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. 2006 Oct 6;2(10):e165.
doi: 10.1371/journal.pgen.0020165. Epub 2006 Aug 17.

Heat-shock promoters: targets for evolution by P transposable elements in Drosophila

Jean-Claude Walser  1 , Bing Chen, Martin E Feder
Affiliations

Heat-shock promoters: targets for evolution by P transposable elements in Drosophila

Jean-Claude Walser et al. PLoS Genet. .

Abstract

Transposable elements are potent agents of genomic change during evolution, but require access to chromatin for insertion-and not all genes provide equivalent access. To test whether the regulatory features of heat-shock genes render their proximal promoters especially susceptible to the insertion of transposable elements in nature, we conducted an unbiased screen of the proximal promoters of 18 heat-shock genes in 48 natural populations of Drosophila. More than 200 distinctive transposable elements had inserted into these promoters; greater than 96% are P elements. By contrast, few or no P element insertions segregate in natural populations in a "negative control" set of proximal promoters lacking the distinctive regulatory features of heat-shock genes. P element transpositions into these same genes during laboratory mutagenesis recapitulate these findings. The natural P element insertions cluster in specific sites in the promoters, with up to eight populations exhibiting P element insertions at the same position; laboratory insertions are into similar sites. By contrast, a "positive control" set of promoters resembling heat-shock promoters in regulatory features harbors few P element insertions in nature, but many insertions after experimental transposition in the laboratory. We conclude that the distinctive regulatory features that typify heat-shock genes (in Drosophila) are especially prone to mutagenesis via P elements in nature. Thus in nature, P elements create significant and distinctive variation in heat-shock genes, upon which evolutionary processes may act.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Geographic Origins of D. melanogaster Populations Screened in This Study
Screens revealed zero to 14 P elements per population (indicated by the number of squares), distinctive by insertion location, in the proximal promoter regions of genes examined (Table 1). Colors of squares correspond to gene set (see Introduction). Inset: Percentages of distinctive P elements discovered in Hsp70 genes and each of the three gene sets screened. A total of 161 P element insertions (the ten P elements in the coding sequence and the five non–P element insertions are not included in the figure). These tallies potentially under-report the actual number of P elements; see Results. F06 (Celera) is the strain whose genome has been sequenced [25] and is the reference strain for the present study. Populations F18, F50, and F52 (in light gray text) were removed from the analysis after screens failed for multiple genes and primer sets.
Figure 2
Figure 2. Locations of TEs Integrating into the Proximal Promoters of Hsp70 Genes
Six nearly identical Hsp70 genes are present in the sequenced Drosophila genome, but only five copies in natural populations. The locations of selected promoter elements and sites are indicated for all copies. (A) Previously discovered TEs and experimental transpositions relative to the conserved Hsp70 sequence. a, Jockey element in Hsp70Ba [16]; b, c, and d, P elements in Hsp70Ba [14,15,21]. An S element is present between the oppositely oriented paralogs Hsp70Aa and Hsp70Bb [13], and is represented twice, corresponding to its location relative to each paralog, as are the HMS Beagle (e) [16] and “56H8” (f) [88] elements inserted within it. Triangles below the line indicate transgene insertion sites (FlyBase; http://flybase.bio.indiana.edu). (B) and (C) Bottom: newly discovered TEs, with the natural population in which they were discovered (F01–F54, exclusive of F06) indicated for each. (B) TEs other than P elements. Four are Gypsy elements that have integrated into the S element in specific populations, the fifth is a Gypsy that has inserted into a Gypsy, and the sixth is a Jockey that has inserted into a P element. The Gypsys are arbitrarily plotted relative to Hsp70Ab and Hsp70Aa, respectively. (C) Natural P elements in Hsp70. The arrows indicating the number of independent EPgy2 insertion sites recently described by Shilova et al. [10]. Except for the Gypsys, TEs were not mapped to a specific Hsp70 gene. Insertion sites localized within the Hsp70 region were all established by sequencing. For population codes, see Figure 1.
Figure 3
