Regulation of I-transposon activity in Drosophila: evidence for cosuppression of nonhomologous transgenes and possible role of ancestral I-related pericentromeric elements

We have previously shown that the activity of functional I retrotransposons (I factors) introduced into Drosophila devoid of such elements can be repressed by transgenes containing an internal fragment of the I factor itself and that this repressing effect presents the characteristic features of homology-dependent gene silencing or cosuppression. Here we show that the same transgenes can induce silencing of a nonhomologous reporter gene containing as the sole I-factor sequence its 100-bp promoter fragment. Silencing of the nonhomologous reporter gene shows strong similarities to I-factor cosuppression: It does not require any translation product from the regulating transgenes, sense and antisense constructs are equally potent, and the silencing effect is only maternally transmitted and fully reversible. A search for genomic I-like sequences containing domains with similarities to those of both the regulating and the reporter transgenes led to the identification of four such elements, which therefore could act as intermediates-or relays-in the cosuppression machinery. These results strongly suggest that ancestral transposition-defective I-related elements, which are naturally present in the Drosophila genome, may participate per se in the natural conditions of I-factor silencing.     

Repression of Hybrid Dysgenesis in Drosophila Melanogaster by Heat-Shock-Inducible Sense and Antisense P-Element Constructs

Sets of sense and antisense P-element constructs controlled by a heat-shock-inducible promoter were tested for their ability to repress manifestations of P-element activity in vivo. As a group, the antisense constructs repressed pupal lethality, a somatic manifestation of P activity, and this repression was significantly enhanced by heat shock. Three of the 11 antisense constructs also repressed gonadal dysgenesis, a manifestation of P activity in the female germ line; however, none had any effect on P-element-mediated mutability in the male germ line. Among the 13 different heat-shock-inducible sense constructs that were tested, those containing the KP and DP elements were strong repressors of pupal lethality, gonadal dysgenesis and P-element-mediated mutability; however, individual lines carrying these constructs varied in their ability to repress each of these traits, presumably because of genomic position effects. With the exception of the sense construct that contained a complete P element, none of the sense or antisense constructs repressed a lacZ reporter gene driven by the P-element promoter. Overall, the experimental results suggest that in nature, P-element activity could be regulated by P-encoded polypeptides and by antisense P RNAs.     

P-Element repression in Drosophila melanogaster by variegating clusters of P-lacZ-white transgenes.

In Drosophila, clusters of P transgenes (P-lac-w) display a variegating phenotype for the w marker. In addition, X-ray-induced rearrangements of chromosomes bearing such clusters may lead to enhancement of the variegated phenotype. Since P-lacZ transgenes in subtelomeric heterochromatin have some P-element repression abilities, we tested whether P-lac-w clusters also have the capacity to repress P-element activity in the germline. One cluster (T-1), located on a rearranged chromosome (T2;3) and derived from a line bearing a variegating tandem array of seven P-lac-w elements, partially represses the dysgenic sterility (GD sterility) induced by P elements. This cluster also strongly represses in trans the expression of P-lacZ elements in the germline. This latter suppression shows a maternal effect. Finally, the combination of variegating P-lac-w clusters and a single P-lacZ reporter inserted in subtelomeric heterochromatic sequences at the X chromosome telomere (cytological site 1A) leads to strong repression of dysgenic sterility. These results show that repression of P-induced dysgenic sterility can be elicited in the absence of P elements encoding a polypeptide repressor and that a transgene cluster can repress the expression of a single homologous transgene at a nonallelic position. Implications for models of transposable element silencing are discussed.     

Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice

Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.     

Cosuppression of nonhomologous transgenes in Drosophila involves mutually related endogenous sequences.

