Modification of Heat-Shock Gene Expression in Drosophila melanogaster Populations via Transposable Elements

Daniel N. Lerman*,1, Pawel Michalak{dagger},{ddagger},1,2, Amanda B. Helin{dagger}, Brian R. Bettencourt{dagger},3 and Martin E. Feder*,{dagger}

* Committee on Evolutionary Biology
{dagger} Department of Organismal Biology & Anatomy, The University of Chicago, Chicago, Illinois
{ddagger} Institute of Evolution, University of Haifa, Haifa, Israel


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We report multiple cases in which disruption of hsp70 regulatory regions by transposable element (TE) insertions underlies natural variation in expression of the stress-inducible molecular chaperone Hsp70 in Drosophila melanogaster. Three D. melanogaster populations from different continents are polymorphic for jockey or P element insertions in the promoter of the hsp70Ba gene. All three TE insertions are within the same 87-bp region of hsp70Ba promoter, and we suggest that the distinctive promoter architecture of hsp genes may make them vulnerable to TE insertions. Each of the TE insertions reduces Hsp70 levels, and RNase protection assays demonstrate that such insertions can reduce transcription of the hsp70Ba gene. In addition, the TEs alter two measures of organismal fitness, inducible thermotolerance and female reproductive success. Thus, transposition can create quantitative genetic variation in gene expression within populations, on which natural selection can act.

Key Words: Drosophila melanogasterhsp70; heat shock proteins • transposable elements • regulatory evolution


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Transposable elements (TEs) have clearly had a major impact on genome evolution (McDonald 1995; Capy et al. 2000; Gray 2000; Kidwell and Lisch 2001), but their role in the microevolution of adaptation is controversial. One viewpoint is that transposable elements are predominantly deleterious, and thus selection typically opposes their proliferation and maintenance in the genome (Charlesworth, Sniegowski, and Stephan 1994). Another is that TEs are important mechanisms of microevolutionary change and sometimes are selectively advantageous to their hosts (McDonald 1995; Kidwell and Lisch 2001; McCollum et al. 2002). Such advantages could arise from the impact of transposition on gene expression, and indeed naturally occurring TE insertions can affect transcription and protein levels (Dunn and Laurie 1995; White and Jacobson 1996; Wu, Wilks, and Gibson 1998). Few studies, however, directly test the impact of TEs on organism-level function and/or fitness in populations under ecologically relevant conditions (Dunn and Laurie 1995; Wendel and Wessler 2000). Our finding that TEs have repeatedly and independently disrupted the promoter of the Drosophila melanogaster hsp70Ba gene provides an opportunity for such tests.

Hsp70 is the principal heat-inducible molecular chaperone in D. melanogaster, in which it confers inducible stress tolerance (Feder and Krebs 1998) but also participates in the regulation of cell growth and death, intracellular signaling, and diverse other processes (Zatsepina et al. 2001; Gabai and Sherman 2002). Elevated levels of Hsp70 are usually beneficial for inducible thermotolerance but deleterious for growth and development (Krebs and Feder 1997, 1998; Zatsepina et al. 2001). Thus, evolution at Hsp70-inducing temperatures in the absence of heat stress repeatedly results in decreased Hsp70 levels (Bettencourt, Feder, and Cavicchi 1999; Sorensen et al. 1999; Lansing, Justesen, and Loeschcke 2000; Zatsepina et al. 2001). At least five nearly identical genes at two chromosomal loci encode Hsp70 in D. melanogaster (Bettencourt and Feder 2002). Locus 87A7 contains an inverted pair, hsp70Aa and hsp70Ab, and locus 87C1 has three copies: hsp70Ba, and, approximately 40 kb away, hsp70Bb and hsp70Bc are tandemly arrayed (Moran et al. 1979; Bettencourt and Feder 2002). In view of the stringent transcriptional regulation of the hsp70 genes, their proximal promoters are obvious candidates for encoding evolutionary variation in Hsp70 protein levels. Vigorous hsp70 transcription ensues when a common transcription factor, heat-shock factor (HSF), binds to heat-shock response elements (HSEs) in the promoter of each hsp70 gene. While HSEs 1 and 2 are sufficient for some hsp70 transcription, full-strength transcription requires all four (Xiao and Lis 1988; Lee et al. 1992; Shopland et al. 1995). The spacing of the HSEs is also critical: the insertion of spacers 1–5 bps long between HSEs 1 and 2 reduces promoter activity by 90% (Amin et al. 1994).

