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. 2016 Aug 12;12(8):e1006249.
doi: 10.1371/journal.pgen.1006249. eCollection 2016 Aug.

Multiple Independent Retroelement Insertions in the Promoter of a Stress Response Gene Have Variable Molecular and Functional Effects in Drosophila

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Multiple Independent Retroelement Insertions in the Promoter of a Stress Response Gene Have Variable Molecular and Functional Effects in Drosophila

Miriam Merenciano et al. PLoS Genet. .

Abstract

Promoters are structurally and functionally diverse gene regulatory regions. The presence or absence of sequence motifs and the spacing between the motifs defines the properties of promoters. Recent alternative promoter usage analyses in Drosophila melanogaster revealed that transposable elements significantly contribute to promote diversity. In this work, we analyzed in detail one of the transposable element insertions, named FBti0019985, that has been co-opted to drive expression of CG18446, a candidate stress response gene. We analyzed strains from different natural populations and we found that besides FBti0019985, there are another eight independent transposable elements inserted in the proximal promoter region of CG18446. All nine insertions are solo-LTRs that belong to the roo family. We analyzed the sequence of the nine roo insertions and we investigated whether the different insertions were functionally equivalent by performing 5'-RACE, gene expression, and cold-stress survival experiments. We found that different insertions have different molecular and functional consequences. The exact position where the transposable elements are inserted matters, as they all showed highly conserved sequences but only two of the analyzed insertions provided alternative transcription start sites, and only the FBti0019985 insertion consistently affects CG18446 expression. The phenotypic consequences of the different insertions also vary: only FBti0019985 was associated with cold-stress tolerance. Interestingly, the only previous report of transposable elements inserting repeatedly and independently in a promoter region in D. melanogaster, were also located upstream of a stress response gene. Our results suggest that functional validation of individual structural variants is needed to resolve the complexity of insertion clusters.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. FBti0019985 is inserted in the first intron of cbx gene and it overlaps with CG18446 5’-UTR region.
Schematic representation of the genomic region where FBti0019985 is inserted: chromosome 2R: 9,864,510–9,875,072. FBti0019985 is shown in red. Black boxes represent exons and white boxes represent the 5’-UTRs and the 3’-UTRs. Primers used to check for the presence/absence of FBti0019985 are depicted as black arrows (FL, R, and L; see Materials and Methods).
Fig 2
Fig 2. Besides FBti0019985, eight other roo solo-LTR are inserted in the proximal promoter of CG18446.
Schematic representation of the genomic region where the nine roo solo-LTRs are inserted. roo insertions are depicted as red triangles. White boxes represent CG18446 5’-UTR. Regions depicted with dotted lines are not drawn to scale. Target Site Duplications (TSDs) are shown in blue. NC, allele frequency (%) in the North American population; IT, allele frequency (%) in the Italian population; SW, allele frequency (%) in the Swedish population; Out-AF total, allele frequency (%) in all the out-of-Africa populations; ZI, allele frequency (%) in the Zambia population.
Fig 3
Fig 3. Conserved regulatory regions in the the nine roo solo-LTR insertions and in the proximal promoter region of CG18446.
(A) Location of the nine transcription factor binding sites (green boxes), the Inr motif (blue box), and regions with matrix association potential (MARs) (black boxes), in the roo solo-LTR consensus sequence. Deaf1, ara, mirr and caup TFBS have been identified in this work. (B) Location of the eight transcription factor binding sites (green boxes) and the two core promoter motifs (blue boxes) in the proximal promoter region of CG18446. Different roo insertions are depicted as red triangles. The positions of roo+175 and roo+278 are not drawn to scale.
Fig 4
Fig 4. Mapping of piRNA reads and HP1a reads to the FBti0019985 region.
(A) Number of piRNA reads mapped to a 1.4 kb region including FBti0019985. aLi et al (2009) piRNA library, bSatyaki et al (2014) piRNA library and cShpiz et al (2014) piRNA library. (B) Number of HP1a reads mapped to the same 1.4 kb region.
Fig 5
Fig 5. FBti0019985 and roo+7 affect the transcription start site of CG18446.
Schematic representation of the results obtained using the 5’-RACE technique. Red boxes represent different roo insertions and white boxes represent CG18446 5’-UTRs. Partial transcripts obtained by 5’-RACE are depicted as grey lines. The region of the transcript that overlaps with a roo insertion is shown as a red line. The last 50 bp of FBti0019985 and roo+7 are included in the 5’-UTR of CG18446.
Fig 6
Fig 6. FBti0019985 is associated with changes in CG18446 expression.
Normalized CG18446 expression level relative to Act5C in embryos without roo insertion (grey) and in embryos with different roo insertions (red). (A) For FBti0019985, we compared the expression of CG18446 in flies with four different genetic backgrounds. In three backgrounds, the presence of FBti0019985 was associated with CG18446 upregulation. These results were significant in two backgrounds, RAL-810 and IV68, and marginally significant in the third background, RAL-639. (B) roo+7 was only associated with changes of expression in one of the three backgrounds analyzed: RAL-405. (C) roo-90 was also only associated with changes of expression in one of the three backgrounds analyzed: RAL-21. (D) Finally, roo-44 was not associated with changes in expression in any of the two backgrounds analyzed. Error bars represent the standard error of the mean (SEM) for the three biological replicates performed for each experiment.
Fig 7
Fig 7. Flies with FBti0019985 showed increased egg-to-adult viability under nonstress and under cold-stress conditions in three different genetic backgrounds.
Egg-to-adult viability of strains without FBti0019985 (grey) and with the FBti0019985 insertion (red) in nonstress (control) and in cold-stress conditions. Results of the three replicates performed with (A) RAL-783 and RAL-810, (B) RAL-908 and RAL-802, and (C) IV22 and IV68. Error bars represent the SEM of the different vials analyzed in each experiment.
Fig 8
Fig 8. Other roo solo-LTR insertions are not consistently associated with cold-stress resistant phenotypes.
Egg-to-adult viability in nonstress (control) and in cold-stress conditions of the RAL-783 strain without any of the nine roo insertions (grey) and of different strains with roo insertions (red). (A) RAL-405 (roo+7), (B) RAL-911 (roo+7), (C) RAL-21 (roo-90), (D) RAL-820 (roo-90), (E) RAL-195 (roo-44), (F) RAL-383 (roo-44), (G) RAL- 75 (roo-68), and (H) RAL-716 (roo-68). Error bars represent the SEM of the different vials analyzed in each experiment.

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Grants and funding

This work was supported by the Ministerio de Economía y Competitividad (MINECO: http://www.idi.mineco.gob.es) BFU-2011-24397 and RYC-2010-07306 to JG, and BES-2012-052999 to AU, Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional (MINECO/FEDER:http://www.idi.mineco.gob.es) BFU2014-57779-P, the European Commission (https://ec.europa.eu) FP7-PEOPLE-2011-CIG-293860, the Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya (http://agaur.gencat.cat) 2014-SGR-201 to JG, and by the Agence Nationale de la Recherche (http://www.agence-nationale-recherche.fr) ANR-14-CE02-0003 to MARdC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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