei (Michaud et al., 1998) and Sim2 has been implicated in Down Syndrome (Michaud and Fan, 1997). In Drosophila, a homolog of Arnt, called Tango (Tgo), has been shown to dimerize in vivo with the bHLH-PAS proteins Sim and Trachealess (Trh) (So (...truncated)


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R.B. Emmons, D. Duncan, P.A. Estes, P. Kiefel, J.T. Mosher, M. Sonnenfeld, M.P. Ward, I. Duncan, S.T. Crews. The spineless-aristapedia and tango bHLH-PAS proteins interact to control antennal and tarsal development in Drosophila, Development, 1999, pp. 3937-3945, 126/17,