These experimental insertions of sequence have a recurrent counterpart in the hsp70Ba genes of Drosophila populations. We surveyed ten populations founded from collections of Drosophila in the wild and found that three, two previously reported (Michalak et al. 2001; Zatsepina et al. 2001) and one reported here, are polymorphic for naturally occurring TE insertions in the hsp70Ba proximal promoter (fig. 1). The T32 population, originally collected in subequatorial Africa, is polymorphic for a truncated jockey element in the promoter (Zatsepina et al. 2001). This population expresses little Hsp70 and shows poor inducible thermotolerance (Zatsepina et al. 2001). Populations in Evolution Canyon, Israel, are polymorphic for a 1.2 kb P element in the hsp70Ba promoter (Michalak et al. 2001). Drosophila from the cooler, north-facing slope of the canyon accumulate less Hsp70 (Feder and Korol, unpublished data) and have a greater allelic frequency of the P element–bearing allele (Michalak et al. 2001) than those from its warmer, south-facing slope. As we describe below, a P element has independently inserted in the hsp70Ba promoter of a third population ("Arv/Zim"). Because of (1) the correlation between TEs in the hsp70Ba promoter and Hsp70 protein level in Drosophila populations (Zatsepina et al. 2001) and (2) the impact of experimental promoter disruption on hsp70 transcription (Xiao and Lis 1988; Lee et al. 1992; Amin et al. 1994; Shopland et al. 1995), we hypothesized that these TEs could reduce hsp70Ba transcription in whole Drosophila. Here we test this hypothesis with the jockey element in the T32 population, and we show that this element reduces transcription. We also created replicate lines fixed for the presence or absence of each of the three TEs and report that each of these insertions can reduce Hsp70 levels and alter inducible thermotolerance. The two P elements, moreover, affect female reproductive success. Thus, by disrupting hsp70 promoters, TEs can affect whole-organism fitness in natural populations.



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FIG. 1. Transposable elements disrupt the Drosophila melanogaster hsp70Ba promoter. A, Wild-type hsp70Ba promoter. B, A 1447 bp fragment corresponding to the 3' end of the jockey element is inserted 107 bps upstream of the hsp70Ba transcription start site in the T strain. The insertion intervenes between HSEs 2 and 3, displacing HSEs 3 and 4 as well as three GAGA elements (white unlabeled boxes). The GAGA factor affects chromatin structure and gene expression (Wilkins and Lis 1997). C, A 1383 bp nonautonomous P element in the Arv/Zim strain. D, A 1221 bp nonautonomous P element in strains from both slopes of Evolution Canyon

 

    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Fly Populations
The T32 population was collected in Chad, Africa, in 1977, and has been maintained at 31°–32°C (Zatsepina et al. 2001). Evolution Canyon flies were collected from the north-facing slope (NFS) of Evolution Canyon (Lower Nahal Oren, Mt. Carmel, Israel) during August–September, 1997: 10 males and 10 females from 25 isofemale lines were combined in population cages to generate a synthetic population that has been maintained at 25°C (Michalak et al. 2001). The Arv/Zim populations were founded from crosses of parents from California and Zimbabwe and were cultured at 18°C for >5 years before experimentation (D. Rand, personal communication). At least five generations before experimentation, flies were transferred to a 25°C incubator. Additional populations whose hsp70Ba promoters were screened for size polymorphisms (see below) were (Bettencourt and Feder 2002): AUS (Australia), QD18 (Japan), Z(H)1 (Zimbabwe), Z53 (Zimbabwe), LA66 (Zambia), FrV3-1 (France), A28 (derived from Oregon R; Bettencourt, Feder, and Cavicchi 1999).

Transposable Element PCR Screens
Genomic DNA was isolated from mass preparations of approximately 100 flies from each population (above) by phenol/chloroform/isoamyl alcohol extraction and suspended in 50 µl water. The hsp70Ba promoter was amplified by adding 0.5 µl genomic DNA to buffer (10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100) with 1.5–3.0 mM MgCl2, 0.2 mM each dNTP, 5 pmol each primer, and 1.25 units Taq DNA polymerase (Promega) per 25 µl reaction. Primers were: upper, 5'GCAAGCAATCATCATCCAAT3', and lower, 5'ACTGTGTTTCTGGGGTTCAT3'. This amplification yields a ~900 bp (for wild-type) product that starts ~467 bp upstream from the transcription start site and ends ~190 bp downstream from the start of translation, depending on population. Populations with TE insertions were initially detected by gel electrophoresis as larger polymerase chain reaction (PCR) products, ranging from 2099 to 2354 bps, and identified by sequence analysis (see below).

