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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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Validation of PRE Prediction in Four Species(A) Score plots of PRE predictions in the Bithorax complex of fourDrosophila species (genome versions used in thiswork are: D.melanogaster: 4.0, D. simulans: 1.0,D.yakuba: 1.0, and D. pseudoobscura: 2.0).Coordinates of sequences used plotted from left to right of the figureare as follows: D.melanogaster: 12807958–12472096,D.simulans: 8665200–8999760, D. yakuba:12260626–12603034, and D. pseudoobscura:731941–359993. Positions of experimentally verified PREs andhomeotic gene promoters are shown above each plot. Asterisks show theregion of iab3 analysed in Figure 4E to 4G. (D. melanogasterposition 12663000).(B) ChIP analysis of Polycomb (PC; FBgn0003042) and Polyhomeotic (PH;FBgn0004861) enrichments on the bxd PRE in embryos offour species. Region analysed is shown in Figure 3A. Error bars indicatestandard deviation. Horizontal lines represent mean enrichments ofnegative control fragments that were present at detectable levels in allsamples.(C) Transgenic reporter assay for PRE function. All transgenic PREscomprise 1.6 kb centred on the region shown in Figure 3A. Top row: the D.melanogaster bxd PRE [4] was cloned upstream ofthe miniwhite reporter gene. As previously reported,the eyes of transgenic flies show variegation, pairing-sensitivesilencing (left panel), loss of silencing in a PcGmutant background (middle panel), and loss of activation in atrxG mutant background (right panel). Bottom threerows: miniwhite reporter constructs containing 1.6 kbof D.simulans, D.yakuba, and D. pseudoobscurasequences orthologous to the D. melanogaster bxd PREwere injected into D.melanogaster embryos. All show behaviour similar tothe D. melanogaster bxd PRE. Several independent lineswere analysed for each construct. Each photograph shows the line thatdisplayed the strongest effect.(D) The number of total lines tested that showed a given behaviour arelisted. PSS, pairing-sensitive silencing; var, variegation. ForDm, Ds, and Dp,few lines were recovered, due to strong silencing of theminiwhite transformation marker by thebxd PRE.
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pbio-0060261-g001: Validation of PRE Prediction in Four Species(A) Score plots of PRE predictions in the Bithorax complex of fourDrosophila species (genome versions used in thiswork are: D.melanogaster: 4.0, D. simulans: 1.0,D.yakuba: 1.0, and D. pseudoobscura: 2.0).Coordinates of sequences used plotted from left to right of the figureare as follows: D.melanogaster: 12807958–12472096,D.simulans: 8665200–8999760, D. yakuba:12260626–12603034, and D. pseudoobscura:731941–359993. Positions of experimentally verified PREs andhomeotic gene promoters are shown above each plot. Asterisks show theregion of iab3 analysed in Figure 4E to 4G. (D. melanogasterposition 12663000).(B) ChIP analysis of Polycomb (PC; FBgn0003042) and Polyhomeotic (PH;FBgn0004861) enrichments on the bxd PRE in embryos offour species. Region analysed is shown in Figure 3A. Error bars indicatestandard deviation. Horizontal lines represent mean enrichments ofnegative control fragments that were present at detectable levels in allsamples.(C) Transgenic reporter assay for PRE function. All transgenic PREscomprise 1.6 kb centred on the region shown in Figure 3A. Top row: the D.melanogaster bxd PRE [4] was cloned upstream ofthe miniwhite reporter gene. As previously reported,the eyes of transgenic flies show variegation, pairing-sensitivesilencing (left panel), loss of silencing in a PcGmutant background (middle panel), and loss of activation in atrxG mutant background (right panel). Bottom threerows: miniwhite reporter constructs containing 1.6 kbof D.simulans, D.yakuba, and D. pseudoobscurasequences orthologous to the D. melanogaster bxd PREwere injected into D.melanogaster embryos. All show behaviour similar tothe D. melanogaster bxd PRE. Several independent lineswere analysed for each construct. Each photograph shows the line thatdisplayed the strongest effect.(D) The number of total lines tested that showed a given behaviour arelisted. PSS, pairing-sensitive silencing; var, variegation. ForDm, Ds, and Dp,few lines were recovered, due to strong silencing of theminiwhite transformation marker by thebxd PRE.

