Hormonal induction of Dopa decarboxylase in the epidermis of Drosophila is mediated by the Broad-Complex.

The 2B5 early puff locus corresponds to the Broad-Complex BR-C) and encodes a family of transcription factors whose members are induced by the molting hormone ecdysone. Mutations in the br subcomplementation group substantially reduce the levels of Dopa decarboxylase (DDC) in the epidermis of mature third instar larvae but not in mature second instar organisms. Enzyme levels are normal in the central nervous system of the two mutants examined. The specificity of these effects suggests that a product of the BR-C locus mediates the rapid appearance of DDC in mature third instar larvae experiencing an elevated titer of ecdysone. The likely identity of this protein has been confirmed by pursuing the observation that the br28 allele caused by the insertion of a P element into the Z2 DNA-binding domain. Both the transcript and a protein carrying this domain are present in the epidermis and a BR-C recombinant protein carrying the Z2 finger binds to the first intron of the Ddc gene. Five binding sites have been identified within the intron by DNAase I footprinting and a core consensus sequence has been derived which shares some identity with the consensus binding site of the Z2 protein to the Sgs-4 regulatory region. Our demonstration that Ddc is a target of BR-C in the epidermis is the first direct evidence of a role for this early gene in a tissue other than the salivary glands. The data reinforce the idea that BR-C, which clearly mediates a salivary gland-specific response to ecdysone, may play a widespread role in the hormone's activation of gene cascades in other target tissues.

[1]  V. Bedian,et al.  Differential expression of Broad-Complex transcription factors may forecast tissue-specific developmental fates during Drosophila metamorphosis. , 1994, Development.

[2]  C. Thummel,et al.  The Drosophila Broad-Complex plays a key role in controlling ecdysone-regulated gene expression at the onset of metamorphosis. , 1993, Development.

[3]  W. Talbot,et al.  The drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily , 1991, Cell.

[4]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[5]  T. Wright.  The genetics of biogenic amine metabolism, sclerotization, and melanization in Drosophila melanogaster. , 1987, Advances in genetics.

[6]  J. Fristrom,et al.  Differential responses of the dopa decarboxylase gene to 20-OH-ecdysone in Drosophila melanogaster. , 1986, Developmental biology.

[7]  J. Hirsh,et al.  The cloned dopa decarboxylase gene is developmentally regulated when reintegrated into the drosophila genome , 1983, Cell.

[8]  R. Treisman,et al.  The POZ domain: a conserved protein-protein interaction motif. , 1994, Genes & development.

[9]  G. Guild,et al.  The ecdysone-induced puffing cascade in Drosophila salivary glands: a Broad-Complex early gene regulates intermolt and late gene transcription. , 1991, Genetics.

[10]  D. Lindsley,et al.  The Genome of Drosophila Melanogaster , 1992 .

[11]  R. J. Hill,et al.  The reaction with polytene chromosomes of antibodies raised against Drosophila E75A protein. , 1993, Insect biochemistry and molecular biology.

[12]  M. Ashburner,et al.  Temporal control of puffing activity in polytene chromosomes. , 1974, Cold Spring Harbor symposia on quantitative biology.

[13]  P. Karlson,et al.  Zum tyrosinstoffwechsel der insekten IX. Kontrolle des tyrosinstoffwechsels durch ecdyson , 1962 .

[14]  I. Kiss,et al.  Cytogenetic analysis of the 2B3-4-2B11 Region of the X-chromosome of Drosophila melanogaster , 2004, Chromosoma.

[15]  R. Hodgetts,et al.  Epidermal cell-specific quantitation of dopa decarboxylase mRNA in Drosophila by competitive RT-PCR: an effect of Broad-Complex mutants. , 1995, Developmental genetics.

[16]  A. Beaton,et al.  Interactions and developmental effects of mutations in the Broad-Complex of Drosophila melanogaster. , 1988, Genetics.

[17]  J. Marsh,et al.  Sequence and structure of the dopa decarboxylase gene of Drosophila: evidence for novel RNA splicing variants. , 1986, The EMBO journal.

[18]  W. A. Johnson,et al.  Regulated splicing produces different forms of dopa decarboxylase in the central nervous system and hypoderm of Drosophila melanogaster. , 1986, The EMBO journal.

[19]  L. Riddiford,et al.  Characterization of the dopa decarboxylase gene of Manduca sexta and its suppression by 20-hydroxyecdysone. , 1995, Developmental biology.

