Puff Genes Research Articles

Amplification and expression of a salivary gland DNA puff gene in the prothoracic gland of Bradysia hygida (Diptera: Sciaridae)

The DNA puff BhC4-1 gene, located in DNA puff C4 of Bradysiahygida, is amplified and expressed in the salivary gland at the end of the fourth larval instar as a late response to the increase in 20-hydroxyecdysone titer that triggers metamorphosis. Functional studies revealed that the mechanisms that regulate BhC4-1 expression in the salivary gland are conserved in transgenic Drosophila. These studies also led to the identification of a cis-regulatory module that drives developmentally regulated expression of BhC4-1-lacZ in the prothoracic gland cells of the ring gland, a compound organ which in Drosophila results from the fusion of the prothoracic glands, the corpus allatum and the corpus cardiacum. Here we have investigated the occurrence of BhC4-1 expression in B. hygida prothoracic glands. We report the identification of the B. hygida prothoracic gland and demonstrate that it releases ecdysone. Using RT-qPCR, western blots and immunolocalization experiments, we demonstrate that the BhC4-1 mRNA and the BhC4-1 protein are both expressed in the B. hygida prothoracic glands at the same time that DNA puff C4 is formed in the salivary gland. We also show that BhC4-1 is concomitantly amplified 4.8-fold in the prothoracic gland and 23-fold in the salivary gland. Our results reveal the occurrence of stage specific expression of a DNA puff gene in the prothoracic glands of B. hygida, and extend previous studies that have shown that DNA puff genes expression is not restricted to the salivary gland. In addition, the description of stage specific gene amplification in the prothoracic glands of B. hygida constitutes the first demonstration that gene amplification in Diptera might occur concomitantly in two different tissues in the same developmental stage.

Regulation of Drosophila Metamorphosis by Xenobiotic Response Regulators

Mammalian Nrf2-Keap1 and the homologous Drosophila CncC-dKeap1 protein complexes regulate both transcriptional responses to xenobiotic compounds as well as native cellular and developmental processes. The relationships between the functions of these proteins in xenobiotic responses and in development were unknown. We investigated the genes regulated by CncC and dKeap1 during development and the signal transduction pathways that modulate their functions. CncC and dKeap1 were enriched within the nuclei in many tissues, in contrast to the reported cytoplasmic localization of Keap1 and Nrf2 in cultured mammalian cells. CncC and dKeap1 occupied ecdysone-regulated early puffs on polytene chromosomes. Depletion of either CncC or dKeap1 in salivary glands selectively reduced early puff gene transcription. CncC and dKeap1 depletion in the prothoracic gland as well as cncCK6/K6 and dKeap1EY5/EY5 loss of function mutations in embryos reduced ecdysone-biosynthetic gene transcription. In contrast, dKeap1 depletion and the dKeap1EY5/EY5 loss of function mutation enhanced xenobiotic response gene transcription in larvae and embryos, respectively. Depletion of CncC or dKeap1 in the prothoracic gland delayed pupation by decreasing larval ecdysteroid levels. CncC depletion suppressed the premature pupation and developmental arrest caused by constitutive Ras signaling in the prothoracic gland; conversely, constitutive Ras signaling altered the loci occupied by CncC on polytene chromosomes and activated transcription of genes at these loci. The effects of CncC and dKeap1 on both ecdysone-biosynthetic and ecdysone-regulated gene transcription, and the roles of CncC in Ras signaling in the prothoracic gland, establish the functions of these proteins in the neuroendocrine axis that coordinates insect metamorphosis.

The DNA puff 4C expresses a salivary secretion protein in Trichosia pubescens (Diptera; Sciaridae)

DNA puffs are genomic regions of polytene chromosomes that undergo developmentally controlled DNA amplification and transcription in salivary glands of sciarid flies. Here, we tested the hypothesis that DNA puff genes code for salivary proteins in Trichosia pubescens. To do that, we generated antibodies against saliva and immunoscreened a cDNA library made from salivary glands. We isolated clones corresponding to DNA puff regions, including clone D-50 that contained the entire coding sequence of the previously isolated C4B1 gene from puff 4C. Indeed, we showed that puff 4C is a DNA puff region detecting its local transcription and its extra rounds of DNA incorporation compared to neighboring regions. We further confirmed D-50 clone identity in Western blots reacted with the anti-saliva anitiserum. We detected a recombinant protein expressed by this clone that had the expected size for a full-length product of the gene. We end with a discussion of the relationship between DNA puff genes and their products.

