Naturally Occurring Transposable Elements Disrupt hsp70 Promoter Function in Drosophila melanogaster

Daniel N. Lerman* and Martin E. Feder*,{dagger}

* Committee on Evolutionary Biology and Department of Organismal Biology & Anatomy, The University of Chicago; {dagger} Committees on Genetics and Molecular Medicine and Department of Organismal Biology & Anatomy, The University of Chicago

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


    Abstract
 TOP
 Abstract
 Materials and Methods
 RESULTS
 Discussion
 Acknowledgements
 References
 
Naturally occurring transposable element (TE) insertions that disrupt Drosophila promoters are correlated with modified promoter function and are posited to play a significant role in regulatory evolution, but their phenotypes have not been established directly. To establish the functional consequences of these TE insertions, we created constructs with either TE-bearing or TE-lacking hsp70 promoters fused to a luciferase reporter gene and assayed luciferase luminescence in transiently transfected Drosophila cells. Each of the four TEs reduces luciferase signal after heat shock and heat inducibility of the hsp70 promoter. To test if the differences in hsp70 promoter activity are TE-sequence dependent, we replaced each of the TEs with multiple intergenic sequences of equal length. These replacement insertions similarly reduced luciferase signal, suggesting that the TEs affect hsp70 promoter function by altering promoter architecture. These results are consistent with differences in Hsp70 expression levels, inducible thermotolerance, and fecundity previously associated with the TEs. That two different varieties of TEs in two different hsp70 genes have common effects suggests that TE insertion represents a general mechanism through which selection manipulates hsp70 gene expression.

Key Words: transposable element • hsp70 • promoter • transcriptional regulation • gene expression

Although mobile genetic or transposable elements (TEs) have had a massive and ancient impact on genome organization and diversity (Kazazian 2004), the roles of TEs in ongoing adaptive evolution in eukaryotes are less clear, because transposition is typically both deleterious and effectively marginalized by evolutionary and genetic mechanisms (Franchini, Ganko, and McDonald 2004). Recent discoveries, however, distinctively correlate transposition with adaptive microevolution (Franchini, Ganko, and McDonald 2004). The components of this correlation are as follows: (1) transposition into regulatory regions of genes; (2) such genes segregating as alleles of TE-less genes in natural populations; (3) allelic variation in the regulation of gene expression; (4) allelic variation in gene expression affecting fitness, and thus exposing allelic frequency to natural selection; and (5) covariation of allelic frequency with natural variation in the agent of selection. Although each component has numerous examples, the experimental verification of all components in a single system has been elusive. For instance, the best recent examples (Kalendar et al. 2000; Daborn et al. 2002; Lerman et al. 2003; Schlenke and Begun 2004) are complete except for demonstrating that the TEs themselves (rather than sequence to which they are linked) encode the adaptive phenotype and how they do so. Here we supply this missing link by experimentally excising or replacing naturally occurring TEs in a common genetic background and showing that the expected changes in gene expression result.

