Transposon regulation in Drosophila: piRNA-producing P elements facilitate repression of hybrid dysgenesis by a P element that encodes a repressor polypeptide.

The transposons of Drosophila melanogaster are regulated by small RNAs that interact with the Piwi family of proteins. These piRNAs are generated from transposons inserted in special loci such as the telomere-associated sequences at the left end of the X chromosome. Drosophila's P transposons can also be regulated by a polypeptide encoded by the KP element, a 1.15-kb-long member of the P family. Using piRNA-generating telomeric P elements (TPs) and repressor-producing transgenic KP elements, we demonstrate a functional connection between these two modes of regulation. By themselves, the TPs partially repress gonadal dysgenesis, a trait caused by rampant P-element activity in the germ line. This repression is manifested as a strictly maternal effect arising from the cytoplasmic transmission of P-specific piRNAs from mother to offspring. The repression is enhanced by genetic interactions between the TPs and other, non-telomeric P elements-a phenomenon attributable to ping-pong amplification of maternal piRNAs. KP elements, like other kinds of non-telomeric P elements, enhance regulation anchored in the TPs. However, with some TPs, the enhanced regulation is manifested as a strictly zygotic effect of the KP element. This effect is seen when the TP has few sequences in common with the KP element, a condition not conducive to ping-pong amplification of piRNAs; it can be attributed to the action of the KP repressor polypeptide. Because the effect is seen only when a TP was present in the mother's genotype, maternally generated P-element piRNAs could facilitate regulation by the KP repressor polypeptide.

A test for enhancement of cytotype regulation in Drosophila melanogaster by the transposase-encoding P element ∆2-3.

Transposable P elements are regulated in the germ line by piRNAs, which are small RNAs that associate with the Piwi class of proteins. This regulation, called the P cytotype, is enhanced by genetic interactions between P elements that are primary sources of these RNAs and other P elements. The enhanced regulation is thought to reflect amplification of the primary piRNAs by cleavage of mRNAs derived from the other P elements through a mechanism called the ping-pong cycle. We tested the transposase-encoding P element known as ∆2-3 for its ability to enhance cytotype regulation anchored in P elements inserted at the telomere of the left arm of the X chromosome (TP elements). The ∆2-3 P element lacks the intron between exons 2 and 3 in the structurally complete P element (CP). Unlike the CP element, it does not markedly enhance cytotype regulation anchored in TP elements, nor does it transmit transposase activity through the egg cytoplasm. However, mRNAs from both the CP and ∆2-3 elements are maternally deposited in embryos. These observations suggest that maternally transmitted CP mRNA enhances cytotype regulation by participating in the ping-pong cycle and that it encodes the P transposase in the embryonic germ line, whereas maternally transmitted ∆2-3 mRNA does not, possibly because it is not efficiently directed into the primordial embryonic germ line. Strong transposon regulation may, therefore, require ping-pong cycling with maternally inherited mRNAs in the embryo.

Maternal enhancement of cytotype regulation in Drosophila melanogaster by genetic interactions between telomeric P elements and non-telomeric transgenic P elements.

The X-linked telomeric P elements (TPs) TP5 and TP6 regulate the activity of the entire P element family because they are inserted in a major locus for the production of Piwi-interacting RNAs (piRNAs). The potential for this cytotype regulation is significantly strengthened when either TP5 or TP6 is combined with a non-telomeric X-linked or autosomal transgene that contains a P element. By themselves, none of the transgenic P elements have any regulatory ability. Synergism between the telomeric and transgenic P elements is much greater when the TP is derived from a female. Once an enhanced regulatory state is established in a female, it is transmitted to her offspring independently of either the telomeric or transgenic P elements - that is, it works through a strictly maternal effect. Synergistic regulation collapses when either the telomeric or the transgenic P element is removed from the maternal genotype, and it is significantly impaired when the TPs come from stocks heterozygous for mutations in the genes aubergine, piwi or Su(var)205. The synergism between telomeric and transgenic P elements is consistent with a model in which P piRNAs are amplified by alternating, or ping-pong, targeting of primary piRNAs to sense and antisense P transcripts, with the sense transcripts being derived from the transgenic P element and the antisense transcripts being derived from the TP.

