Cytotype Regulation by Telomeric P Elements in Drosophila melanogaster : Interactions With P Elements From M9 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 M9 strains, which, like P strains, carry P elements, but have little or no ability to repress dysgenesis. The telomeric and M9 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 M9 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 M9 P elements. This synergism between TP and M9 P elements also appears to persist for at least one generation after the TP has been removed from the genotype. HYBRID dysgenesis is a syndrome of abnormal traits that occurs in the offspring of crosses between certain kinds of Drosophila melanogaster strains (Kidwell et al. 1977; Bregliano and Kidwell 1983; Engels 1989). This syndrome is characterized by high mutation rates, the occurrence of chromosome rearrangements, and sterility. Different families of transposable elements are responsible for hybrid dysgenesis. However, most research on this phenomenon has focused on the P elements, which are cut-and-paste transposons whose movement is restricted to germline cells. P-element movement is catalyzed by an 87-kDa polypeptide, the P transposase, which is encoded by structurally complete members of the P-element family (Karess and Rubin 1984; Rio et al. 1986); these elements are 2907 bp long. Many different types of incomplete P elements are also found in D. melanogaster genomes. Incomplete P elements cannot produce the transposase, but they can be mobilized by it as long as they have transposase target sequences in both their left and right ends (Rio 1990). P-element movement is restricted to the germline because the introns present in the transposase gene are fully removed from P transcripts only in germline cells (Laski et al. 1986). In somatic cells, the last P intron remains in the P RNA and prevents the synthesis of the catalytically active transposase. In its place, a shorter polypeptide is produced. This 66-kDa polypeptide is also made in germline cells, where it partially represses P-element activity (Misra and Rio 1990; Gloor et al. 1993; Simmons et al. 2002a). Polypeptides encoded by some incomplete P elements—in particular, the protein product of a 1.2-kb P element called KP—also function as partial repressors of hybrid dysgenesis (Black et al. 1987; Andrews and Gloor 1995; Simmons et al. 2002b). For 3 decades, D. melanogaster strains have been classified into two broad categories, M and P, according to whether or not they yield dysgenic hybrids when they are crossed (Kidwell et al. 1977). Crosses between M females and P males produce dysgenic hybrids, whereas the reciprocal crosses, P females 3 M males, usually do not and neither do crosses between two different M strains or between two different P strains. These observations imply that P strains possess an ability to induce hybrid dysgenesis when they contribute paternally to crosses with M strains and that they also possess an ability to repress hybrid dysgenesis when they contribute maternally in crosses to other P strains (or to themselves). P–M hybrid dysgenesis is most easily detected by noting the occurrence of sterility in females (Engels and Preston 1979; Kidwell and Novy 1979). This sterility is due to the failure of the germline tissues Corresponding author: Department of Genetics, Cell Biology and Development, 250 BioScience Center, University of Minnesota, 1445 Gortner Ave., St. Paul, MN 55108-1095. E-mail: [email protected] Genetics 176: 1957–1966 (August 2007) to develop. Females with this defect, called gonadal dysgenesis (GD), cannot produce eggs—a trait that can be readily scored in each individual examined. The classification of D. melanogaster strains on the basis of the results of crosses roughly coincides with a classification based on the presence or absence of P elements in genomes—that is, P strains possess P elements and M strains lack them (Bingham et al. 1982). Furthermore, P strains possess a state called the P cytotype, which strongly represses P-element movement, and M strains have a complementary state called the M cytotype, which permits it (Engels 1979a, 1989). Genetic analyses have indicated that the ability to repress hybrid dysgenesis (i.e., the P cytotype) depends on the presence of P elements in the genome (Engels 1979a; Kidwell 1981; Sved 1987). The P-element family is therefore autoregulated. There are, however, many exceptions to the simple classification of strains as P or M. Some strains with P elements in their genomes do not induce hybrid dysgenesis, or induce it very weakly, when they contribute paternally in crosses to M strains; however, they do repress hybrid dysgenesis when they contribute maternally in crosses to P