Broad‐Complex, E74, and E75 early genes control DNA puff BhC4‐1 expression in prepupal salivary glands

The DNA puff BhC4-1 gene of the sciarid Bradysia hygida is induced in salivary glands prior to the pupal molt as a secondary response to the increase in ecdysone titers. Previous studies demonstrated that the BhC4-1 promoter is activated in transgenic Drosophila melanogaster salivary glands as a late response to the ecdysone peak that triggers metamorphosis, revealing that this aspect of BhC4-1 transcriptional regulation is conserved in the Drosophila background. To identify regulators of BhC4-1 expression, we utilized a candidate gene approach and tested the roles of the ecdysone-induced genes BR-C, E74, and E75. Our results reveal that the BR-C Z3 isoform is essential for BhC4-1-lacZ induction in prepupal salivary glands and constitute the first demonstration of the participation of early genes products on DNA puff genes regulation.

Molecular characterization of the B-2 DNA puff gene of Rhynchosciara americana.

We have sequenced a 2.5-kb DNA fragment of the B-2 DNA puff from the sciarid Rhynchosciara americana and have defined its transcription unit. This puff is active during the formation of the communal cocoon, which is important for successful metamorphosis of this species and coincides with the final cycle of polytenization in its salivary glands. The B-2 polypeptide, together with the products of two other previously characterized DNA puffs, seems to be engaged in an interaction that results in a gradual modification and hardening of the cocoon structure. The B-2 messenger is temporally regulated in apparent coordination with the other puff products. The predicted polypeptide has characteristics similar to polypeptides from previously sequenced DNA puff genes, in particular those from the R. americana C-8 gene and the Bradysia hygida C-4 gene. The cloned sequence of the B-2 puff is differentially amplified in the three gland regions examined, achieving its highest amplification level of approximately fourfold (two extra cycles) in the anterior segment of the gland. The C-3 DNA puff sequence was also found to be differentially amplified in the different gland regions. Implications of the widespread presence of DNA amplification as a form of gene regulation in the Sciaridae are discussed.

Ecdysone-regulated puff genes 2000

The Ashburner model for the hormonal control of polytene chromosome puffing has provided a strong foundation for understanding the basic mechanisms of steroid-regulated gene expression (Cold Spring Harbor Symp. Quant. Biol. 38 (1974) 655). According to this model, the steroid hormone 20-hydroxyecdysone (referred here as ecdysone) directly induces the expression of a small set of early regulatory genes. These genes, in turn, induce a much larger set of late target genes that play a more direct role in controlling the biological responses to the hormone. The recent characterization of two early puff genes, E63-1 and E23, and three late puff genes, D-spinophilin, L63, and L82, provide further confirmation of the Ashburner model. In addition, these studies provide exciting new directions for our understanding of ecdysone signaling. Overexpression studies of E63-1 implicate this gene in directing calcium-dependent salivary gland glue secretion. In contrast, overexpression of E23 indicates that this ABC transporter family member may negatively regulate ecdysone signaling by actively transporting the hormone out of target cells. Finally, genetic studies of the L63 and L82 late genes reveal unexpected possible functions for ecdysone in controlling developmental timing and growth. This review surveys the recent characterization of these ecdysone-inducible genes and provides an overview of how they expand our understanding of ecdysone functions during development.

C7, a Novel Nucleolar Protein, Is the Mouse Homologue of the Drosophila Late Puff Product L82 and an Isoform of Human OXR1

The C7 gene was identified in a project aimed to characterize differential gene expression upon attachment of cells to extracellular matrix proteins in vitro. C7 is the homologue of Drosophila L82, a late puff gene (Stowers et al. (1999) Dev. Biol. 213, 116–130) and human OXR1, a gene, which protects cells against oxidation (Volkert et al. (2000) Proc. Natl. Acad. Sci. USA 97, 14530–14535). All are transcribed into multiple splice forms with a common 3′ domain. Additional members of this novel gene family are found in a number of eukaryotic species. In the mouse, the C7 gene is highly and broadly expressed during development in at least 4 splice forms, 3 of which were sequenced. In the adult, the C7 gene is most highly expressed in brain and testis. Antibodies to recombinant C7 protein localized to nucleoli in a variety of cell types, suggesting that C7 may be involved in the formation or function of this important organelle.

The 82F Late Puff Contains the L82 Gene, an Essential Member of a Novel Gene Family

Metamorphosis in Drosophila results from a hierarchy of ecdysone-induced gene expression initiated at the end of the third larval instar. A now classical model of this hierarchy was proposed based on observations of the activity of polytene chromosome “puffs” which distinguished “early” puffs as those directly induced by ecdysone and “late” puffs as those which become active as a secondary response to the hormone. We report here the isolation and characterization of the L82 gene corresponding to the extensively characterized late puff at 82F. L82 is a complex gene that spans at least 50 kb of genomic DNA, produces at least seven different nested mRNAs, and has homology to a novel gene family. In contrast to most previously characterized puff genes, the broad developmental expression pattern of L82 suggests that it is controlled by both ecdysone-dependent and ecdysone-independent regulatory mechanisms. L82 mutations were identified by transgene rescue of developmental delay and eclosion lethal phenotypes.