Maternal impairment of transposon regulation in Drosophila melanogaster by mutations in the genes aubergine, piwi and Suppressor of variegation 205.

TP5, a P element inserted in the telomere-associated sequences of the X chromosome, represses the excision of other P elements in the germ line through a combination of maternal and zygotic effects. The maternal component of this repression is impaired by heterozygous mutations in the aubergine and Suppressor of variegation 205 genes; one mutation in the piwi gene also appears to impair repression. In the female germ line, the level of TP5 mRNA is increased by these impairing mutations. The impairing aubergine and piwi mutations also increase the level of germ-line mRNA from CP, a transgene that encodes the P-element transposase; however, the Suppressor of variegation 205 mutation does not. These findings are discussed in terms of a model of P-element regulation that involves post-transcriptional and chromatin re-organizing events mediated by maternally transmitted small RNAs derived from the telomeric P element.

Cytotype regulation of P transposable elements in Drosophila melanogaster: repressor polypeptides or piRNAs?

The telomeric P elements TP5 and TP6 are associated with the P cytotype, a maternally inherited condition that represses P-element-induced hybrid dysgenesis in the Drosophila germ line. To see if cytotype repression by TP5 and TP6 might be mediated by the polypeptides they could encode, hobo transgenes carrying these elements were tested for expression of mRNA in the female germ line and for repression of hybrid dysgenesis. The TP5 and TP6 transgenes expressed more germ-line mRNA than the native telomeric P elements, but they were decidedly inferior to the native elements in their ability to repress hybrid dysgenesis. These paradoxical results are inconsistent with the repressor polypeptide model of cytotype. An alternative model based on the destruction of P transposase mRNA by Piwi-interacting (pi) RNAs was supported by finding reduced P mRNA levels in flies that carried the native telomeric P elements, which are inserted in a known major piRNA locus.

Cytotype regulation by telomeric P elements in Drosophila melanogaster: variation in regulatory strength and maternal effects.

Strains carrying the X-linked telomeric P elements TP5 or TP6 varied in their ability to repress hybrid dysgenesis. The rank ordering of these strains was consistent across different genetic assays and was not related to the type of telomeric P element (TP5 or TP6) present. Strong repression of dysgenesis was associated with weak expression of mRNA from the telomeric P element and also with a reduced amount of mRNA from a transposase-producing P element contained within a transgene inserted on an autosome. A strictly maternal component of repression, transmitted independently of the telomeric P element, was detected in the daughters but not the sons of females from the strongest repressing strains. However, this effect was seen only when dysgenesis was induced by crossing these females to males from a P strain, not when it was induced by crossing them to males homozygous for a single transposase-producing P element contained within a transgene. These findings are consistent with the hypothesis that the P cytotype, the condition that regulates P elements, involves an RNA interference mechanism mediated by piRNAs produced by telomeric P elements such as TP5 and TP6 and amplified by RNAs produced by other P elements.

Cytotype regulation in Drosophila melanogaster: synergism between telomeric and non-telomeric P elements.

The X-linked telomeric P elements TP5 and TP6 interact synergistically with non-telomeric P elements to repress hybrid dysgenesis. In this repression, the telomeric P elements exert maternal effects, which, however, are not sufficient to establish synergism with the non-telomeric P elements. Once synergism is established, the capacity to repress dysgenesis in the offspring of a cross persists for at least two generations after removing the telomeric P element from the genotype. At the molecular level, synergism between telomeric and non-telomeric P elements is correlated with effective elimination of P-element mRNA in the germ line. Maternally transmitted mutations in the genes aubergine, piwi and Suppressor of variegation 205 [Su(var)205] block the establishment of synergism between telomeric and non-telomeric P elements, and paternally transmitted mutations in piwi and Su(var)205 disrupt synergism that has already been established. These findings are discussed in terms of a model of cytotype regulation of P elements based on Piwi-interacting RNAs (piRNAs) that are amplified by cycling between sense and antisense species.