strains—that is, they have the P cytotype. These strains have therefore been considered to be versions of P strains that do not induce hybrid dysgenesis effectively. They have been termed Q strains (Simmons et al. 1980; Engels and Preston 1981; Kidwell 1981; Bingham et al. 1982). Other strains have P elements in their genomes but they do not repress hybrid dysgenesis effectively when they contribute maternally in crosses to P strains, and neither do they induce hybrid dysgenesis when they contribute paternally in crosses to M strains (Bingham et al. 1982). Because these strains behave somewhat like M strains, they have been termed M9 or pseudo-M (Kidwell 1985; Simmons and Bucholz 1985). Both Q and M9 types are prevalent in surveys of strains derived within the past few decades from natural populations; see, for example, Anxolabéhère et al. (1985). The history of genetics is replete with examples in which exceptions to a rule have provided key insights into biological phenomena. In this article, we use the Q and M9 exceptions to the simple P–M dichotomy to investigate the nature of cytotype regulation. In previous work, single P elements with the ability to repress hybrid dysgenesis were isolated from the genomes of two Q strains, n6 and Mt. Carmel (Stuart et al. 2002). These elements are inserted in the telomere-associated sequences (TASs) at the left end of the X chromosome. A large body of work by Stéphane Ronsseray, Dominique Anxolabéhère, and colleagues has shown that strains carrying only P elements inserted in the X-linked TAS repress hybrid dysgenesis, sometimes strongly (Ronsseray et al. 1991, 1993, 1996, 1998; Marin et al. 2000). The telomeric P elements isolated from n6 and Mt. Carmel repress hybrid dysgenesis only when they are transmitted maternally in crosses (Simmons et al. 2004). Because maternal transmission is a key feature of the P cytotype, these (and other) telomeric P elements may play an important role in establishing this powerful system of P-element regulation. Two M9 strains, Sexi and Muller-5 Birmingham, have also been shown to repress hybrid dysgenesis, albeit weakly (Kidwell 1985; Simmons and Bucholz 1985; Simmons et al. 1987, 1990). These strains may have an incipient or latent version of the P cytotype, or they may have some other feature that enables them to repress P-element activity. In this article, we report the effect of combining the isolated telomeric P elements (TP s) from n6 and Mt. Carmel with the plethora of P elements from the M9 strains Sexi and Muller-5 Birmingham. Our study was motivated by the work of Ronsseray et al. (1998), who discovered interactions between telomeric P elements, telomeric P transgenes, and P elements from different P strains. However, one important difference between our study and theirs is that none of the interacting strains, either TP or M9, in our experiments carried complete P elements. Thus, there was no possibility for the synthesis of either the P transposase or the 66-kDa repressor polypeptide. We find that hybrid dysgenesis is repressed much more strongly by the TP–M9 combinations than by the TP or M9 P elements themselves—that is, telomeric P elements interact with other P elements to create the strong system of repression that we call the P cytotype. At a mechanistic level, these interactions might reflect physical contact between the TP and M9 P elements so that a repressive factor—perhaps an imprint of telomeric heterochromatin—is transferred from the telomere to P elements scattered throughout the genome, or they might reflect the interplay of molecules produced separately by the TP and M9 P elements. On this latter hypothesis, the TP and M9 P elements might encode different polypeptides that work together to repress P-element activity, or they might generate P RNAs that trigger and sustain an RNA interference (RNAi) response. The evidence that we report here and in the accompanying article in this issue (Simmons et al. 2007) is consistent with the latter idea. MATERIALS AND METHODS Drosophila stocks and husbandry: The stocks, genetic markers, and special chromosomes are described on the FlyBase website (http://flybase.bio.indiana.edu/), in Lindsley and Zimm (1992), and in other references cited in the text. The TP5 and TP6 stocks have P elements inserted in the TASs at the left end of the X chromosome. The TP5 element, originally isolated from the n6 Q strain, is 1.8 kb long and the TP6 element, originally isolated from the Mt. Carmel Q strain, is 1.9 kb long (Stuart et al. 2002). These are the only P elements present in these stocks. Sexi.4 and Sexi.7 (Rasmusson et al. 1990) are highly inbred stocks derived from the M9 strain Sexi (Kidwell 1985). Neither of these stocks 1958 M. J. Simmons et al.