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Evolutionary plasticity of polycomb/trithorax response elements in Drosophila species.

Hauenschild A, Ringrose L, Altmutter C, Paro R, Rehmsmeier M - PLoS Biol. (2008)

Bottom Line: Our results show that PRE evolution is extraordinarily dynamic.Finally, although it is theoretically possible that new elements can arise out of nonfunctional sequence, evidence that they do so is lacking.By demonstrating that PRE evolution is not limited to the adaptation of preexisting elements, these findings document a novel dimension of cis-regulatory evolution.

View Article: PubMed Central - PubMed

Affiliation: Universität Bielefeld, Center for Biotechnology CeBiTec, Bielefeld, Germany.

ABSTRACT
cis-Regulatory DNA elements contain multiple binding sites for activators and repressors of transcription. Among these elements are enhancers, which establish gene expression states, and Polycomb/Trithorax response elements (PREs), which take over from enhancers and maintain transcription states of several hundred developmentally important genes. PREs are essential to the correct identities of both stem cells and differentiated cells. Evolutionary differences in cis-regulatory elements are a rich source of phenotypic diversity, and functional binding sites within regulatory elements turn over rapidly in evolution. However, more radical evolutionary changes that go beyond motif turnover have been difficult to assess. We used a combination of genome-wide bioinformatic prediction and experimental validation at specific loci, to evaluate PRE evolution across four Drosophila species. Our results show that PRE evolution is extraordinarily dynamic. First, we show that the numbers of PREs differ dramatically between species. Second, we demonstrate that functional binding sites within PREs at conserved positions turn over rapidly in evolution, as has been observed for enhancer elements. Finally, although it is theoretically possible that new elements can arise out of nonfunctional sequence, evidence that they do so is lacking. We show here that functional PREs are found at nonorthologous sites in conserved gene loci. By demonstrating that PRE evolution is not limited to the adaptation of preexisting elements, these findings document a novel dimension of cis-regulatory evolution.