Cytotype regulation by telomeric P elements in Drosophila melanogaster: interactions with P elements from M' strains.

P strains of Drosophila are distinguished from M strains by having P elements in their genomes and also by having the P cytotype, a maternally inherited condition that strongly represses P-element-induced hybrid dysgenesis. The P cytotype is associated with P elements inserted near the left telomere of the X chromosome. Repression by the telomeric P elements TP5 and TP6 is significantly enhanced when these elements are crossed into M' strains, which, like P strains, carry P elements, but have little or no ability to repress dysgenesis. The telomeric and M' P elements must coexist in females for this enhanced repression ability to develop. However, once established, it is transmitted maternally to the immediate offspring independently of the telomeric P elements themselves. Females that carry a telomeric P element but that do not carry M' P elements may also transmit an ability to repress dysgenesis to their offspring independently of the telomeric P element. Cytotype regulation therefore involves a maternally transmissible product of telomeric P elements that can interact synergistically with products from paternally inherited M' P elements. This synergism between TP and M' P elements also appears to persist for at least one generation after the TP has been removed from the genotype.

Cytotype regulation by telomeric P elements in Drosophila melanogaster: evidence for involvement of an RNA interference gene.

P elements inserted at the left telomere of the X chromosome evoke the P cytotype, a maternally inherited condition that regulates the P-element family in the Drosophila germline. This regulation is completely disrupted in stocks heterozygous for mutations in aubergine, a gene whose protein product is involved in RNA interference. However, cytotype is not disrupted in stocks heterozygous for mutations in two other RNAi genes, piwi and homeless (spindle-E), or in a stock heterozygous for a mutation in the chromatin protein gene Enhancer of zeste. aubergine mutations exert their effects in the female germline, where the P cytotype is normally established and through which it is maintained. These effects are transmitted maternally to offspring of both sexes independently of the mutations themselves. Lines derived from mutant aubergine stocks reestablish the P cytotype quickly, unlike lines derived from stocks heterozygous for a mutation in Suppressor of variegation 205, the gene that encodes the telomere-capping protein HP1. Cytotype regulation by telomeric P elements may be tied to a system that uses RNAi to regulate the activities of telomeric retrotransposons in Drosophila.

Does RNA interference influence meiotic crossing over in Drosophila melanogaster?

Mutations in the RNA interference (RNAi) genes aubergine (aub), homeless and piwi were tested for effects on the frequency, distribution and coincidence of meiotic crossovers in the long arm of the X chromosome. Some increases in crossover frequency were seen in these tests, but they may have been due to a maternal effect of the balancer chromosomes that were used to maintain the RNAi mutations in stocks rather than to the RNAi mutations themselves. These same balancers produced strong zygotic interchromosomal effects when tested separately. Mutations in aub and piwi did not affect the frequency of crossing over in the centric heterochromatin of chromosome II; nor did a balancer chromosome III.

Impairment of cytotype regulation of P-element activity in Drosophila melanogaster by mutations in the Su(var)205 gene.

Cytotype regulation of transposable P elements in the germ line of Drosophila melanogaster is associated with maternal transmission of P elements inserted at the left telomere of the X chromosome. This regulation is impaired in long-term stocks heterozygous for mutations in Suppressor of variegation 205 [Su(var)205], a gene implicated in the control of telomere length. Regulation by TP5, a structurally incomplete P element at the X telomere, is more profoundly impaired than regulation by TP6, a different incomplete P element inserted at the same site in a TAS repeat at the X telomere. Genetic analysis with the TP5 element indicates that its regulatory ability is not impaired in flies whose fathers came directly from a stock heterozygous for a Su(var)205 mutation, even when the flies themselves carry this mutation. However, it is impaired in flies whose grandfathers came from such a stock. Furthermore, this impairment occurs even when the Su(var)205 mutation is not present in the flies themselves or in their mothers. The impaired regulatory ability of TP5 persists for at least several generations after TP5 X chromosomes extracted from a long-term mutant Su(var)205 stock are made homozygous in the absence of the Su(var)205 mutation. Impairment of TP5-mediated regulation is therefore not directly dependent on the Su(var)205 mutation. However, it is characteristic of the six mutant Su(var)205 stocks that were tested and may be related to the elongated telomeres that develop in these stocks. Impairment of regulation by TP5 is also seen in a stock derived from Gaiano, a wild-type strain that has elongated telomeres due to a dominant mutation in the Telomere elongation (Tel) gene. Regulation by TP6 is not impaired in the Gaiano genetic background. The regulatory abilities of the TP5 and TP6 elements are therefore not equally susceptible to the effects of elongated telomeres in the mutant Su(var)205 and Gaiano stocks.