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)

Validation of PRE Prediction in Four Species(A) Score plots of PRE predictions in the Bithorax complex of fourDrosophila species (genome versions used in thiswork are: D.melanogaster: 4.0, D. simulans: 1.0,D.yakuba: 1.0, and D. pseudoobscura: 2.0).Coordinates of sequences used plotted from left to right of the figureare as follows: D.melanogaster: 12807958–12472096,D.simulans: 8665200–8999760, D. yakuba:12260626–12603034, and D. pseudoobscura:731941–359993. Positions of experimentally verified PREs andhomeotic gene promoters are shown above each plot. Asterisks show theregion of iab3 analysed in Figure 4E to 4G. (D. melanogasterposition 12663000).(B) ChIP analysis of Polycomb (PC; FBgn0003042) and Polyhomeotic (PH;FBgn0004861) enrichments on the bxd PRE in embryos offour species. Region analysed is shown in Figure 3A. Error bars indicatestandard deviation. Horizontal lines represent mean enrichments ofnegative control fragments that were present at detectable levels in allsamples.(C) Transgenic reporter assay for PRE function. All transgenic PREscomprise 1.6 kb centred on the region shown in Figure 3A. Top row: the D.melanogaster bxd PRE [4] was cloned upstream ofthe miniwhite reporter gene. As previously reported,the eyes of transgenic flies show variegation, pairing-sensitivesilencing (left panel), loss of silencing in a PcGmutant background (middle panel), and loss of activation in atrxG mutant background (right panel). Bottom threerows: miniwhite reporter constructs containing 1.6 kbof D.simulans, D.yakuba, and D. pseudoobscurasequences orthologous to the D. melanogaster bxd PREwere injected into D.melanogaster embryos. All show behaviour similar tothe D. melanogaster bxd PRE. Several independent lineswere analysed for each construct. Each photograph shows the line thatdisplayed the strongest effect.(D) The number of total lines tested that showed a given behaviour arelisted. PSS, pairing-sensitive silencing; var, variegation. ForDm, Ds, and Dp,few lines were recovered, due to strong silencing of theminiwhite transformation marker by thebxd PRE.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0060261-g001: Validation of PRE Prediction in Four Species(A) Score plots of PRE predictions in the Bithorax complex of fourDrosophila species (genome versions used in thiswork are: D.melanogaster: 4.0, D. simulans: 1.0,D.yakuba: 1.0, and D. pseudoobscura: 2.0).Coordinates of sequences used plotted from left to right of the figureare as follows: D.melanogaster: 12807958–12472096,D.simulans: 8665200–8999760, D. yakuba:12260626–12603034, and D. pseudoobscura:731941–359993. Positions of experimentally verified PREs andhomeotic gene promoters are shown above each plot. Asterisks show theregion of iab3 analysed in Figure 4E to 4G. (D. melanogasterposition 12663000).(B) ChIP analysis of Polycomb (PC; FBgn0003042) and Polyhomeotic (PH;FBgn0004861) enrichments on the bxd PRE in embryos offour species. Region analysed is shown in Figure 3A. Error bars indicatestandard deviation. Horizontal lines represent mean enrichments ofnegative control fragments that were present at detectable levels in allsamples.(C) Transgenic reporter assay for PRE function. All transgenic PREscomprise 1.6 kb centred on the region shown in Figure 3A. Top row: the D.melanogaster bxd PRE [4] was cloned upstream ofthe miniwhite reporter gene. As previously reported,the eyes of transgenic flies show variegation, pairing-sensitivesilencing (left panel), loss of silencing in a PcGmutant background (middle panel), and loss of activation in atrxG mutant background (right panel). Bottom threerows: miniwhite reporter constructs containing 1.6 kbof D.simulans, D.yakuba, and D. pseudoobscurasequences orthologous to the D. melanogaster bxd PREwere injected into D.melanogaster embryos. All show behaviour similar tothe D. melanogaster bxd PRE. Several independent lineswere analysed for each construct. Each photograph shows the line thatdisplayed the strongest effect.(D) The number of total lines tested that showed a given behaviour arelisted. PSS, pairing-sensitive silencing; var, variegation. ForDm, Ds, and Dp,few lines were recovered, due to strong silencing of theminiwhite transformation marker by thebxd PRE.
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