The present study uses the Hsp70-encoding genes of Drosophila melanogaster as a model system. In this species, Hsp70 is a molecular chaperone that contributes significantly to inducible thermotolerance, which is key to reproductive success in the face of natural thermal stress (Feder et al. 1996; Feder, Blair, and Figueras 1997; Lerman et al. 2003). Hsp70, however, plays numerous additional roles in the cell, for which stringent regulation at low concentrations is essential (Lerman et al. 2003). Thus, repeatably and depending on circumstances, either high or low levels of Hsp70 expression evolve in both natural and experimental populations (Sorensen, Dahlgaard, and Loeschcke 2001; Feder et al. 2002). These levels are inversely correlated with the frequencies of hsp70 alleles in which distinct TEs have inserted in the proximal promoter, suggesting selection for or against TE-bearing alleles as a mechanism of action (Michalak et al. 2001; Zatsepina et al. 2001; Bettencourt et al. 2002; Lerman et al. 2003). Indeed, alleles with a Jockey insertion are associated with reduced hsp70 mRNA, and both Jockey and P-bearing alleles are associated with reduced Hsp70 protein (Lerman et al. 2003). The natural or evolutionary variation in Hsp70 level could be due to the transposable elements themselves, and/or to sequence variation that is linked to the TEs but external to them. If the former, the TEs could affect Hsp70 level via two non-exclusive mechanisms: First, diverse components of the transcriptional apparatus associate with the hsp70 promoter, and the proper organization and spacing of their binding sites are essential for full-strength transcription (O'Brien and Lis 1991; Lis and Wu 1993; Amin et al. 1994; Li et al. 1996; Mason and Lis 1997; Weber et al. 1997). Hence disruption of promoter architecture by TE insertion could reduce transcriptional activation (Xiao and Lis 1988; Lee et al. 1992; Amin et al. 1994; Shopland et al. 1995). Alternatively, variation in hsp70 transcription could stem from introduction of novel regulatory elements in the TEs themselves (Kazazian 2004). To characterize the effect of the TEs and elucidate the mechanism by which they affect Hsp70 levels, we placed hsp70 promoter alleles with or without TE insertions in luciferase expression constructs, replaced TE insertions in these constructs with intergenic DNA of equal length, and excised these sequences from constructs. Each TE reduced luciferase signal, indicating that the TEs can be sufficient to reduce gene transcription. Furthermore, each replacement insert similarly reduced luciferase signal in all four hsp70 constructs, suggesting that the corresponding TEs affect hsp70 promoter function primarily by altering promoter architecture.


    Materials and Methods
 TOP
 Abstract
 Materials and Methods
 RESULTS
 Discussion
 Acknowledgements
 References
 
Fly Populations and hsp70Bb Promoter Sequencing
We obtained promoter sequence from three Drosophila populations harboring hsp70Ba alleles with or without TEs in the proximal promoter (T32, collected in subequatorial Chad and maintained at 30°–31°C (henceforth referred to as the T strain; Zatsepina et al. 2001); Arv/Zim, founded from crosses of parents from California and Zimbabwe (Lerman et al. 2003); and 1997 Evolution Canyon, collected from the North-facing slope (NFS) of Evolution Canyon, Israel, during August–September, 1997 [Michalak et al. 2001]) and a fourth population with similar allelism in hsp70Bb (2000 Evolution Canyon, collected from both the North-facing and South-facing [SFS] slopes of Evolution Canyon during the months of August and September 2000). In the 2000 Evolution Canyon population, hsp70Bb-specific primers (table 1A in the Supplementary Material online) were used to amplify a fragment (wild-type, 3,644 bp) containing the hsp70Bb promoter, coding sequence, and 3' UTR, and sequenced as previously described (Lerman et al. 2003).

Creation of Wild-type and TE-Bearing Reporter Constructs
The hsp70Ba promoter region from TE-bearing (TE+) and TE-lacking (TE–) flies from the T, Arv/Zim, and 1997 Evolution Canyon ("Canyon 97") populations was amplified from single-fly DNA in a 100-µl reaction, as above (see table 1B in the Supplementary Material online). This amplification yields a ~900 bp (for wild-type; 2,099–2,354 bp for TE-disrupted promoters) product containing the promoter, 5' UTR, and first 190 bp of the hsp70Ba coding sequence.

To create luciferase reporter constructs, hsp70 promoters were re-amplified from the polymerase chain reaction (PCR) products described above (Canyon 2000 whole hsp70Bb gene and hsp70Ba promoter and partial coding sequence for the other three populations) with primers designed to introduce restriction sites to facilitate cloning (table 1C in the Supplementary Material online). Re-amplification of gene-specific PCR products ensured that the wild-type and TE-disrupted promoter constructs were derived from the same gene (rather than from different hsp70 gene copies). A 2–3 µg portion of these new PCR products was digested for 3 h at 37°C with 15 U each of Kpn1 and Bgl2 in a 40-µl reaction. Then, 4 µg of the pGL-3-Basic Vector (Promega; ~4.8 kb), which contains the cDNA-encoding firefly luciferase (luc) but lacks eukaryotic promoter or enhancer sequences, was also digested with the same restriction enzymes. Digests were purified with the QIAquick Gel-Extraction kit (Qiagen), promoters were ligated into the pGL-3 vector, and maxi preps were purified with the Qiagen Plasmid Maxi Kit, yielding 300-µl purified plasmid at a concentration of 1–2 µg/µl. TE+ and TE– constructs were confirmed by gel electrophoresis and sequencing. All constructs begin upstream of HSE4 and end 60 bp downstream of the transcription start site (fig. 1).