Cytotype Regulation Facilitates Repression of Hybrid Dysgenesis by Naturally Occurring KP Elements in Drosophila melanogaster.

P elements inserted in the Telomere Associated Sequences (TAS) at the left end of the X chromosome are determiners of cytotype regulation of the entire P family of transposons. This regulation is mediated by Piwi-interacting (pi) RNAs derived from the telomeric P elements (TPs). Because these piRNAs are transmitted maternally, cytotype regulation is manifested as a maternal effect of the TPs. When a TP is combined with a transgenic P element inserted at another locus, this maternal effect is strengthened. However, when certain TPs are combined with transgenes that contain the small P element known as KP, stronger regulation arises from a zygotic effect of the KP element. This zygotic effect is observed with transgenic KP elements that are structurally intact, as well as with KP elements that are fused to an ancillary promoter from the hsp70 gene. Zygotic regulation by a KP element occurs only when a TP was present in the maternal germ line, and it is more pronounced when the TP was also present in the grand-maternal germ line. However, this regulation does not require zygotic expression of the TP These observations can be explained if maternally transmitted piRNAs from TPs enable a polypeptide encoded by KP elements to repress P element transposition in zygotes that contain a KP element. In nature, repression by the KP polypeptide may therefore be facilitated by cytotype-mediating piRNAs.

Establishment and maintenance of the P cytotype associated with telomeric P elements in Drosophila melanogaster.

P elements inserted near the left telomere of the X chromosome are associated with the P cytotype, a maternally transmitted condition that strongly regulates the activity of the P transposon family in some strains of Drosophila. The regulatory abilities of two such elements, TP5 and TP6, are stable in homozygous stocks over many generations. However, these regulatory abilities are attenuated when the telomeric P elements are transmitted through heterozygous females, and they are utterly lost when the elements are transmitted through males. Paternally transmitted telomeric P elements reacquire regulatory ability when they pass through a female germ line. This reacquisition is enhanced if the females in which it occurs came from mothers who carried a telomeric P element. The enhancement has two components: (1). a strictly maternal effect that is transmitted to the females independently of the mother's telomeric P element ("presetting" or the "pre-P cytotype") and (2). a zygotic effect associated with inheritance of the mother's telomeric P element. One telomeric P element can enhance the reacquisition of another's regulatory ability. When X chromosomes that carry telomeric P elements are extracted through males and made homozygous by using a balancer chromosome, most of the resulting stocks develop strong regulatory abilities in a few generations. However, some of the stocks do not attain the regulatory ability of the original population.

A hobo transgene that encodes the P-element transposase in Drosophila melanogaster: autoregulation and cytotype control of transposase activity.

Drosophila were genetically transformed with a hobo transgene that contains a terminally truncated but otherwise complete P element fused to the promoter from the Drosophila hsp70 gene. Insertions of this H(hsp/CP) transgene on either of the major autosomes produced the P transposase in both the male and female germlines, but not in the soma. Heat-shock treatments significantly increased transposase activity in the female germline; in the male germline, these treatments had little effect. The transposase activity of two insertions of the H(hsp/CP) transgene was not significantly greater than their separate activities, and one insertion of this transgene reduced the transposase activity of P(ry(+), Delta2-3)99B, a stable P transgene, in the germline as well as in the soma. These observations suggest that, through alternate splicing, the H(hsp/CP) transgene produces a repressor that feeds back negatively to regulate transposase expression or function in both the somatic and germline tissues. The H(hsp/CP) transgenes are able to induce gonadal dysgenesis when the transposase they encode has P-element targets to attack. However, this ability and the ability to induce P-element excisions are repressed by the P cytotype, a chromosomal/cytoplasmic state that regulates P elements in the germline.