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PRE Function at Nonorthologous Regions(A and B) trachealess (trh). (A) PREprediction score plots for orthologous regions of thetrh locus in four species. Coordinates of sequencesshown from left to right of the figure are as follows: D. melanogaster:362619–382619, D.simulans: 8587236–8607236, D. yakuba:368751–388751, and D. pseudoobscura: 1070270–1066807. Thetrh transcription unit is shown. Black boxes 1 and2 at the top of each plot show sites analysed by ChIP in (B). (B) ChIPanalysis of PcG enrichments on sites 1 and 2, performed as in Figure 1. PCR primerswere designed to detect orthologous regions in all four species for eachsite.(C and D) decapentaplegic (dpp). (C)Score plots, as for (A), with boxes 1, 2, and 3 indicated. Coordinatesof sequences shown from left to right of the figure are as follows:D.melanogaster: 2454316–2459382, D. simulans:2391870–2418870, D. yakuba: 2451500–2478500, andD.pseudoobscura: 1720818–1747818. (D) ChIPanalysis of sites 1, 2, and 3 as for (B).(E, F, and G) D.pseudoobscura has a functional PRE in theiab3 region of the Bithorax complex that is absentin the other three species. Asterisks in Figure 1A indicate the region inquestion. (E) The predicted D. pseudoobscura PRE is shown (top). Theorthologous region was identified in the three other species by sequencealignment. Coordinates of sequences shown from left to right of thefigure are as follows: D.melanogaster: 12663856–12663307,D.simulans: 8809340–8809937. D. yakuba:12410998–12411614, and D. pseudoobscura:567475–566926. Motifs as in Figure 3A. Motifs that are notpresent in the D.melanogaster sequence are shown in red for the otherthree species. Conservation between D. melanogaster andD.pseudoobscura is marked as in Figure 3A. The D. melanogaster,D.simulans, and D. yakuba sequences areover 90% identical. Short insertions in the D. simulans andD.yakuba sequences with respect to D. melanogaster areshown as white boxes.(F, G, and H) the D.pseudoobscura PRE is functional, the orthologoussequences from other species are not. (F) ChIP analysis of PcGenrichments in embryos of four species on the regions shown in (E). (Gand H) transgenic reporter assay; 1.6 kb of either the predictedD.pseudoobscura PRE or the orthologous region fromD.melanogaster, centred on the region shown in (E), werecloned upstream of the miniwhite reporter gene andinjected into D.melanogaster embryos. (G) Top row: theD.pseudoobscura PRE shows variegation (middle panel, top),pairing-sensitive silencing (left panel), loss of silencing in aPcG mutant background (middle panel) and loss ofactivation in a trxG mutant background (right panel).Several independent lines were analysed for each construct. Eachphotograph shows the line that displayed the strongest effect. (H) Toprow: this behaviour was observed in several independent transgeniclines. PSS, pairing-sensitive silencing; var, variegation. (G and H)Bottom rows: the orthologous region from D. melanogaster hasnone of these properties.
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pbio-0060261-g004: PRE Function at Nonorthologous Regions(A and B) trachealess (trh). (A) PREprediction score plots for orthologous regions of thetrh locus in four species. Coordinates of sequencesshown from left to right of the figure are as follows: D. melanogaster:362619–382619, D.simulans: 8587236–8607236, D. yakuba:368751–388751, and D. pseudoobscura: 1070270–1066807. Thetrh transcription unit is shown. Black boxes 1 and2 at the top of each plot show sites analysed by ChIP in (B). (B) ChIPanalysis of PcG enrichments on sites 1 and 2, performed as in Figure 1. PCR primerswere designed to detect orthologous regions in all four species for eachsite.(C and D) decapentaplegic (dpp). (C)Score plots, as for (A), with boxes 1, 2, and 3 indicated. Coordinatesof sequences shown from left to right of the figure are as follows:D.melanogaster: 2454316–2459382, D. simulans:2391870–2418870, D. yakuba: 2451500–2478500, andD.pseudoobscura: 1720818–1747818. (D) ChIPanalysis of sites 1, 2, and 3 as for (B).(E, F, and G) D.pseudoobscura has a functional PRE in theiab3 region of the Bithorax complex that is absentin the other three species. Asterisks in Figure 1A indicate the region inquestion. (E) The predicted D. pseudoobscura PRE is shown (top). Theorthologous region was identified in the three other species by sequencealignment. Coordinates of sequences shown from left to right of thefigure are as follows: D.melanogaster: 12663856–12663307,D.simulans: 8809340–8809937. D. yakuba:12410998–12411614, and D. pseudoobscura:567475–566926. Motifs as in Figure 3A. Motifs that are notpresent in the D.melanogaster sequence are shown in red for the otherthree species. Conservation between D. melanogaster andD.pseudoobscura is marked as in Figure 3A. The D. melanogaster,D.simulans, and D. yakuba sequences areover 90% identical. Short insertions in the D. simulans andD.yakuba sequences with respect to D. melanogaster areshown as white boxes.(F, G, and H) the D.pseudoobscura PRE is functional, the orthologoussequences from other species are not. (F) ChIP analysis of PcGenrichments in embryos of four species on the regions shown in (E). (Gand H) transgenic reporter assay; 1.6 kb of either the predictedD.pseudoobscura PRE or the orthologous region fromD.melanogaster, centred on the region shown in (E), werecloned upstream of the miniwhite reporter gene andinjected into D.melanogaster embryos. (G) Top row: theD.pseudoobscura PRE shows variegation (middle panel, top),pairing-sensitive silencing (left panel), loss of silencing in aPcG mutant background (middle panel) and loss ofactivation in a trxG mutant background (right panel).Several independent lines were analysed for each construct. Eachphotograph shows the line that displayed the strongest effect. (H) Toprow: this behaviour was observed in several independent transgeniclines. PSS, pairing-sensitive silencing; var, variegation. (G and H)Bottom rows: the orthologous region from D. melanogaster hasnone of these properties.