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FIG. 1.— hsp70-luciferase reporter constructs. TE+ and TE– promoters from each fly population were amplified, digested with Kpn1 and Bgl2, and ligated into the pGL3-basic vector as described in the text. TE insertion sites in the A. hsp70BaT; B. hsp70BaArv/Zim; C. hsp70BaCanyon 9; and D. hsp70BbCanyon 2000 promoters. HSEs, TATA box, GAGA elements (striped boxes), and transcription start site are as shown. Duplicated host DNA at TE insertion sites flank TEs as shown; wild-type (TE–) promoters contain a single copy of this sequence. Insertion site duplication in hsp70BaT (A) results in an additional putative GAGA element (underlined in sequence), defined as the pentamer consensus sequence GAGAG (Omichinski et al. 1997; Wilkins and Lis 1997). The last T in the duplicated hsp70BbCanyon 2000 sequence (D) is the first T of the TATA box. Constructs are not drawn to scale.

 
Creation of Replacement Inserts
To remove the TEs and facilitate their replacement with random DNA sequences, two hsp70 promoter fragments were amplified from the TE-containing constructs described above. The portion of the hsp70 promoter upstream from the TE was amplified using the aforementioned Kpn1-containing upper primer and a lower primer containing a Pac1 restriction site. This lower primer starts just upstream of the TE, and was therefore specific for each TE-containing promoter (table 1D in the Supplementary Material online). The part of the hsp70 promoter downstream of the TE was amplified using the aforementioned Bgl2-containing lower primer and gene-specific Pac1-containing upper primers, which began just downstream of the TE insertions (table 1D in the Supplementary Material online).

A 2-µg portion of each of the amplified promoter fragments was digested for 3 h at 37°C with Pac1, and the upstream and downstream promoter pieces (150 µg each) were ligated to each other at the Pac1 site with 3 U T4 Ligase. Successful upstream-downstream ligants were amplified with the aforementioned Kpn1- and Bgl2-containing primers and confirmed by gel electrophoresis. To create "Excision" reporter constructs, these ligants were digested with Kpn1 and Bgl2, ligated into pGL-3 (cut with Kpn1 and Bgl2), and purified as above. Preparations were confirmed by DNA sequencing. The excision constructs contained an 8-bp Pac1 site at the excision site, which was not present in wild-type promoters (the insertion of this site did not significantly reduce luciferase signal in the Canyon 2000, Arv/Zim, and T populations (see Results). The excision constructs differ from the TE-containing constructs in that the former contain an 8-bp Pac1 site where the TE was, and lack the 8 bp sequence duplicated at the TE insertion site.

Four random DNA sequences used to replace each of the naturally occurring TEs were obtained by amplification (table 1E in the Supplementary Material online) of intergenic sequences from D. melanogaster chromosome 3R (GenBank accession number AE003602). Designated by letter, these are: A (positions 64618–66588 of the GenBank sequence), B (248199–249655), C (253707–255437), and D (262490–263956). These positions are the limits of the respective intergenic sequences; each 3'-complimentary primer (table 1E in the Supplementary Material online) was designed so that the resulting amplicon would be the same length as the TE it was replacing.

Intergenic PCR products and the excision luciferase constructs described above were digested with Pac1 and cleaned with Qiagen spin columns. Three of the four random DNA sequences eventually were successfully ligated into the site from which each naturally occurring TE had previously been excised, and ligants (henceforth "Replacement" constructs) were purified and confirmed by gel electrophoresis. Because the Replacement constructs were the same length as the TEs they replaced, Replacement sequences from the same intergenic region were not identical (e.g., insert A for Canyon 97 was not identical to replacement sequence A for Arv/Zim, and so on).

