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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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Differences in Number and Genomic Position of PREs betweenDrosophila Species(A) Dynamic scoring system for PRE predictions in pairs of genomes. Toppanel: for each predicted PRE from one species, its orthologous regionin another species, if present, is determined by BLAST search (see Materials and Methods). Around thisorthologous region, PREs are predicted in sequence areas of increasingsizes (1 kb, 10 kb, 20 kb, and chromosome-wide) and with increasingscore cutoffs (70, 104, 114, and 157). The diagram shows 1 kb and 10 kbas examples. Score plot, bottom right: PRE scores are calculated aroundthe orthologous region (marked “BLAST”), and theclosest region that scores above the relevant threshold is taken as theputative functional analog of the original PRE. In the example shown, noPRE scoring over 70 is found within 1 kb of the BLAST site. The searchis extended to 10 kb, and the closest PRE scoring over 104 (asterisk) istaken. Table, bottom left: score cutoffs increase with increasing searchradii (columns) and decreasing E-values (rows). Thecutoffs of 70, 104, and 114 correspond to an E-value of0.005 per search, such that 200 searches (about the number of PREspredicted in a genome-wide scan in D. melanogaster)correspond to an overall E-value of 1.0 (bottom row oftable).(B) Phylogenetic tree showing divergence times betweenDrosophila species. Adapted from http://flybase.bio.indiana.edu/static_pages/species/muller_synteny.html(C), Immunostaining with anti-PC antibody and (D) anti-H3K27me3antibody, on polytene chromosomes from four species.(E) Average band numbers from polytene chromosomes stained as in (C andD). Seven to ten genomes were counted per species and per antibody.Error bars show standard deviation.(F) Western blot with PC, PH, and H3K27me3 antibodies on equivalentquantities of protein extract from embryos of four species: D.melanogaster (Dm), D. pseudoobscura(Dp), D. simulans (Ds), and D.yakuba (Dy). Anti-histone H3 (FBgn0001199)is shown as loading control.(G) Distances between orthologous regions and predicted analogs (seeFigure 2A).Triangles: genome-wide–predicted D. melanogaster PREsversus D.yakuba analogs. Boxes: genome-wide–predictedD.melanogaster PREs versus D. pseudoobscuraanalogs. Diamonds: 1-kb sequences randomly chosen from theD.melanogaster genome versus D. pseudoobscura PREs.The numbers of random sequences on each chromosome equal the numbers ofPREs on that chromosome. Horizontal dotted line indicates a BLASTdistance of 1 kb. Vertical dotted lines indicate the number of PRE pairsin each category that have a BLAST distance of 1 kb.
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pbio-0060261-g002: Differences in Number and Genomic Position of PREs betweenDrosophila Species(A) Dynamic scoring system for PRE predictions in pairs of genomes. Toppanel: for each predicted PRE from one species, its orthologous regionin another species, if present, is determined by BLAST search (see Materials and Methods). Around thisorthologous region, PREs are predicted in sequence areas of increasingsizes (1 kb, 10 kb, 20 kb, and chromosome-wide) and with increasingscore cutoffs (70, 104, 114, and 157). The diagram shows 1 kb and 10 kbas examples. Score plot, bottom right: PRE scores are calculated aroundthe orthologous region (marked “BLAST”), and theclosest region that scores above the relevant threshold is taken as theputative functional analog of the original PRE. In the example shown, noPRE scoring over 70 is found within 1 kb of the BLAST site. The searchis extended to 10 kb, and the closest PRE scoring over 104 (asterisk) istaken. Table, bottom left: score cutoffs increase with increasing searchradii (columns) and decreasing E-values (rows). Thecutoffs of 70, 104, and 114 correspond to an E-value of0.005 per search, such that 200 searches (about the number of PREspredicted in a genome-wide scan in D. melanogaster)correspond to an overall E-value of 1.0 (bottom row oftable).(B) Phylogenetic tree showing divergence times betweenDrosophila species. Adapted from http://flybase.bio.indiana.edu/static_pages/species/muller_synteny.html(C), Immunostaining with anti-PC antibody and (D) anti-H3K27me3antibody, on polytene chromosomes from four species.(E) Average band numbers from polytene chromosomes stained as in (C andD). Seven to ten genomes were counted per species and per antibody.Error bars show standard deviation.(F) Western blot with PC, PH, and H3K27me3 antibodies on equivalentquantities of protein extract from embryos of four species: D.melanogaster (Dm), D. pseudoobscura(Dp), D. simulans (Ds), and D.yakuba (Dy). Anti-histone H3 (FBgn0001199)is shown as loading control.(G) Distances between orthologous regions and predicted analogs (seeFigure 2A).Triangles: genome-wide–predicted D. melanogaster PREsversus D.yakuba analogs. Boxes: genome-wide–predictedD.melanogaster PREs versus D. pseudoobscuraanalogs. Diamonds: 1-kb sequences randomly chosen from theD.melanogaster genome versus D. pseudoobscura PREs.The numbers of random sequences on each chromosome equal the numbers ofPREs on that chromosome. Horizontal dotted line indicates a BLASTdistance of 1 kb. Vertical dotted lines indicate the number of PRE pairsin each category that have a BLAST distance of 1 kb.