Mentions: To assess the evolutionary behaviour of PREs independent of genome alignment, weapplied the algorithm to four Drosophila genomes:D. melanogaster,D. simulans,D. yakuba, andD. pseudoobscura. The algorithm was trained onD. melanogasterPRE sequences. Its performance on other Drosophila genomes wasconfirmed by comparison of PRE predictions in the homeotic Bithorax complexes ofall four species, showing that well-characterised PREs in D. melanogaster are alsopredicted with high significance at orthologous sites in the three other genomes(Figure 1A). Inaddition, antibodies raised against D. melanogaster PcG proteins were confirmed in the otherthree species by western blot (Figure 2F) and were used for ChIP. This analysis showed that PcGproteins were enriched on the predicted PREs of the Bithorax complex in embryosof all four species (Figures1B and S1, and unpublished data). Interestingly, Polycomb protein (PC) andPolyhomeotic protein (PH) were detected at similar levels on thebxd PRE in D.melanogaster, but at different levels on thebxd PRE in the other species (Figure 1B). Similar behaviour was alsodetected in other ChIP experiments (Figures 3C, 4B,and 4D). It is unlikely thatthese differences arise from different antibody affinities in the differentspecies, because both the PC and PH antibodies gave essentially identicalresults in western blots on embryonic extracts of the four species (Figure 2F). Furthermore, thedifferences in ChIP enrichments are not consistently higher for a given antibodyor species (see, for example, Figure 4B and 4D). We reason that these differences may arise from the fact that weused embryos for the ChIP experiments. The ChIP results represent an average ofbinding levels for a mixture of cell types, and a range of embryonic stages from0–16 h. We observed that embryonic development in the four speciesproceeds at slightly different rates, which would affect the distribution ofembryonic stages in a 0–16-h collection, and may therefore affect theobserved binding levels of PC and PH. Alternatively, the different binding of PCand PH may reflect different species-specific compositions of PcG complexes atdifferent PREs.


Evolutionary plasticity of polycomb/trithorax response elements in Drosophila species.

Hauenschild A, Ringrose L, Altmutter C, Paro R, Rehmsmeier M - PLoS Biol. (2008)

PRE Function at Nonorthologous Regions(A and B) trachealess (trh). (A) PREprediction score plots for orthologous regions of thetrh locus in four species. Coordinates of sequencesshown from left to right of the figure are as follows: D. melanogaster:362619–382619, D.simulans: 8587236–8607236, D. yakuba:368751–388751, and D. pseudoobscura: 1070270–1066807. Thetrh transcription unit is shown. Black boxes 1 and2 at the top of each plot show sites analysed by ChIP in (B). (B) ChIPanalysis of PcG enrichments on sites 1 and 2, performed as in Figure 1. PCR primerswere designed to detect orthologous regions in all four species for eachsite.(C and D) decapentaplegic (dpp). (C)Score plots, as for (A), with boxes 1, 2, and 3 indicated. Coordinatesof sequences shown from left to right of the figure are as follows:D.melanogaster: 2454316–2459382, D. simulans:2391870–2418870, D. yakuba: 2451500–2478500, andD.pseudoobscura: 1720818–1747818. (D) ChIPanalysis of sites 1, 2, and 3 as for (B).(E, F, and G) D.pseudoobscura has a functional PRE in theiab3 region of the Bithorax complex that is absentin the other three species. Asterisks in Figure 1A indicate the region inquestion. (E) The predicted D. pseudoobscura PRE is shown (top). Theorthologous region was identified in the three other species by sequencealignment. Coordinates of sequences shown from left to right of thefigure are as follows: D.melanogaster: 12663856–12663307,D.simulans: 8809340–8809937. D. yakuba:12410998–12411614, and D. pseudoobscura:567475–566926. Motifs as in Figure 3A. Motifs that are notpresent in the D.melanogaster sequence are shown in red for the otherthree species. Conservation between D. melanogaster andD.pseudoobscura is marked as in Figure 3A. The D. melanogaster,D.simulans, and D. yakuba sequences areover 90% identical. Short insertions in the D. simulans andD.yakuba sequences with respect to D. melanogaster areshown as white boxes.(F, G, and H) the D.pseudoobscura PRE is functional, the orthologoussequences from other species are not. (F) ChIP analysis of PcGenrichments in embryos of four species on the regions shown in (E). (Gand H) transgenic reporter assay; 1.6 kb of either the predictedD.pseudoobscura PRE or the orthologous region fromD.melanogaster, centred on the region shown in (E), werecloned upstream of the miniwhite reporter gene andinjected into D.melanogaster embryos. (G) Top row: theD.pseudoobscura PRE shows variegation (middle panel, top),pairing-sensitive silencing (left panel), loss of silencing in aPcG mutant background (middle panel) and loss ofactivation in a trxG mutant background (right panel).Several independent lines were analysed for each construct. Eachphotograph shows the line that displayed the strongest effect. (H) Toprow: this behaviour was observed in several independent transgeniclines. PSS, pairing-sensitive silencing; var, variegation. (G and H)Bottom rows: the orthologous region from D. melanogaster hasnone of these properties.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2573935&req=5