Transient Transfection and Luciferase Assay
Drosophila Schneider S2 cells were cultured at 25°C in Sheilds and Sang M3 medium (Sigma) containing 10% heat-inactivated fetal bovine serum (Sigma); 3.5 ± 105 cells were seeded in 350 µl medium per well in 24-well plates the day before transfection. Cells were transiently transfected with Superfect Transfection Reagent (Qiagen). Then 1 µg plasmid DNA was co-transfected with 0.05 µg pRL-CMV (Promega) per well. The pRL-CMV vector contains the cDNA encoding Renilla luciferase (Rluc) and the CMV enhancer and early promoter.

After transfection, cells were incubated at 25°C for 6 h, transferred to 1.5-ml microcentrifuge tubes and placed in thermostatted waterbaths at either 36.5°C (heat shock) or 25°C (control) for 5 min, transferred to a 25°C waterbath for 1 h, and harvested by centrifugation. Eight samples per promoter (i.e., TE+, TE–, Excision, Replacements) were subjected to heat shock, and four samples/promoter were subjected to control treatment for a total of 72 separate transfections for each of the populations (except in the T strains, for which only two replacement inserts were created). Cells were washed in phosphate-buffered saline (PBS), lysed in 150 µl Passive Lysis Buffer (Promega), and stored at –80°C overnight for analysis.

Luciferase signal was quantitated with the Dual-Luciferase Reporter Assay System (Promega), which allows sequential measurement of firefly and Renilla luciferase activity from the same sample of lysate. For each assay, 5 µl lysate (+ 5 µl PBS) was analyzed in a manual luminometer programmed to perform a 10-s measurement. Firefly luciferase background was determined by measuring luminescence activity of nontransfected control cells. Before quantitation of Renilla luminescent signal, firefly signal was quenched; nonetheless, residual luminescence from the firefly luciferase reaction can affect Renilla luminescence measurements. Accordingly, Renilla background luminescence was determined by preparing a lysate of heat-shocked cells transfected with the appropriate hsp70-luc reporter vector only (i.e., no pRL-CMV) and measuring firefly luciferase luminescence followed by apparent Renilla luminescence. Backgrounds were subtracted from raw firefly and Renilla luminescence measurements, respectively, and luciferase activity was expressed as the ratio of firefly/Renilla luminescence.

Several sets of S2 cells were used for transformations. These differed systematically in their normalized luminescence signal (data not shown). Accordingly, luminescence signals were compared only in transfected cells from the same source cell population, and all comparisons within a fly population (e.g., Arv/Zim etc.) were performed in a single experiment.

Statistics
Statistical analyses used StatView 4.5 (Abacus Concepts). Factorial analyses of variance (ANOVA) tested for group effect. Post hoc analyses tested for effect of promoter after heat-shock and control treatments, as well as for effect of heat shock in TE+, TE–, Excision, and Replacement samples.


    RESULTS
 TOP
 Abstract
 Materials and Methods
 RESULTS
 Discussion
 Acknowledgements
 References
 
Sequencing
The Canyon 2000 NFS population is polymorphic for a nonautonomous P element insertion in hsp70Bb promoter (fig. 1; GenBank accession number AY370940). While the other TEs described in Lerman et al. (2003) inserted between HSEs 2 and 3 in the hsp70Ba promoter, this element is located between the TATA box and the first HSE (41 bps upstream from the transcription start site) and disrupts the hsp70Bb gene promoter. This P element (1,050 bp) is smaller than those found in the Arv/Zim (1,383 bp) and Canyon 2000 (1,221 bp) populations. Like these P elements, the Canyon 2000 P element has a large internal deletion and is missing part of exon 1, all of exon 2, and part of exon 3; these P elements are therefore similar to the type II repressor-making KP element (Ohare and Rubin 1983; Black et al. 1987; Rasmusson, Raymond, and Simmons 1993). The Canyon 2000 P element is at an allelic frequency of approximately 20% in the 2000 NFS population and 0% in the 2000 SFS population.

Constructs, Controls, and Repeatability of Luciferase Assays
Renilla luciferase activity was positively correlated with the amount of transfected pRL-CMV. Luminescence in cells transfected with the pGL3-Basic vector only (i.e., with no promoter) was equal to firefly luciferase background measurements from nontransfected control cells or blank tubes after both heat-shock and control treatments (data not shown).