[20]  D. Withers,et al.  The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers. , 1991, Genetics.

[21]  M. Ashburner,et al.  Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. III. Consequences of ecdysone withdrawal. , 1976, Developmental Biology.

[22]  E. Hochuli,et al.  Purification of recombinant proteins with metal chelate adsorbent. , 1990, Genetic engineering.

[23]  R. Hodgetts,et al.  Stage-specific mechanisms regulate the expression of the dopa decarboxylase gene during Drosophila development , 1986 .

[24]  C. Thummel,et al.  Methods for quantitative analysis of transcription in larvae and prepupae. , 1994, Methods in cell biology.

[25]  M. Ashburner,et al.  Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. II. The effects of inhibitors of protein synthesis. , 1976, Developmental biology.

[26]  D. Nash,et al.  Larval age and the pattern of DNA synthesis in polytene chromosomes. , 1968, Canadian journal of genetics and cytology. Journal canadien de genetique et de cytologie.

[27]  J. Hirsh,et al.  cis-regulatory sequences responsible for alternative splicing of the Drosophila dopa decarboxylase gene , 1994, Molecular and cellular biology.

[28]  T. Südhof,et al.  Membrane fusion machinery: Insights from synaptic proteins , 1993, Cell.

[29]  F. Huet,et al.  Sequential gene activation by ecdysone in Drosophila melanogaster: the hierarchical equivalence of early and early late genes. , 1995, Development.

[30]  D. Hogness,et al.  The E75 ecdysone-inducible gene responsible for the 75B early puff in Drosophila encodes two new members of the steroid receptor superfamily. , 1990, Genes & development.

[31]  C. Thummel,et al.  The Drosophila 78C early late puff contains E78, an ecdysone-inducible gene that encodes a novel member of the nuclear hormone receptor superfamily , 1993, Cell.

[32]  G. Fraenkel.  A Hormone Causing Pupation in the Blowfly Calliphora erythrocephala , 1935 .

[33]  W. Terzaghi,et al.  DNA binding site preferences and transcriptional activation properties of the Arabidopsis transcription factor GBF1. , 1992, The EMBO journal.

[34]  C. Thummel,et al.  Molecular analysis of the initiation of insect metamorphosis: a comparative study of Drosophila ecdysteroid-regulated transcription. , 1993, Developmental biology.

[35]  J. Marsh,et al.  Developmental expression and spatial distribution of dopa decarboxylase in Drosophila. , 1987, Developmental biology.

[36]  P. Karlson,et al.  Zum Tyrosinstoffwechsel der Insekten—VIII , 1962 .

[37]  J. Hirsh,et al.  Regulation of the Drosophila dopa decarboxylase gene in neuronal and glial cells. , 1987, Genes & development.

[38]  J. Natzle.  Temporal regulation of Drosophila imaginal disc morphogenesis: a hierarchy of primary and secondary 20-hydroxyecdysone-responsive loci. , 1993, Developmental biology.

[39]  R. D. Gietz,et al.  Induction of translatable mRNA for dopa decarboxylase in Drosophila: an early response to ecdysterone. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[40]  D. Hogness,et al.  The Drosophila 74EF early puff contains E74, a complex ecdysone-inducible gene that encodes two ets-related proteins , 1990, Cell.

[41]  H. Biessmann,et al.  Temporal and spatial expression of the yellow gene in correlation with cuticle formation and dopa decarboxylase activity in Drosophila development. , 1991, Developmental biology.

[42]  S. Beckendorf,et al.  The Broad‐Complex directly controls a tissue‐specific response to the steroid hormone ecdysone at the onset of Drosophila metamorphosis. , 1994, The EMBO journal.

[43]  L. Riddiford,et al.  Regulation of dopa decarboxylase gene expression in the larval epidermis of the tobacco hornworm by 20-hydroxyecdysone and juvenile hormone. , 1990, Developmental biology.

[44]  C. Thummel,et al.  Molecular interactions within the ecdysone regulatory hierarchy: DNA binding properties of the Drosophila ecdysone-inducible E74A protein , 1990, Cell.

[45]  L. Cherbas,et al.  Identification of ecdysone response elements by analysis of the Drosophila Eip28/29 gene. , 1991, Genes & development.

[46]  G. Privé,et al.  The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. , 1994, Proceedings of the National Academy of Sciences of the United States of America.