Single-fly DNA for TE-specific screens (for allele frequency data) was prepared by homogenizing individual flies in 50 µl buffer (10 mM Tris-HCl, pH 8.2, 1 mM EDTA, 25 mM NaCl) with 0.2 µg/µl proteinase K (Gloor et al. 1993). To screen T32 individuals for the jockey element, the foregoing primers and a jockey-specific internal primer (lower, 5'-AAGAAGACTCAAGCGACACC-3') were used in a single PCR (using 2.5 µl template DNA). To screen Arv/Zim and Evolution Canyon individuals for the P element, the hsp70Ba promoter amplification was performed along with an additional PCR that contained the same lower primer as above and a P element–specific upper primer (5'GCCTTCTTTTATCTTTTCTGG3'). Individuals with and without TEs were distinguishable via agarose gel electrophoresis. Reaction conditions for all PCRs consisted of an initial 1.5 min at 94°C, 35 cycles of 92°C for 1 min, 54°C for 1 min, and 72°C for 2.5 min, and a final step at 72°C for 5 min.

DNA Sequencing
Promoter and TE sequences from the Evolution Canyon and T32 populations were reported previously (GenBank accession numbers: T32, AY032740; Evolution Canyon, AF377341). The GenBank accession number for the Arv/Zim Hsp70Ba promoter and P element sequence is AF377953. To amplify Arv/Zim hsp70Ba promoters for sequencing analysis, 1 µl template DNA from single-fly preparations (as above) was added to buffer [50 mM KCl, 50 mM Tris-HCl (pH 8.3)] with 1.5 mM MgCl2, 0.2 mM each dNTP, 5 pmol each primer (flanking primers as above), and 2.5 units MasterAmp Extra-Long DNA Polymerase Mix (Epicentre Technologies) per 100 µl reaction. Polymerase chain reaction products were cleaned with Qiagen spin columns, cloned into the pGEM-T Easy vector (Promega), prepared with Qiagen Miniprep spin columns, and sequenced. Sequencing reactions were performed with ABI Prism cycle sequencing kits and ABI 377 sequencers (PerkinElmer). All samples were sequenced with pUC/M13 forward and reverse vector primers. Internal sequencing primers were used to provide complete coverage.

Construction of Replicate Lines
Because each of the three populations is polymorphic for TE-disrupted hsp70Ba promoters, we were able to create replicate lines from each of these populations that were fixed for either the presence or absence of the TE-disrupted promoter. Males and virgin females from each source population were paired and allowed to mate in glass vials with 7 ml of standard medium at 25°C; 30 to 50 mated pairs were established for each of the three populations. Adults were cleared after 5 days and genotyped as described above. Progeny homozygous for TEs or their absence were used to found expanded replicate lines. Numbers of replicates were: T32 strains, 6 jockey-present and 6 jockey-absent; Evolution Canyon strains, 5 P-present and 5 P-absent; Arv/Zim, 4 P-present and 4 P-absent replicates.

Determination of Hsp70 Protein Levels
Adults from each replicate line were collected 1 day ± 4 h after eclosion and placed individually in cryotubes (humidified with 10 µl PBS) that were submerged in a thermostatted water bath at 36°C for 30 min and then at 25°C for 30 min, immediately frozen in liquid nitrogen, and stored at -80°C. Flies from the T32 lines underwent an additional 5 min heat shock at 40.7°C before freezing. Hsp70 levels in each fly were determined by ELISA (Feder et al. 1996). Samples were lysed by homogenization with a motorized pestle in ice-cold phosphate-buffered saline with Complete Protease Inhibitor (Roche) and centrifuged at 20,000xg and 4°C for 30 min. Total protein concentration was determined by BCA (Pierce) analysis, and Pro-Bind 96-well assay plates (Falcon) were coated with 20 µg protein overnight at 4°C. After blocking and washing, bound Hsp70 was detected with the Drosophila-inducible Hsp70-specific antibody 7FB (Velazquez, DiDomenico, and Lindquist 1980) coupled to alkaline phosphatase (AP) via secondary (rabbit anti-rat IgG [Cappel]) and tertiary (AP-conjugated goat anti-rabbit IgG [Sigma]) antibodies. Incubation with the phosphatase substrate yielded a colored product that was quantified with a microplate reader. Hsp70 concentration is expressed as a percentage of an Hsp70 standard included on each plate. Males and females were analyzed separately. Sample sizes were 24 to 44 flies per sex.