Figure 3. Number of Natural P Element Insertions (161 Total) Distinctive by Population and Location into the “Proximal Promoter Region” of Each of the Screened Genes (Table 1)
Genes without any such insertions are not represented in the main figure. These tallies and estimates are conservative in three ways: (1) P elements inserting within 1,000 bp of the transcription start site of Bsg25D and CG6396 are included although they actually insert into neighboring genes (see Figure 4); (2) The tally for Hsp70 excludes non–P elements and those previously discovered (Figure 2), and divides the remaining total (44, light gray bar in background) by five, the Hsp70 copy number for natural populations [17]; and (3) Re-screening of a subset of insertions implies an underestimation of the tally at the 161 P insertion sites (see Results and Figure 7). Inset: frequencies of genes in each Gene Set (I, including Hsp70, II, and III) in which 0, 1, or >1 P elements had inserted.
Figure 4
Figure 4. Locations of P Elements Integrating into the Proximal Promoters of Heat-Shock Genes Other than Hsp70 (Gene Set I)
Data are plotted as in Figure 2 except as follows: F-numbers in columns refer to natural populations with transposons integrating at identical sites. Primer sets used in the screens amplified regions 3′ to transcription start site of different length; P elements discovered upstream of the initiator are plotted (pale), but not included in comparative analyses (i.e., in Hsp22 in population F31, Hsp68 in F05, and in Hsrω in F14). In Hsrω, numerous P elements were discovered in one region (box); a randomly chosen subset of these were localized (by sequencing) within that region (see enlargement). Several putative deletions were also discovered, and are plotted. For population codes see Figure 1. Table S1 provides additional information about Gene Set I and relevant sources for the organization of promoter regions.
Figure 5
Figure 5. Locations of P Elements Integrating into the Proximal Promoters of Non–Heat-Shock Genes Resembling Heat-Shock Genes in Relevant Features of Their Proximal Promoters (Gene Set II), and in Genes Dissimilar to Heat-Shock Genes (Gene Set III)
Data are plotted as in Figure 2 except as follows: Primer sets used in the screens amplified regions 3′ to transcription start site of different length; P elements discovered upstream of the initiator are plotted (pale), but not included in comparative analyses (i.e., in su(s) in population F17, in Act5C in F03 and F31, and in Elf in F43 and F54). Note that in Gene Set III, the two P elements discovered are not clearly associated with their focal genes, integrating into or just upstream of genes neighboring the focal genes. For population codes, see Figure 1. Table 1 provides additional information about the gene sets.
Figure 6
Figure 6. Frequencies of Experimental P Element Insertions Reported by FlyBase Database into the Proximal Promoter Regions of Each of the Genes Screened in Natural Populations in the Present Study
Data are plotted as in Figure 3. Note that insertions in the different Hsp70 copies are not combined as in Figure 3. The FlyBase database (http://flybase.bio.indiana.edu/transposons/) terms all tallied P element insertions as “transgene insertions.”
Figure 7
Figure 7. Distinctive P Elements Revealed by Re-screening a Random Sample of P Element Insertion Sites in Natural Populations for Four Genes, Hsp23, Hsp27, Hsrω, and Hsp70
The P element insertion sites were selected from Gene Set I. A plus sign (+) indicates successful PCR amplification with one primer complementary to the focal gene and another complementary to a unique sequence in the P element (top), and thus reports the size and orientation of the P element; a minus sign (−) indicates no amplification. Table S7 provides sequences of these primers. At each insertion site in a population, one to six distinctive P elements segregated; these are designated a–f. For Hsp23, Hsp27, and Hsrω, nine insertion sites shared by two or more natural populations (indicated by boxes) and 17 unique insertion sites were re-screened. Amplicons that share a symbol (filled square [█], filled triangle [▴], filled circle •], etc.) occurred at the same integration site in different populations and were indistinguishable by size or orientation. For Hsp70, a five-copy gene in natural populations [17], the specific gene of insertion was not determined; thus, each distinctive amplicon (a–f) could represent insertion(s) at the same site in one to five of the Hsp70 genes. For population codes see Figure 1. ORF, open reading frame.
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