pbio-0060261-g004: PRE Function at Nonorthologous Regions(A and B) trachealess (trh). (A) PREprediction score plots for orthologous regions of thetrh locus in four species. Coordinates of sequencesshown from left to right of the figure are as follows: D. melanogaster:362619–382619, D.simulans: 8587236–8607236, D. yakuba:368751–388751, and D. pseudoobscura: 1070270–1066807. Thetrh transcription unit is shown. Black boxes 1 and2 at the top of each plot show sites analysed by ChIP in (B). (B) ChIPanalysis of PcG enrichments on sites 1 and 2, performed as in Figure 1. PCR primerswere designed to detect orthologous regions in all four species for eachsite.(C and D) decapentaplegic (dpp). (C)Score plots, as for (A), with boxes 1, 2, and 3 indicated. Coordinatesof sequences shown from left to right of the figure are as follows:D.melanogaster: 2454316–2459382, D. simulans:2391870–2418870, D. yakuba: 2451500–2478500, andD.pseudoobscura: 1720818–1747818. (D) ChIPanalysis of sites 1, 2, and 3 as for (B).(E, F, and G) D.pseudoobscura has a functional PRE in theiab3 region of the Bithorax complex that is absentin the other three species. Asterisks in Figure 1A indicate the region inquestion. (E) The predicted D. pseudoobscura PRE is shown (top). Theorthologous region was identified in the three other species by sequencealignment. Coordinates of sequences shown from left to right of thefigure are as follows: D.melanogaster: 12663856–12663307,D.simulans: 8809340–8809937. D. yakuba:12410998–12411614, and D. pseudoobscura:567475–566926. Motifs as in Figure 3A. Motifs that are notpresent in the D.melanogaster sequence are shown in red for the otherthree species. Conservation between D. melanogaster andD.pseudoobscura is marked as in Figure 3A. The D. melanogaster,D.simulans, and D. yakuba sequences areover 90% identical. Short insertions in the D. simulans andD.yakuba sequences with respect to D. melanogaster areshown as white boxes.(F, G, and H) the D.pseudoobscura PRE is functional, the orthologoussequences from other species are not. (F) ChIP analysis of PcGenrichments in embryos of four species on the regions shown in (E). (Gand H) transgenic reporter assay; 1.6 kb of either the predictedD.pseudoobscura PRE or the orthologous region fromD.melanogaster, centred on the region shown in (E), werecloned upstream of the miniwhite reporter gene andinjected into D.melanogaster embryos. (G) Top row: theD.pseudoobscura PRE shows variegation (middle panel, top),pairing-sensitive silencing (left panel), loss of silencing in aPcG mutant background (middle panel) and loss ofactivation in a trxG mutant background (right panel).Several independent lines were analysed for each construct. Eachphotograph shows the line that displayed the strongest effect. (H) Toprow: this behaviour was observed in several independent transgeniclines. PSS, pairing-sensitive silencing; var, variegation. (G and H)Bottom rows: the orthologous region from D. melanogaster hasnone of these properties.
Mentions: To assess the evolutionary behaviour of PREs independent of genome alignment, weapplied the algorithm to four Drosophila genomes:D. melanogaster,D. simulans,D. yakuba, andD. pseudoobscura. The algorithm was trained onD. melanogasterPRE sequences. Its performance on other Drosophila genomes wasconfirmed by comparison of PRE predictions in the homeotic Bithorax complexes ofall four species, showing that well-characterised PREs in D. melanogaster are alsopredicted with high significance at orthologous sites in the three other genomes(Figure 1A). Inaddition, antibodies raised against D. melanogaster PcG proteins were confirmed in the otherthree species by western blot (Figure 2F) and were used for ChIP. This analysis showed that PcGproteins were enriched on the predicted PREs of the Bithorax complex in embryosof all four species (Figures1B and S1, and unpublished data). Interestingly, Polycomb protein (PC) andPolyhomeotic protein (PH) were detected at similar levels on thebxd PRE in D.melanogaster, but at different levels on thebxd PRE in the other species (Figure 1B). Similar behaviour was alsodetected in other ChIP experiments (Figures 3C, 4B,and 4D). It is unlikely thatthese differences arise from different antibody affinities in the differentspecies, because both the PC and PH antibodies gave essentially identicalresults in western blots on embryonic extracts of the four species (Figure 2F). Furthermore, thedifferences in ChIP enrichments are not consistently higher for a given antibodyor species (see, for example, Figure 4B and 4D). We reason that these differences may arise from the fact that weused embryos for the ChIP experiments. The ChIP results represent an average ofbinding levels for a mixture of cell types, and a range of embryonic stages from0–16 h. We observed that embryonic development in the four speciesproceeds at slightly different rates, which would affect the distribution ofembryonic stages in a 0–16-h collection, and may therefore affect theobserved binding levels of PC and PH. Alternatively, the different binding of PCand PH may reflect different species-specific compositions of PcG complexes atdifferent PREs.