Inducible Thermotolerance Measurements
Inducible thermotolerance was assayed separately for day-old male and female adults. For each assay, 20 flies from each replicate line were placed in glass vials containing 7 ml of standard Drosophila medium. Vials were submerged in thermostatted water baths to deliver the following temperature regime: 30 min at 36°C and 30 min at 25°C (termed "pretreatment"), followed by a 30 min heat shock. Heat-shock temperature varied according to population (T32, 40.7°C; Arv/Zim, 40.6°C; Evolution Canyon, 40.4°C) to produce mortality of 20% to 80%; in the absence of pretreatment, mortality was 100%. Sample sizes were 64 to 160 vials per sex, 20 flies per vial.

Ribonuclease Protection Assay
Wandering third instar larvae were collected from jockey-present and jockey-absent lines (seven larvae from each replicate line), placed in cryotubes with 10 µl water, pretreated for 30 min at 36°C, allowed to recover for 30 min at 25°C, and stored at -80°C. Control larvae were kept at 25°C for 1 h. Total RNA was isolated with RNAqueous (Ambion) by homogenizing tissue on ice with a motorized plastic pestle. RNA yield was determined by UV spectrophotometry and samples were stored at -80°C.

The extensive homogenization of the hsp70 genes (Bettencourt and Feder 2002) precluded the design of a single riboprobe that could simultaneously distinguish between hsp70Ba alleles and the other hsp70 genes. For example, in the ~2.2 kb of transcribed hsp70 sequence, a single SNP and a 7 bp indel polymorphism (both in the 5' UTR) distinguish the hsp70Ba gene in jockey+ strains from the other hsp70 gene copies. These sites, however, could not be used to distinguish the hsp70Ba gene from the other hsp70 genes in jockey- flies, as the hsp70 gene copies do not vary at these sites in jockey- strains. Similarly, the pair of SNPs (located in the 3' end of the gene) that distinguishes the hsp70Ba gene from the others in the jockey- strains were not present in the hsp70Ba gene in jockey+ strains. Accordingly, we designed two probes: one that distinguishes between the hsp70Ba gene and the other hsp70 genes in hsp70Bajockey/jockey homozygotes, and another that distinguishes between the hsp70Ba and other hsp70 genes in the hsp70Ba+/+ flies. A 103-bp fragment of the transcribed region of each hsp70Ba allele and a 141-bp region of the actin5c gene (control probe) were cloned in a pGEM-T Easy vector (Promega) and prepared with a Qiagen Plasmid Maxi Kit. Plasmids were linearized with Sac2, and 3' overhangs were recessed with 2.5 U DNA Polymerase I, large (Klenow) fragment (Promega). Riboprobes were synthesized by transcription from 0.5 µg of plasmid with Sp6 RNA polymerase in the presence of [32P]CTP. Next, 2 x 105 CPM of gel-purified radiolabeled probe was hybridized at 50°C overnight to 15 µg sample RNA; hybridization reactions were treated with 500 µg Ribonuclease A (Roche) and 5000 Units of RNase T1 (Ambion), and the recovered RNA was assayed by electrophoresis on a denaturing polyacrylamide/urea gel and PhosphorImager analysis (Molecular Dynamics). Gels were analyzed with ImageQuant software (Molecular Dynamics), and mRNA levels were approximated by quantifying the average intensity of pixels within equivalent rectangles surrounding the bands. Background density was subtracted separately for each of the two probes used. To compare message levels determined in multiple assays, hsp70Ba levels were expressed as a proportion of combined hsp70Bb and hsp70Bc levels for each genotype. For both genotypes, the hsp70Bb and hsp70Bc transcripts yielded a single detectable band. Because some of the bands differed in size—and therefore in number of 32P-labeled nucleotides—message levels were standardized by multiplying by a size factor (the ratio of labeled cytosine nucleotides) before comparison.

Reproductive Success Measurements
At 24 h after eclosion, individual females were placed with two males in vials containing 7 ml standard medium and transferred together to fresh vials every 24 h for 3 days. As new adults eclosed, total numbers of offspring per female were determined. Females that produced no adult offspring were equally common in each experimental group. These were excluded from the analysis on the assumption that these failures were due to erroneous sexing, poor-quality medium, and other factors unrelated to genotype. Sample sizes: 10 to 20 females per replicate for each genotype.