Control (no heat shock) Treatment
In the absence of heat-shock luciferase luminescence was low and unaffected by TE insertions in promoters extracted from each of the four populations (i.e., post-hoc comparisons are not significant [statistics not shown]; fig 2). Under these conditions, luciferase luminescence was also low and did not differ among constructs in which the TEs had been excised or replaced with the intergenic DNA amplicons A-D (fig. 2).



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FIG. 2.— hsp70 promoter activity. Drosophila S2 cells were transfected with TE+ and TE–hsp70-luc reporter constructs and subjected to control ("Con") or heat-shock ("HS") treatments, as described in the text. Firefly luciferase signal is expressed as the ratio of firefly to Renilla luciferase luminescence. A. T; B. Arv/Zim; C. Canyon 97; and D. Canyon 2000 populations.

 
Heat Inducibility (comparison of control and heat shock treatments)
To determine the impact of the TE insertions on heat inducibility, we compared luciferase levels for a given construct before and after heat shock. Heat shock increased luciferase luminescence above that of non-heat-shocked cells for all TE– promoters, but it did not significantly increase luminescence for TE+ promoters (fig 2; table 1). That is, the TEs reduced heat inducibility of these promoters. Heat inducibility of luciferase luminescence was also abolished when TEs were replaced with several intergenic DNA sequences of similar length (fig 2; table 1). Excising the TEs rescued wild-type heat inducibility in three of the four hsp70 promoter constructs (Canyon 2000, Canyon 97, and Arv/Zim).


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Table 1 Analysis of Variance of Normalized Luminescence Measurements

 
Luciferase Activity in Heat-Shocked Cells
Whereas above we compare each promoter variant to itself under heat-shock or control conditions, here we compare promoter variants to one another, all after heat shock:
  1. TE+ vs. TE– (wild-type). All constructs with TE+ promoters had lower luciferase luminescence than those with TE– promoters (fig 2; table 1). This difference in luciferase activity was similar for each of the TEs, ranging from 52% (for Canyon 97 promoters) to 72% (for Canyon 2000 promoters).
  2. TE+ vs. Replacement. Luciferase signal did not differ between any Replacement construct and its corresponding TE+ construct for any of the four fly populations (fig 2; statistics not shown). Thus replacement of the naturally occurring TEs with intergenic DNA amplicons reduces luciferase luminescence to a similar extent as do the naturally occurring TEs themselves.
  3. TE– vs. Excision. TE– hsp70 promoters and their corresponding Excision constructs resulted in similar luciferase activity in the Canyon 2000, Arv/Zim, and T populations. In the Canyon 97 samples, however, luciferase luminescence was 32% lower in the Excision constructs than in TE– (fig 2; table 1).
  4. Replacement vs. Excision. For the Canyon 2000, Canyon 97, and Arv/Zim hsp70 promoters, hsp70-luc constructs with each of the three replacement insertions expressed less luciferase than did the corresponding Excision constructs. These differences in luminescence were similar to those between TE+ and TE– constructs, ranging from 44% (in Canyon 97 Insert A) to 92% (in Canyon 2000 Insert D). For the T strain, only the D insert had significantly lower luciferase levels than its corresponding Excision construct, although both C and D Replacements had significantly lower luciferase levels than the corresponding wild-type (TE–) construct (fig 2; table 1; Replacement versus TE– post-hocs not shown).


    Discussion
 TOP
 Abstract
 Materials and Methods
 RESULTS
 Discussion
 Acknowledgements
 References
 
While noncoding regulatory variation is thought to underlie important phenotypic changes, distinguishing the impact of specific nucleotide variants from those of linked loci is often difficult (Rockman and Wray 2002). Here we tested the functional consequences of naturally occurring TE insertions that disrupt Drosophila hsp70 promoters. To mitigate the confounding influence of linkage disequilibrium, we measured luciferase luminescence in Drosphila S2 cells transfected with hsp70luc reporter constructs. These experiments clearly demonstrate that the each of the TEs reduces hsp70 promoter activity, and they suggest that the differences in Hsp70 expression, thermotolerance, and reproductive success previously associated with these insertions (Lerman et al 2003) are caused by TE disruption of the hsp70 promoters. Furthermore, replacement of the TEs with intergenic sequences establishes that disruption of hsp promoter architecture is sufficient for reduced hsp transcription