The DNA puff gene BhC4-1 of Bradysia hygida is specifically transcribed in early prepupal salivary glands of Drosophila melanogaster.

The BhC4-1 gene of the sciarid Bradysia hygida is located at DNA puff C4 and is amplified and actively transcribed in the salivary gland at the end of the last larval instar. We show here that a 3.6 kb fragment from the upstream region of the BhC4-1 gene is able to drive transcription in transgenic Drosophila specifically in prepupal salivary gland in a temporally regulated manner. The mRNA is present in maximal amounts in prepupae +3 h; in prepupae +9 h the levels of BhC4-1 mRNA decline, and it is no longer detected in pupae +24 h. Taken together these results suggest that most, if not all, of the key promoter regulatory elements were included in the DNA fragments employed to transform Drosophila. Moreover, strong expression of the transgenes implies conservation of the regulatory elements involved, since Drosophila transcription factors appear to recognize B. hygida regulatory DNA sequences. Quantitative Southern blot hybridization indicates that the sequences from DNA puff C4 are not amplified at detectable levels in salivary glands of transgenic prepupae when the BhC4-1 gene is transcribed. Transcription of a DNA puff in the absence of amplification indicates that the induction of these processes involves distinct mechanisms.

The DHR78 Nuclear Receptor Is Required for Ecdysteroid Signaling during the Onset of Drosophila Metamorphosis

Pulses of ecdysteroids direct Drosophila through its life cycle by activating stage- and tissue-specific genetic regulatory hierarchies. Here we show that an orphan nuclear receptor, DHR78, functions at the top of the ecdysteroid regulatory hierarchies. Null mutations in DHR78 lead to lethality during the third larval instar with defects in ecdysteroid-triggered developmental responses. Consistent with these phenotypes, DHR78 mutants fail to activate the mid-third instar regulatory hierarchy that prepares the animal for metamorphosis. DHR78 protein is bound to many ecdysteroid-regulated puff loci, suggesting that DHR78 directly regulates puff gene expression. In addition, ectopic expression of DHR78 has no effects on development, indicating that its activity is regulated posttranslationally. We propose that DHR78 is a ligand-activated receptor that plays a central role in directing the onset of Drosophila metamorphosis.

Molecular characterization of the C-3 DNA puff gene of Rhynchosciara americana

We have mapped a region of about 33 kb which includes the transcription unit of the C-3 DNA puff gene of Rhynchosciara americana. The C-3 TU and a region extending approximately 800 bp upstream of the C-3 promoter were characterized. The TU is composed of three exons and produces a 1.1-kb mRNA whose level in salivary glands increases with the expansion of the C-3 puff. The C-3 messenger appears to undergo rapid deadenylation resulting in an RNA of about 0.95 kb which can still be observed in gland cells 15 h after the puff has regressed. The 1.1-kb mRNA codes for a 32.4-kDa, predominantly alpha-helical polypeptide with three conserved parallel coiled-coil stretches. The aa composition and structure of this polypeptide suggests that it is secreted and contributes to the formation of the cocoon in which the larvae pupate. The region upstream of the promoter contains several A-rich sequences with similarity to the ACS of yeast which might have a role in the initiation of replication/amplification.

Coordination of larval and prepupal gene expression by the DHR3 orphan receptor during Drosophila metamorphosis.

The DHR3 orphan receptor gene is induced directly by the steroid hormone ecdysone at the onset of Drosophila metamorphosis. DHR3 expression peaks in early prepupae, as the early puff genes are repressed and betaFTZ-F1 is induced. Here we provide evidence that DHR3 directly contributes to both of these regulatory responses. DHR3 protein is bound to many ecdysone-induced puffs in the polytene chromosomes, including the early puffs that encode the BR-C and E74 regulatory genes, as well as the E75, E78 and betaFTZ-F1 orphan receptor loci. Three DHR3 binding sites were identified downstream from the start site of betaFTZ-F1 transcription, further indicating that this gene is a direct target of DHR3 regulation. Ectopic expression of DHR3 revealed that the polytene chromosome binding pattern is of functional significance. DHR3 is sufficient to repress BR-C, E74A, E75A and E78B transcription as well as induce betaFTZ-F1. DHR3 thus appears to function as a switch that defines the larval-prepupal transition by arresting the early regulatory response to ecdysone at puparium formation and facilitating the induction of the betaFTZ-F1 competence factor in mid-prepupae. This study also provides evidence for direct cross-regulation among orphan members of the nuclear receptor superfamily and further implicates these genes as critical transducers of the hormonal signal during the onset of Drosophila metamorphosis.