Statistical Analysis
Strains and sexes were analyzed separately. After checking for among-group variance homogeneity (Levene's test) and normality (Kolmogorov-Smirnov-Lilliefors test), a simple hierarchical ANOVA with fixed factors was used to analyze differences in Hsp70 expression levels and thermotolerance, in which "line replicates" were nested within "genotypes" (TE-presence vs. TE-absence).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Sequencing
The hsp70Ba proximal promoter fragments amplified from the AUS, QD18, Z(H)1, Z53, LA66, FrV3-1, and A28 populations were invariant in size, consistent with the absence of TEs in these fragments. By contrast, the Arv/Zim hsp70Ba promoter is polymorphic for a nonautonomous P element that has inserted 97 bp upstream of the transcription start site (fig. 1C). As is the case in the T32 and Evolution Canyon populations, the Arv/Zim P element intervenes between the second and third HSEs. The P elements in the Arv/Zim and Evolution Canyon strains are similar in size (1383 vs. 1221 bp) and nucleotide sequence (99.8% identity excluding indels). The hsp70BaPallele is at a frequency of 60% in the Arv/Zim strain (n = 96 flies sampled).

Effect of jockey Insertion on hsp70Ba Transcription in the T32 Population
In control (non-stressed) larvae, hsp70 mRNA was not detectable. The actin5c control probe hybridized equally to samples from both control and heat-shocked larvae (fig. 2A). By contrast, hsp70Ba mRNA levels were lower in hsp70Bajockey/jockey larvae than in hsp70Ba+/+ larvae (fig. 2A). Indeed, this effect is repeatable in multiple replicate lines (fig. 2B). Standardized hsp70Ba message levels (i.e., the ratio of hsp70Ba-specific signal to that for the adjacent hsp70 genes, hsp70Bb and hsp70Bc) ± SE are 1.372 ± 0.100 in the hsp70Ba+/+ replicates and 0.135 ± 0.003 in the hsp70Bajockey/jockey replicates. Thus, hsp70Ba message abundance is approximately 90% lower in the hsp70Bajockey/jockey lines than in the wild-type, a significant difference (t = 12.41; P < 0.0001).



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FIG. 2. RNase protection assays show that the jockey insertion in the hsp70Ba promoter reduces transcriptional activation of hsp70Ba in T strains. A, The jockey-specific probe (J+) distinguishes between hsp70Ba (lane 3; arrowhead) and Hsp70Bb + hsp70Bc mRNA (lane 3; arrow). This 103 bp probe contains a 7 bp indel in the 5' UTR that is present in hsp70Bb and hsp70Bc but not in hsp70Ba. After hybridization to RNA from hsp70Bajockey/jockey flies and subsequent digestion, the protected fragments are 103 bp for hsp70Bb and hsp70Bc (which contain the insertion) and 96 bp for hsp70Ba (which does not contain the insertion). Another probe (J-) distinguishes between hsp70Ba and Hsp70Bb + hsp70Bc transcripts in hsp70Ba+/+ flies. The full 96 bp probe is protected in hsp70Ba (lane 4; arrowhead). Whereas the probe contains an A at position 49, Hsp70Bb + hsp70Bc contain a C at that position. Accordingly, after digestion two labeled fragments (of 47 and 49 bps) corresponding to Hsp70Bb + hsp70Bc are protected (lane 4; arrow), and these run at almost the same size on the gel. A probe specific for transcripts from the actin5c gene (Act) is cohybridized with every sample: the protected doublet is 141 bps. The hsp70Aa and hsp70Ab genes differ from those at the 87C locus at several sites: digestion of probes bound to transcripts from these genes will result in smaller fragments (low doublet, lane 3; not shown in lane 4). RNA from control larvae (not heat-shocked) was hybridized to both the Act and J+ probes (lane 1). Free probes (which are larger because they contain a vector sequence that is not homologous to hsp70 and thus digested in the protection assay) are in lanes 2, 5, and 6. B, RNase protection assay with larvae from five additional replicate lines per genotype. Lanes 1–5, hsp70Ba+/+ lines; lanes 6–10, hsp70Bajockey/jockeylines. Relative hsp70Ba mRNA levels were quantified by obtaining the ratio of hsp70Ba-specific signal (arrowhead) to that for the hsp70Bb + hsp70Bc genes (arrow) for all six replicates per group after corrections for background and protected fragment size, as described in the text

 
Hsp70 Expression
Because Hsp70 levels and inducible thermotolerance vary with gender (Dahlgaard et al. 1998; Krebs, Feder, and Lee 1998; Sorensen et al. 1999), we analyzed these variables independently for male and female flies. For both sexes in all three populations, replicate lines with TE-disrupted promoters exhibited lower Hsp70 levels after standard Hsp70-inducing heat shocks than matched replicate lines in which the TEs were absent from the promoters (fig. 3A). These differences, ranging between 7% and 28%, were significant for both sexes in the Evolution Canyon and Arv/Zim populations, and for males in the T32 population (table 1). For each sex, replicate lines within treatments (presence or absence of a TE in the hsp70Ba promoter) affected Hsp70 level only in Arv/Zim. For clarity, data for replicate lines within population are pooled in figure 3, but all statistical analyses were performed on replicate lines nested within genotype (table 1).