Cosuppression refers to the phenomenon in which silencing among dispersed homologous genes occurs. Here we demonstrate that two nonhomologous reciprocal fusion genes, white-Alcohol dehydrogenase (w-Adh) and Adh-w, exhibit cosuppression using the endogenous Adh sequence as an intermediary. Deletion of the endogenous Adh gene eliminates the interaction, while reintroduction of an 8.6 kb Adh fragment restores the silencing. Using truncated Adh constructs, a nontranscribed segment in the Adh regulatory region was found to be one of the sequences required for homology recognition. The silencing interaction is initiated during early development. The silenced transgenes are associated with the Polycomb group complex of chromatin proteins.     

Simultaneous suppression of multiple genes by single transgenes. Down-regulation of three unrelated lignin biosynthetic genes in tobacco

Many reports now describe the manipulation of plant metabolism by suppressing the expression of single genes. The potential of such work could be greatly expanded if multiple genes could be coordinately suppressed. In the work presented here, we test a novel method for achieving this by using single chimeric constructs incorporating partial sense sequences for multiple genes to target suppression of two or three lignin biosynthetic enzymes. We compare this method with a more conventional approach to achieving the same end by crossing plants harboring different antisense transgenes. Our results indicate that crossing antisense plants is less straightforward and predictable in outcome than anticipated. Most progeny had higher levels of target enzyme activity than predicted and had lost the expected modifications to lignin structure. In comparison, plants transformed with the chimeric partial sense constructs had more consistent high level suppression of target enzymes and had significant changes to lignin content, structure, and composition. It was possible to suppress three target genes coordinately using a single chimeric construct. Our results indicate that chimeric silencing constructs offer great potential for the rapid and coordinate suppression of multiple genes on diverse biochemical pathways and that the technique therefore deserves to be adopted by other researchers.     

The mcp element from the Drosophila melanogaster bithorax complex mediates long-distance regulatory interactions.

In the studies reported here, we have examined the properties of the Mcp element from the Drosophila melanogaster bithorax complex (BX-C). We have found that sequences from the Mcp region of BX-C have properties characteristic of Polycomb response elements (PREs), and that they silence adjacent reporters by a mechanism that requires trans-interactions between two copies of the transgene. However, Mcp trans-regulatory interactions have several novel features. In contrast to classical transvection, homolog pairing does not seem to be required. Thus, trans-regulatory interactions can be observed not only between Mcp transgenes inserted at the same site, but also between Mcp transgenes inserted at distant sites on the same chromosomal arm, or even on different arms. Trans-regulation can even be observed between transgenes inserted on different chromosomes. A small 800-bp Mcp sequence is sufficient to mediate these long-distance trans-regulatory interactions. This small fragment has little silencing activity on its own and must be combined with other Polycomb-Group-responsive elements to function as a "pairing-sensitive" silencer. Finally, this pairing element can also mediate long-distance interactions between enhancers and promoters, activating mini-white expression.     

P element-encoded regulatory products enhance Repeat-Induced Gene Silencing (RIGS) of P-lacZ-white clusters in Drosophila melanogaster

In Drosophila melanogaster, some clusters of P transgenes ( P-lacZ-white) display a variegating phenotype for the white marker in the eye, a phenomenon termed "Repeat-Induced Gene Silencing" (RIGS). We have tested the influence of the P element repression state (P cytotype) on the eye phenotype of several P-lac-w clusters that differ in transgene copy number or genomic insertion site. P element-encoded regulatory products strongly enhance RIGS. The effect occurs in both sexes, is detectable with clusters having at least three copies and is observed at both genomic locations tested (cytogenetic regions 50C and 92E). Single variegating P-lac-w transgenes located in pericentromeric heterochromatin are not affected by P regulatory products. All P strain backgrounds tested enhance RIGS, including chromosomes bearing a single P element encoding a truncated P transposase or carrying a single internally deleted KP element. Therefore, clusters are highly sensitive to different types of P repressors. Finally, a chimeric gene in which the 5' portion of the P element is fused to the polyhomeotic coding sequence (ph(p1)) also strongly enhances silencing of P-lac-w clusters. These results have implications for the mechanism of action of the P repressors and show that P transgene clusters represent a new class of P-sensitive alleles, providing a simple assay for somatic P repression that can be completed in one generation.