Transposition events are frequent at both hsp70 gene clusters (Zatsepina et al. 2001; Bettencourt et al. 2002; Maside, Bartolome, and Charlesworth 2002), and, as the present study shows, TEs have interrupted the proximal promoters of two hsp70 genes. Lerman et al. (2003) hypothesized that the distinctive open chromatin conformation of hsp70 promoters facilitates their transpositional disruption.

While high levels of Hsp70 are important for tolerance of extreme temperatures (see Introduction), high levels of Hsp70 can be deleterious at moderately high but non-lethal temperatures (Krebs and Feder 1997a; 1997b; Bettencourt, Feder, and Cavicchi 1999; Zatsepina et al. 2001), presumably as a result of over-activation of its multiple functions in the cell (Lerman et al. 2003). Thus Lerman et al. (2003) hypothesized that selection maintains the TEs to reduce Hsp70 levels in Drosophila in non-stressful environments. The reduction in hsp70BaP allele frequency on the hotter and more variable south-facing slope (SFS; 1.2%) compared with the NFS (33.9%) in the 1997 Evolution Canyon populations is consistent with this hypothesis (Michalak et al. 2001), as is the lower hsp70BbP allele frequency for the SFS population (0%) than for the NFS (20%) population in the Evolution Canyon 2000 collection. An alternative hypothesis, that the absence of the hsp70BbP allele in the Canyon 2000 SFS population results from drift/founder effect, requires data on gene flow between these populations for testing.

Full-strength transcription of heat-shock genes requires the orderly interaction of the transcriptional holoenzyme/polymerase complex, the heat-shock transcription factor (HSF), and conserved elements in the heat-shock promoter. hsp70 genes normally have an open chromatin conformation (Costlow and Lis 1984), and TFIID, RNA polymerase II (Pol II), and the GAGA factor (GFA) all associate with the hsp70 promoter prior to heat shock (Lis and Wu 1993). While this constitutive binding and pre-assembly primes hsp70 genes for rapid response to heat shock, it is not sufficient for hsp70 induction because the Pol II complex pauses 20–40 nucleotides downstream of the transcription start site (Weber et al. 1997). Resumption of elongation by Pol II, the rate-limiting step in hsp70 transcription (O'Brien and Lis 1991), requires cooperative binding of HSF trimers to HSEs (Amin et al. 1994). Heat-shock transcription factor and the TATA binding protein also interact cooperatively in vivo (Mason and Lis 1997), and GFA further stabilizes HSF-HSE engagement (Li et al. 1996; Mason and Lis 1997). Hence, disruption of promoter architecture, by TE insertion or any other means, might reduce transcriptional activation by altering cooperative protein-protein interactions at the hsp70 promoter. Prior work supports this hypothesis. Although 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 between and stereoalignment of HSEs is also critical. Insertion of spacers of 1–5 and 11–14 nucleotides between HSEs 1 and 2 reduces promoter activity by 90%, whereas spacers of 6–10 and 16–18 nucleotides largely restore promoter activity (Amin et al. 1994).

As we show, all of the TEs affect hsp70 promoter function, despite differences in insertion site, length, and sequence. This effect was not a foregone conclusion, however, as the experimental manipulations of hsp70 promoter structure discussed above differ from the disruptions caused by the naturally occurring TEs in several important ways. For example, Amin et al. (1994) used constructs that contained only the minimal hsp70 promoter—i.e., HSEs 1 and 2. Thus, changing the spacing between HSEs 2 and 3, where three of the TEs have inserted, was previously unexamined. In addition, while the maximum spacer length previously tested was 25 bp, the TEs described here are all much larger (>1 kb). Finally, the TEs are natural—not anthropogenic—mutagens.