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FIG. 3. Transposable element insertions alter Hsp70 expression and inducible thermotolerance. A, Hsp70 levels (means ± SE) measured by ELISA. Hierarchical analysis of variance (ANOVA) of replicate lines nested within group (i.e., TE-present vs. TE-absent) shows a significant reduction of Hsp70 in lines with TE insertions for T strain males and for Evolution Canyon and Arv/Zim males and females (*P <= 0.05, **P <= 0.01). B, Inducible thermotolerance measurements (±SE). T strain and Evolution Canyon lines with TE insertions have reduced inducible thermotolerance, and Arv/Zim lines with the P element insertion have increased thermotolerance (nested ANOVA, *P <= 0.05, **P <= 0.001, ***P <= 0.001)

 

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Table 1 Nested ANOVA Analysis of Hsp70 Expression (Line Replicates Nested Within Genotypes)

 
Inducible Thermotolerance
For both sexes in all three populations, TE insertions altered inducible thermotolerance, measured as the survival of a heat shock, after an Hsp-inducing pretreatment, in the replicate lines (fig. 3B). Transposable element insertions reduced thermotolerance in the T32 and Evolution Canyon lines by 16% to 22% but increased thermotolerance in the Arv/Zim lines by 62% to 65%. These differences are significant for both sexes in the Evolution Canyon and Arv/Zim populations, and for males in the T32 population (table 2). Interestingly, males consistently show greater thermotolerance than females despite having lower Hsp70 levels (fig. 2A). In all cases, replicate lines within treatments affected inducible thermotolerance, but that influence obviously did not obscure the effect of treatment.


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Table 2 Nested ANOVA Analysis of Thermotolerance (Line Replicates Nested Within Genotypes)

 
Reproductive Success
Reproductive success was measured as the total number of eclosing adults resulting from 3 days of egg-laying by a single female. Mean (±SE) offspring per strain were: Evolution Canyon (P-absent, 16.8 ± 0.9; P-present, 22.7 ± 1.0) and Arv/Zim (P-absent, 10.6 ± 1.3 SE; P-present 13.2 ± 1.4). Thus, the P elements are correlated with increased reproductive success of 24% to 36% (ANOVAs of replicate lines nested within genotype: Evolution Canyon, P < 0.001; Arv/Zim, P = 0.048). Transposable element effects are not evident in the T32 population, however, whose reproductive success is relatively greater in the laboratory (jockey-, 49.3 ± 1.8; jockey+, 50.3 ± 2.1).


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Phenotypic Consequences of TE Insertions
Change in the regulation of gene expression is a principal mechanism of phenotypic evolution (Wilson, Carlson, and White 1977; Dickinson 1991). While it has been proposed that TEs can cause regulatory changes that are of evolutionary importance, this process has been difficult to observe. We show here that three D. melanogaster populations are polymorphic for TE insertions that disrupt the hsp70Ba promoter. One of these elements chosen for detailed analysis, the jockey insertion in the T32 population, reduces hsp70Ba transcription. Each TE insertion reduces Hsp70 expression levels (although not significantly so in male T32 flies) and alters inducible thermotolerance. In two populations, lines with TEs present in the hsp70Ba promoter have greater reproductive success than lines with TEs absent. Thus, although not unanimous, the results clearly indicate that these naturally occurring polymorphisms in TE insertions can have diverse effects on transcription of the gene in which they occur and the phenotypes to which it contributes. TEs may thus play an important role in regulatory evolution.

Because hsp70Ba is but one of at least five Hsp70-encoding genes in D. melanogaster, its mutation by transposition might not have an impact on overall Hsp70 protein levels. As measurements of Hsp70 protein levels demonstrate, however, the effect of TE insertions in hsp70Ba is repeatedly evident even against the background of the other hsp70 genes. In this and other sets of duplicate genes, TE insertions in one copy may afford an opportunity to fine tune protein levels without abolishing overall transcription. Indeed, the hsp70 genes at 87A7 and 87C1 have different morphological and developmental expression domains (Lakhotia and Prasanth 2002), so the phenotypic impact of TEs in hsp70Ba may be even greater than is evident from bulk Hsp70 levels.