Regulation of P-element transposase activity in Drosophila melanogaster by hobo transgenes that contain KP elements.

Fusions between the Drosophila hsp70 promoter and three different incomplete P elements, KP, SP, and BP1, were inserted into the Drosophila genome by means of hobo transformation vectors and the resulting transgenic stocks were tested for repression of P-element transposase activity. Only the H(hsp/KP) transgenes repressed transposase activity, and the degree of repression was comparable to that of a naturally occurring KP element. The KP transgenes repressed transposase activity both with and without heat-shock treatments. Both the KP element and H(hsp/KP) transgenes repressed the transposase activity encoded by the modified P element in the P(ry(+), Delta2-3)99B transgene more effectively than that encoded by the complete P element in the H(hsp/CP)2 transgene even though the P(ry(+), Delta2-3)99B transgene was the stronger transposase source. Repression of both transposase sources appeared to be due to a zygotic effect of the KP element or transgene. There was no evidence for repression by a strictly maternal effect; nor was there any evidence for enhancement of KP repression by the joint maternal transmission of H(hsp/KP) and H(hsp/CP) transgenes. These results are consistent with the idea that KP-mediated repression of P-element activity involves a KP-repressor polypeptide that is not maternally transmitted and that KP-mediated repression is not strengthened by the 66-kD repressor produced by complete P elements through alternate splicing of their RNA.

The P cytotype in Drosophila melanogaster: a maternally transmitted regulatory state of the germ line associated with telomeric P elements.

The incomplete P elements TP5 and TP6 are inserted in the TAS repeats near the left telomere of the Drosophila melanogaster X chromosome. These telomeric P elements repress P-induced gonadal dysgenesis and germ-line hypermutability in both sexes. However, their capacity to repress hypermutability is lost when they are transmitted patroclinously in a cross. TP5 and TP6 do not repress P-element activity in somatic cells, nor do they alter the somatic or germ-line phenotypes of P-insertion alleles. In the germ line, these elements suppress the phenotype of a P-insertion allele of the singed gene that is evoked by other P elements, presumably because these other elements encode repressor polypeptides. This suppression is more effective when the telomeric P elements are inherited maternally. Regulation by telomeric P elements parallels that of the P cytotype, a state that represses P-element activity in some strains of Drosophila. This state exists only in the germ line and is maternally transmitted along with the P elements themselves. Regulation by known repressor P polypeptides is not restricted to the germ line and does not require maternal transmission of the relevant P elements. Regulation by telomeric P elements appears to be epistatic to regulation by repressor P polypeptides.

Telomeric P elements associated with cytotype regulation of the P transposon family in Drosophila melanogaster.

P elements inserted at the left end of the Drosophila X chromosome were isolated genetically from wild-type P strains. Stocks carrying these elements were tested for repression of P-strain-induced gonadal dysgenesis in females and for repression of transposase-catalyzed P-element excision in males and females. Both traits were repressed by stocks carrying either complete or incomplete P elements inserted near the telomere of the X chromosome in cytological region 1A, but not by stocks carrying only nontelomeric X-linked P elements. All three of the telomeric P elements that were analyzed at the molecular level were inserted in one of the 1.8-kb telomere-associated sequence (TAS) repeats near the end of the X chromosome. Stocks with these telomeric P elements strongly repressed P-element excision induced in the male germline by a P strain or by the transposase-producing transgenes H(hsp/CP)2, H(hsp/CP)3, a combination of these two transgenes, and P(ry(+), delta2-3)99B. For H(hsp/CP)2 and P(ry(+), delta2-3)99B, the repression was also effective when the flies were subjected to heat-shock treatments. However, these stocks did not repress the somatic transposase activity of P(ry(+), delta2-3)99B. Repression of transposase activity in the germline required maternal transmission of the telomeric P elements themselves. Paternal transmission of these elements, or maternal transmission of the cytoplasm from carriers, both were insufficient to repress transposase activity. Collectively, these findings indicate that the regulatory abilities of telomeric P elements are similar to those of the P cytotype.

piRNA-mediated transposon regulation and the germ-line mutation rate in Drosophila melanogaster males.