Bottom Line: Our results show that PRE evolution is extraordinarily dynamic.Finally, although it is theoretically possible that new elements can arise out of nonfunctional sequence, evidence that they do so is lacking.By demonstrating that PRE evolution is not limited to the adaptation of preexisting elements, these findings document a novel dimension of cis-regulatory evolution.

View Article: PubMed Central - PubMed

Affiliation: Universität Bielefeld, Center for Biotechnology CeBiTec, Bielefeld, Germany.

ABSTRACT
cis-Regulatory DNA elements contain multiple binding sites for activators and repressors of transcription. Among these elements are enhancers, which establish gene expression states, and Polycomb/Trithorax response elements (PREs), which take over from enhancers and maintain transcription states of several hundred developmentally important genes. PREs are essential to the correct identities of both stem cells and differentiated cells. Evolutionary differences in cis-regulatory elements are a rich source of phenotypic diversity, and functional binding sites within regulatory elements turn over rapidly in evolution. However, more radical evolutionary changes that go beyond motif turnover have been difficult to assess. We used a combination of genome-wide bioinformatic prediction and experimental validation at specific loci, to evaluate PRE evolution across four Drosophila species. Our results show that PRE evolution is extraordinarily dynamic. First, we show that the numbers of PREs differ dramatically between species. Second, we demonstrate that functional binding sites within PREs at conserved positions turn over rapidly in evolution, as has been observed for enhancer elements. Finally, although it is theoretically possible that new elements can arise out of nonfunctional sequence, evidence that they do so is lacking. We show here that functional PREs are found at nonorthologous sites in conserved gene loci. By demonstrating that PRE evolution is not limited to the adaptation of preexisting elements, these findings document a novel dimension of cis-regulatory evolution.

Show MeSH
Related in: MedlinePlus