These TE insertions can affect inducible thermotolerance, and thus one component of whole-organism fitness. Hsp70 is sufficient for increased inducible thermotolerance, which is critical for survival of natural heat stress (Feder et al. 1996; Roberts and Feder 2000). Interestingly, the TE-associated decreases in Hsp70 levels corresponded to less inducible thermotolerance in the T32 and Evolution Canyon lines but more inducible thermotolerance in the Arv/Zim lines (fig. 2). Clearly genetic background can affect the relationship between Hsp70 expression and thermotolerance, which may not always be straightforward. Indeed, while the overall TE effect on inducible thermotolerance was significant, so too was the replicate line effect—i.e., that of genetic background within population. Hsp70 is not the only mechanism of inducible thermotolerance (Feder et al. 2002), and extremely high Hsp70 levels actually decrease inducible thermotolerance (Krebs and Feder 1998). The present results also indicate that TEs in the hsp70 promoter are not always omnipotent in their impact, and the genetic or epigenetic factors that modify their impact (e.g., Hsp70 co-chaperones and others [Feder et al. 2002]) are worthy of study.

Furthermore, the TEs are not the only ways in which the regions flanking the hsp70Ba coding sequences vary. For example, in hsp70Bajockey/jockey flies, the hsp70Ba 5'-untranslated region (UTR) has a 7 bp deletion that is not present in hsp70Ba+/+ flies. Because the hsp70 UTRs regulate hsp70 translational efficiency and message stability (Lindquist 1993) this deletion could in principle affect message and protein levels. We suggest that this UTR polymorphism does not contribute to such phenotypic differences for two reasons. First, this polymorphism, which is common in Drosophila, does not covary with Hsp70 expression in other populations (Bettencourt and Feder 2002), and Hsp70 levels differ in the Evolution Canyon and Arv/Zim populations, neither of which has this deletion in the hsp70Ba 5'UTR. Second, in Drosophila S2 cells hsp70Ba promoter-luciferase constructs differing in only the presence or absence of the jockey TE in the promoter exhibit the same differences in expression as the native alleles do (D. N. Lerman, unpublished data). Nonetheless, we cannot exclude that other, unknown variation linked to the TE insertions contributes to variation in hsp70 expression and/or protein levels.

We and others have hypothesized that the magnitude of Hsp70 expression results from a trade-off of the context-specific phenotypes of hsp70 (Krebs and Feder 1998; Lansing, Justesen, and Loeschcke 2000; Zatsepina et al. 2001), which include inducible tolerance of heat and other stresses, deterrence of protein aggregation, protein quality control, intracellular and extracellular signaling, and regulation of growth (Gabai et al. 1997; Mosser et al. 2000; Tang et al. 2001). In environments in which thermal stress is frequent and unpredictable, increases in stress-induced Hsp70 expression should be beneficial (Krebs and Feder 1998) and selection results in increased Hsp70 levels (Feder et al. 2002). In such environments, TE-bearing alleles that reduce inducible thermotolerance should not be favored. Indeed, the dramatic reduction in hsp70BaP allele frequency on the hotter and more variable south-facing slope (SFS) of Evolution Canyon (1.2%) compared with the NFS (33.9%; Michalak et al. 2001) suggests that the non-TE bearing hsp70Ba allele is favored in SFS populations. These differences in allele frequency are consistent with increased Hsp70 levels in SFS populations (Feder and Korol, unpublished data). By contrast, in environments with infrequent extreme stress and/or chronic mild hyperthermia, elevated Hsp70 levels can be deleterious because enhanced stress tolerance is irrelevant and excessive amounts of Hsp70 can harm cells (see Introduction). Indeed, laboratory and wild Drosophila populations at moderately high but nonlethal temperatures repeatedly evolve reduced inducible thermotolerance and Hsp70 expression (Bettencourt, Feder, and Cavicchi 1999; Sorensen et al. 1999; Lansing, Justesen, and Loeschcke 2000; Zatsepina et al. 2001).