Transposons, especially retrotransposons, are abundant in the genome of Drosophila melanogaster. These mobile elements are regulated by small RNAs that interact with the Piwi family of proteins-the piwi-interacting or piRNAs. The Piwi proteins are encoded by the genes argonaute3 (ago3), aubergine (aub), and piwi. Heterochromatin Protein 1 (HP1), a chromatin-organizing protein encoded by the Suppressor of variegation 205 [Su(var)205] gene, also plays a role in this regulation. To assess the mutational impact of weakening the system for transposon regulation, we measured the frequency of recessive X-linked lethal mutations occurring in the germ lines of males from stocks that were heterozygous for mutant alleles of the ago3, aub, piwi, or Su(var)205 genes. These mutant alleles are expected to deplete the wild-type proteins encoded by these genes by as much as 50%. The mutant alleles of piwi and Su(var)205 significantly increased the X-linked lethal mutation frequency, whereas the mutant alleles of ago3 did not. An increased mutation frequency was also observed in males from one of two mutant aub stocks, but this increase may not have been due to the aub mutant. The increased mutation frequency caused by depleting Piwi or HP1suggests that chromatin-organizing proteins play important roles in minimizing the germ-line mutation rate, possibly by stabilizing the structure of the heterochromatin in which many transposons are situated.

Effects of Invasive Winter Moth Defoliation on Tree Radial Growth in Eastern Massachusetts, USA.

Winter moth, Operophtera brumata L. (Lepidoptera: Geometridae), has been defoliating hardwood trees in eastern Massachusetts since the 1990s. Native to Europe, winter moth has also been detected in Rhode Island, Connecticut, eastern Long Island (NY), New Hampshire, and Maine. Individual tree impacts of winter moth defoliation in New England are currently unknown. Using dendroecological techniques, this study related annual radial growth of individual host (Quercus spp. and Acer spp.) trees to detailed defoliation estimates. Winter moth defoliation was associated with up to a 47% reduction in annual radial growth of Quercus trees. Latewood production of Quercus was reduced by up to 67% in the same year as defoliation, while earlywood production was reduced by up to 24% in the year following defoliation. Winter moth defoliation was not a strong predictor of radial growth in Acer species. This study is the first to document impacts of novel invasions of winter moth into New England.

Genetic interactions between P elements involved in piRNA-mediated repression of hybrid dysgenesis in Drosophila melanogaster.

Previous studies have shown that telomeric P elements inserted at the left end of the X chromosome are anchors of the P cytotype, the maternally inherited state that regulates P-element activity in the germ line of Drosophila melanogaster. This regulation is mediated by small RNAs that associate with the Piwi family of proteins (piRNAs). We extend the analysis of cytotype regulation by studying new combinations of telomeric and nontelomeric P elements (TPs and non-TPs). TPs interact with each other to enhance cytotype regulation. This synergism involves a strictly maternal effect, called presetting, which is apparently mediated by piRNAs transmitted through the egg. Presetting by a maternal TP can elicit regulation by an inactive paternally inherited TP, possibly by stimulating its production of primary piRNAs. When one TP has come from a stock heterozygous for a mutation in the aubergine, piwi, or Suppressor of variegation 205 genes, the synergism between two TPs is impaired. TPs also interact with non-TPs to enhance cytotype regulation, even though the non-TPs lack regulatory ability on their own. Non-TPs are not susceptible to presetting by a TP, nor is a TP susceptible to presetting by a non-TP. The synergism between TPs and non-TPs is stronger when the TP was inherited maternally. This synergism may be due to the accumulation of secondary piRNAs created by ping-pong cycling between primary piRNAs from the TPs and mRNAs from the non-TPs. Maternal transmission of P-element piRNAs plays an important role in the maintenance of strong cytotype regulation over generations.