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)

Differences in Number and Genomic Position of PREs betweenDrosophila Species(A) Dynamic scoring system for PRE predictions in pairs of genomes. Toppanel: for each predicted PRE from one species, its orthologous regionin another species, if present, is determined by BLAST search (see Materials and Methods). Around thisorthologous region, PREs are predicted in sequence areas of increasingsizes (1 kb, 10 kb, 20 kb, and chromosome-wide) and with increasingscore cutoffs (70, 104, 114, and 157). The diagram shows 1 kb and 10 kbas examples. Score plot, bottom right: PRE scores are calculated aroundthe orthologous region (marked “BLAST”), and theclosest region that scores above the relevant threshold is taken as theputative functional analog of the original PRE. In the example shown, noPRE scoring over 70 is found within 1 kb of the BLAST site. The searchis extended to 10 kb, and the closest PRE scoring over 104 (asterisk) istaken. Table, bottom left: score cutoffs increase with increasing searchradii (columns) and decreasing E-values (rows). Thecutoffs of 70, 104, and 114 correspond to an E-value of0.005 per search, such that 200 searches (about the number of PREspredicted in a genome-wide scan in D. melanogaster)correspond to an overall E-value of 1.0 (bottom row oftable).(B) Phylogenetic tree showing divergence times betweenDrosophila species. Adapted from http://flybase.bio.indiana.edu/static_pages/species/muller_synteny.html(C), Immunostaining with anti-PC antibody and (D) anti-H3K27me3antibody, on polytene chromosomes from four species.(E) Average band numbers from polytene chromosomes stained as in (C andD). Seven to ten genomes were counted per species and per antibody.Error bars show standard deviation.(F) Western blot with PC, PH, and H3K27me3 antibodies on equivalentquantities of protein extract from embryos of four species: D.melanogaster (Dm), D. pseudoobscura(Dp), D. simulans (Ds), and D.yakuba (Dy). Anti-histone H3 (FBgn0001199)is shown as loading control.(G) Distances between orthologous regions and predicted analogs (seeFigure 2A).Triangles: genome-wide–predicted D. melanogaster PREsversus D.yakuba analogs. Boxes: genome-wide–predictedD.melanogaster PREs versus D. pseudoobscuraanalogs. Diamonds: 1-kb sequences randomly chosen from theD.melanogaster genome versus D. pseudoobscura PREs.The numbers of random sequences on each chromosome equal the numbers ofPREs on that chromosome. Horizontal dotted line indicates a BLASTdistance of 1 kb. Vertical dotted lines indicate the number of PRE pairsin each category that have a BLAST distance of 1 kb.
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Related In: Results  -  Collection

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

pbio-0060261-g002: Differences in Number and Genomic Position of PREs betweenDrosophila Species(A) Dynamic scoring system for PRE predictions in pairs of genomes. Toppanel: for each predicted PRE from one species, its orthologous regionin another species, if present, is determined by BLAST search (see Materials and Methods). Around thisorthologous region, PREs are predicted in sequence areas of increasingsizes (1 kb, 10 kb, 20 kb, and chromosome-wide) and with increasingscore cutoffs (70, 104, 114, and 157). The diagram shows 1 kb and 10 kbas examples. Score plot, bottom right: PRE scores are calculated aroundthe orthologous region (marked “BLAST”), and theclosest region that scores above the relevant threshold is taken as theputative functional analog of the original PRE. In the example shown, noPRE scoring over 70 is found within 1 kb of the BLAST site. The searchis extended to 10 kb, and the closest PRE scoring over 104 (asterisk) istaken. Table, bottom left: score cutoffs increase with increasing searchradii (columns) and decreasing E-values (rows). Thecutoffs of 70, 104, and 114 correspond to an E-value of0.005 per search, such that 200 searches (about the number of PREspredicted in a genome-wide scan in D. melanogaster)correspond to an overall E-value of 1.0 (bottom row oftable).(B) Phylogenetic tree showing divergence times betweenDrosophila species. Adapted from http://flybase.bio.indiana.edu/static_pages/species/muller_synteny.html(C), Immunostaining with anti-PC antibody and (D) anti-H3K27me3antibody, on polytene chromosomes from four species.(E) Average band numbers from polytene chromosomes stained as in (C andD). Seven to ten genomes were counted per species and per antibody.Error bars show standard deviation.(F) Western blot with PC, PH, and H3K27me3 antibodies on equivalentquantities of protein extract from embryos of four species: D.melanogaster (Dm), D. pseudoobscura(Dp), D. simulans (Ds), and D.yakuba (Dy). Anti-histone H3 (FBgn0001199)is shown as loading control.(G) Distances between orthologous regions and predicted analogs (seeFigure 2A).Triangles: genome-wide–predicted D. melanogaster PREsversus D.yakuba analogs. Boxes: genome-wide–predictedD.melanogaster PREs versus D. pseudoobscuraanalogs. Diamonds: 1-kb sequences randomly chosen from theD.melanogaster genome versus D. pseudoobscura PREs.The numbers of random sequences on each chromosome equal the numbers ofPREs on that chromosome. Horizontal dotted line indicates a BLASTdistance of 1 kb. Vertical dotted lines indicate the number of PRE pairsin each category that have a BLAST distance of 1 kb.
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