In addition, that the TE insertions would decrease, rather than increase, expression of adjacent genes was unforeseen. Indeed, other TEs thought to play an adaptive role in regulatory evolution may increase gene expression. For example, Daborn et al. (2002) demonstrate that an Accord insertion in the 5' end of the D. melanogaster cytochrome P450 gene Cyp6g1 is associated with Cyp6g1 overtranscription, which confers insecticide resistance. Schlenke and Begun (2004) show that a 4,803-bp Doc insertion in the 5' flanking region of the D. simulans Cyp6g1 gene is also correlated with increased transcriptional abundance. Yet despite differences in insertion site, length, and sequence, all the TEs described here similarly reduced hsp70 promoter function. This reduction, and the absence of TE insertions that increase Hsp70 expression, may result from selection against high levels of Hsp70, which can be harmful in environments with infrequent extreme stress (Lerman et al. 2003).

Particular sequence elements within each of the TEs could in principle also be responsible for the impact of TEs on promoter activity. Some TEs carry regulatory motifs that alter expression of nearby genes. For example, insertion of the gypsy element, which contains an insulator that can disrupt regulatory interactions, is responsible for many spontaneous mutations in Drosophila (Modolell, Bender, and Meselson 1983; Geyer and Corces 1992; Hogga et al. 2001). Further, TE+ and TE– promoters contain differences in addition to the TE that may contribute to the differences observed in promoter activity. For example, duplicated host DNA flanks each of the TE insertion sites. In the T population, this duplication results in an additional putative GAGA element, which may alter promoter activity, and the other insertion site duplications may also contain putative regulatory sequences. In addition, the Canyon 2000 and Arv/Zim populations contain single-nucleotide polymorphisms (SNPs; two and four SNPs, respectively) that are linked to the P element insertion. Although such noncoding regulatory variation is thought to underlie important phenotypic changes, distinguishing the impact of specific nucleotide variants from those of linked loci is often difficult (Rockman and Wray 2002). Indeed, differences in luciferase luminescence observed between TE+ and TE– promoters might result from these SNPs, rather than from the TEs that they are linked to.

To distinguish between these alternative impacts of TEs, spatial disruption of the promoter versus addition of novel regulatory motifs—and to remove the potentially confounding affect of the insertion site duplications and SNPs—we created the Excision and Replacement luc constructs. All three intergenic replacement inserts in the Canyon 2000, Canyon 97, and Arv/Zim hsp70-luc promoters and one of the two inserts in the T promoter significantly reduced luciferase signal compared with both the wild-type (TE–) and Excision constructs. These reductions were comparable to those observed in the TE+ constructs. The intergenic inserts also reduced heat inducibility of the hsp70-luc promoters, as the TEs themselves did. Taken together, 10 of 11 replacement inserts significantly reduced luciferase luminescence, as did all four TEs, indicating that the reductions in promoter activity caused by the TE insertions did not result primarily from TE-specific sequence, insertion site duplications, or from SNPs linked to the TEs. Rather, these results suggest that disruption of hsp promoter architecture is sufficient for reduced hsp transcription.

We have characterized naturally occurring TE insertions that disrupt the Drosophila hsp70 promoter and show that each of these insertions reduces hsp70 promoter activity. These TEs, moreover, reduce Hsp70 expression levels and affect organismal fitness measurements (Lerman et al. 2003). This study is the first direct demonstration that specific natural nucleotide polymorphisms underlie differences in Hsp70 expression. More broadly, these results suggest that TEs can underlie adaptive regulatory evolution in natural populations.


    Acknowledgements
 TOP
 Abstract
 Materials and Methods
 RESULTS
 Discussion
 Acknowledgements
 References
 
We thank Harinder Singh and his laboratory for use of his luminometer, and David Rand for providing the Arv/Zim populations. This work was supported by National Science Foundation awards IBN99-86158, IBN02-06582, and IBN03-16627, and by a Howard Hughes Medical Institute Predoctoral Fellowship (D.N.L.).


    Footnotes
 
1 Present address: Office of Science Policy & Planning, Office of the Director, National Institutes of Health, 1 Center Drive, Bldg. 1, Rm. 218, Bethesda, MD 20892. Back

Claudia Schmidt-Dannert, Associate Editor


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 Materials and Methods
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 Discussion
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Accepted for publication November 25, 2004.





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