The Drosophila Eip78C gene is not vital but has a role in regulating chromosome puffs.

We have generated a number of chromosomal aberrations that disrupt the early-late ecdysone-induced 78C puff gene (Eip78C, ecdysone-induced protein, FlyBase name for the E78 gene of Stone and Thummel 1993), which encodes the two members of the nuclear hormone receptor superfamily Eip78C-A and Eip78C-B. The aberrations include deletions of the ligand-binding/dimerization domain of both, inversions that split Eip78C-A but retain residual Eip78C-B expression, and a small deletion specific for Eip78C-B. We find that wild-type Eip78C functions are completely dispensable for normal development under laboratory conditions. However, we show that Eip78C-B is required for the maximal puffing activity of a subset of late puffs (63E and 82F) since these puffs are reduced in size in Eip78C-B mutant backgrounds. Paradoxically the same late puffs are reduced, as well as at least one other, when the Eip78C-B cDNA is overexpressed from a heat shock promoter. These data indicate either that Eip78C function is redundant or that it plays a subtle modulating role in the regulation of chromosome puffing.

Two-step regulation of ecdysone-inducible late puffs in salivary glands of Drosophila melanogaster.

Our previous study showed that some ecdysone-inducible late puffs could also be induced by a mild detergent (digitonin) in Drosophila salivary glands. However, they could only be induced at the stage immediately prior to when developmentally programmed puffing occurred, suggesting that these late puff loci were under two-step regulation. Using an in vitro culture of salivary glands, we have examined whether ecdysone or the protein products of early puff genes participate in either of the two steps of late puff regulation. This study has revealed that (i) the acquisition of digitonin-responsiveness (the first step) could be induced in vitro by incubating salivary glands with ecdysone; (ii) the first step could also be induced by protein synthesis inhibition even in the absence of ecdysone; (iii) the second step required both ecdysone and protein synthesis unless treated with digitonin; and (iv) the first step, rather than the second step, determines the timing of normal puff formation in the loci. These results suggest that, during normal development, ecdysone controls both steps by activating two types of early genes; the first type, whose function can be mimicked by cycloheximide, renders the loci responsive to digitonin and the second type, whose function can be mimicked by digitonin, activates the loci to form puffs.

The Drosophila E93 Gene from the 93F Early Puff Displays Stage- and Tissue-Specific Regulation by 20-Hydroxyecdysone

Pulses of ecdysteroids induce dramatic changes in gene expression that direct the early stages of Drosophila metamorphosis. This gene activity is reflected by the appearance of early and late puffs in the salivary gland polytene chromosomes. Curiously, the early puff genes that have been studied to date are induced by both the late larval and prepupal pulses of ecdysteroids and are expressed in many ecdysteroid target tissues, raising the question of how the hormone directs the complex stage- and tissue-specific responses associated with metamorphosis. In an effort to address this question, we have isolated and characterized the E93 gene responsible for the stage-specific 93F early puff. The E93 mRNA displays no response to ecdysteroids in late larval salivary glands but is directly induced 12 hr later by the prepupal ecdysteroid pulse, identical to the response of the 93F puff. In tissues other than the salivary gland, however, E93 displays complex spatial and temporal regulation. E93 spans at least 55 kb of genomic DNA and encodes a 146-kDa protein that has no matches in the sequence databases, but displays several characteristics of known Drosophila transcription factors. We propose that E93 acts in a stage-specific regulatory hierarchy in the salivary gland to direct its histolysis in response to the prepupal ecdysteroid pulse.

The promoter of DNA puff gene II/9-1 of Sciara coprophila is inducible by ecdysone in late prepupal salivary glands of Drosophila melanogaster.

DNA puffs occur in Sciarid salivary gland chromosomes; they are sites of DNA amplification and intense transcription and they appear to encode secreted structural proteins needed for pupation. In this report we have used P-element transformation of Drosophila to study regulation of a Sciara DNA puff gene. We found that a 718-bp promoter fragment of DNA puff gene II/9-1 from Sciara coprophila directs expression of the bacterial reporter gene CAT in late prepupal salivary glands of transgenic Drosophila melanogaster. The identical tissue and analogous stage specificity indicate that some aspects of the ecdysone response are evolutionarily conserved between Drosophila and Sciara. When transgenic salivary glands are cultured in vitro, CAT activity is rapidly induced by ecdysone, suggesting direct control of gene expression by the ecdysone receptor. Putative stage-specific factors limit expression of the chimeric Sciara-CAT gene in transgenic Drosophila to late prepupae but not to third instar larvae when ecdysone titers are also high.