Our determinations of female reproductive success in replicate lines with TEs present or absent in the hsp70 promoter suggest that such a trade-off can contribute to the maintenance of these TEs in natural populations. In lines founded from both the Evolution Canyon and Arv/Zim populations, the P element was correlated with a large increase in reproductive success in nonstressful environments. Thus, natural selection may favor these P elements in some environments and explain the high allelic frequency of the P element in the NFS strain (33.9%). Given that the P element in the Arv/Zim strains is also correlated with an increase in thermotolerance, its high frequency in this line (60%) is not surprising. The high frequency (60%) of the jockey element in the T32 population may be due to its suppression of Hsp70 expression at its distinctively warm culture temperature (31° to 32°) or to other causes, including drift or neutrality. These suggestions should be falsifiable in laboratory evolution or artificial selection experiments. We expect that TEs in hsp70 promoters should be favored in selection for high reproductive rate (Silbermann and Tatar 2000) and/or in warm environments that are not acutely lethal (< 37°C), but that they will be disfavored in environments with periodic exposure to acutely lethal temperatures.

Multiple Insertions in the hsp70Ba Promoter
All three TEs discussed above have inserted within the same 87-bp region of the promoter, suggesting that this region may be a "lightning rod" for TE insertion. Many TEs demonstrate bias in genome distribution, whether due to target site selectivity (Craig 1997), purifying selection against deleterious transposition, or other causes. P elements in particular are more prevalent in the 5' ends of genes than in coding regions (Spradling et al. 1995). The clustering of the TE insertions in the hsp70Ba promoter, however, is also consistent with the promoter's distinctive chromatin conformation: uninduced hsp70 genes have an open chromatin configuration (Costlow and Lis 1984), which facilitates the constitutive recruitment of components of the transcriptional apparatus and poises the gene for immediate transcription upon heat shock. P elements can insert preferentially in or near DNase I hypersensitive (DH) sites, suggesting that an open chromatin configuration favors TE insertion (Voelker et al. 1990). Indeed, high-resolution mapping of DH sites in the hsp70 promoter (Costlow and Lis 1984; Weber et al. 1997) shows that two DH regions coincide almost precisely with the insertion sites of the TEs reported here, and the promoters of other Drosophila hsp genes are a preferred target of local P element insertions (Timakov et al. 2002). Taken together, these data suggest that the unique chromatin configuration of hsp70 promoters contributes to the repeated presence of TEs in this region.

Although the insertions described here are all in the hsp70Ba gene, transposition events have been frequent at both hsp70 gene clusters, located at 87A7 and 87C1. D. melanogaster is polymorphic for several TE-derived insertions or deletions in the region between hsp70Aa and hsp70Ab at 87A7 (Zatsepina et al. 2001; Bettencourt et al. 2002), and the ~40 kb between the hsp70Ba and hsp70Bb genes contains 16 to 21 tandemly arrayed heat-inducible repetitive elements of heterochromatic origin that transposed independently into 87C1 and subsequently spread through the locus (Mason et al. 1982). Analysis of the sequences flanking both hsp70 gene clusters suggests that the 87A7-to-87C1 cluster duplication was likely a retrotransposition (Bettencourt and Feder 2001). Further, S element fragments have inserted upstream of hsp70Aa, hsp70Ab and Hsp70Bb in D. melanogaster. These insertions appear to be fixed in the species, and analysis of sequence variation at the hsp70Bb insertion site suggests that selection favors this insertion (Maside, Bartolome, and Charlesworth 2002).

Conclusion
TEs constitute approximately half of the human genome (Lander et al. 2001) and 10% of the Drosophila genome (Charlesworth and Langley 1989), and are key agents of genome-wide reorganization and large-scale evolution (Shapiro 1992; Brosius 1999a, 1999b). We show here that TEs can also play an adaptive role in microevolution within natural populations by manipulating the expression of genes critical for fitness, and suggest that this may be an additional factor (Rizzon et al. 2002) shaping TE abundance and distribution. The distinctive organization and regulation of the hsp70 promoter may make it an accessible target for transposition and thus the origin of novel alleles, which selection may then maintain or purge as circumstances dictate.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We thank Harinder Singh and Eric Bertolino for their assistance with RNase protection assays, John Lis for useful discussion, David Rand for providing the Arv/Zim strains, and Soojin Yi and two anonymous reviewers for their comments on the manuscript. Supported by Natonal Science Foundation grants IBN9972678, 9986158, and 0072944, United States-Israel Binational Science Foundation Grant 4556, and Howard Hughes Medical Institute predoctoral fellowships.


    Footnotes
 
1 Contributed equally to this work. Back

2 Present address: Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana. Back

3 Present address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts. Back

E-mail: m-feder{at}uchicago.edu. Back


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Accepted for publication September 24, 2002.