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Identification and analysis of evolutionary selection pressures acting at the molecular level in five forkhead subfamilies.

Fetterman CD, Rannala B, Walter MA - BMC Evol. Biol. (2008)

Bottom Line: Branch-site models, which allow estimated selection pressures along specified lineages to vary as compared to the remaining phylogeny, identified positive selection along branches leading to the FoxA3 and Protostomia clades in the FoxA cluster and the branch leading to the FoxO3 clade in the FoxO cluster.Consideration of selection pressures observed in conjunction with known functional information allowed prediction of residue function and refinement of domain boundaries.Identification of residues that differentiate orthologs and paralogs provided insight into the development and functional consequences of paralogs and forkhead subfamily composition differences among species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada. christina.fetterman@ualberta.ca

ABSTRACT

Background: Members of the forkhead gene family act as transcription regulators in biological processes including development and metabolism. The evolution of forkhead genes has not been widely examined and selection pressures at the molecular level influencing subfamily evolution and differentiation have not been explored. Here, in silico methods were used to examine selection pressures acting on the coding sequence of five multi-species FOX protein subfamily clusters; FoxA, FoxD, FoxI, FoxO and FoxP.

Results: Application of site models, which estimate overall selection pressures on individual codons throughout the phylogeny, showed that the amino acid changes observed were either neutral or under negative selection. Branch-site models, which allow estimated selection pressures along specified lineages to vary as compared to the remaining phylogeny, identified positive selection along branches leading to the FoxA3 and Protostomia clades in the FoxA cluster and the branch leading to the FoxO3 clade in the FoxO cluster. Residues that may differentiate paralogs were identified in the FoxA and FoxO clusters and residues that differentiate orthologs were identified in the FoxA cluster. Neutral amino acid changes were identified in the forkhead domain of the FoxA, FoxD and FoxP clusters while positive selection was identified in the forkhead domain of the Protostomia lineage of the FoxA cluster. A series of residues under strong negative selection adjacent to the N- and C-termini of the forkhead domain were identified in all clusters analyzed suggesting a new method for refinement of domain boundaries. Extrapolation of domains among cluster members in conjunction with selection pressure information allowed prediction of residue function in the FoxA, FoxO and FoxP clusters and exclusion of known domain function in residues of the FoxA and FoxI clusters.

Conclusion: Consideration of selection pressures observed in conjunction with known functional information allowed prediction of residue function and refinement of domain boundaries. Identification of residues that differentiate orthologs and paralogs provided insight into the development and functional consequences of paralogs and forkhead subfamily composition differences among species. Overall we found that after gene duplication of forkhead family members, rapid differentiation and subsequent fixation of amino acid changes through negative selection has occurred.

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Selection pressures on amino acids in each of the six clusters analyzed. In each cluster the forkhead domain position was obtained from the NCBI Entrez Protein [11] database record for that sequence (see Additional file 1 for accession numbers). A. FoxA cluster of 31 sequences. Peach: conserved domain IV, site 1 [36,74] Blue: conserved domain V, sites 2–7 [36,74] Green: forkhead domain, sites 25–101 Yellow: conserved domain II, sites 147–160 [72] Pink: conserved domain III, sites 174–177 [72] Black Bar: nuclear localization signal, sites 19–32 and 88–101 [74] Red Bar: EH1 motif, sites 151–160 [33,34]B. FoxD cluster of 24 sequences. Green: forkhead domain, sites 42–119 Acidic and alanine/proline rich regions described by Ernstsson et al. 1996; Sutton et al. 1996; Ernstsson et al. 1997; Freyaldenhoven, Fried, and Wielckens 2002. Red Bar: EH1 motif, sites 163–169 for all of the FOXD4s, sites 176–186 for FOXD1 and FOXD3 [33,34]C. FoxI cluster of 10 sequences. Green: forkhead domain, sites 123–200 Yellow: transactivation domain, sites 196–282 [78]D. FoxO cluster of 12 sequences. Green: forkhead domain, sites 61–141 Orange: nuclear export signal, sites 1–43 and 235–264 for FOXO1a, sites 244–252 and 258–266 for FOXO3a, sites 250–256 for FOXO4 [42,46,47,76] Black Bar: nuclear localization signal, sites 52–60 and 134–180 for FOXO1a, sites 152–154 and 173,174 for FOXO3a, sites 144–183 for FOXO4 [42,45,46] Yellow: transactivation domain, sites 389–428 for FOXO1a, sites 221–326 and 378–428 for FOXO4 [38,39] Blue Bar: phosphorylation site, sites 20, 157 and 216 for FOXO1a, FOXO3a and FOXO4, additionally site 218 for FOXO1a, sites 379 and 383 for FOXO4 [44,48-52,55,56]E. FoxP cluster of 10 sequences. Purple: glutamine rich region, sites 6–182 [58,61,79-81] Blue: zinc finger, sites 288–311 [58,60,61,81] Yellow: leucine zipper, sites 324–349 [58-61] Green: forkhead domain, sites 434–506 Grey Bar: region involved in repression, sites 1–505 [59,80] Red Bar: EH1 motif, sites 398–408 and 501–511 for FOXP1, sites 501–511 for FOXP2.
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Figure 2: Selection pressures on amino acids in each of the six clusters analyzed. In each cluster the forkhead domain position was obtained from the NCBI Entrez Protein [11] database record for that sequence (see Additional file 1 for accession numbers). A. FoxA cluster of 31 sequences. Peach: conserved domain IV, site 1 [36,74] Blue: conserved domain V, sites 2–7 [36,74] Green: forkhead domain, sites 25–101 Yellow: conserved domain II, sites 147–160 [72] Pink: conserved domain III, sites 174–177 [72] Black Bar: nuclear localization signal, sites 19–32 and 88–101 [74] Red Bar: EH1 motif, sites 151–160 [33,34]B. FoxD cluster of 24 sequences. Green: forkhead domain, sites 42–119 Acidic and alanine/proline rich regions described by Ernstsson et al. 1996; Sutton et al. 1996; Ernstsson et al. 1997; Freyaldenhoven, Fried, and Wielckens 2002. Red Bar: EH1 motif, sites 163–169 for all of the FOXD4s, sites 176–186 for FOXD1 and FOXD3 [33,34]C. FoxI cluster of 10 sequences. Green: forkhead domain, sites 123–200 Yellow: transactivation domain, sites 196–282 [78]D. FoxO cluster of 12 sequences. Green: forkhead domain, sites 61–141 Orange: nuclear export signal, sites 1–43 and 235–264 for FOXO1a, sites 244–252 and 258–266 for FOXO3a, sites 250–256 for FOXO4 [42,46,47,76] Black Bar: nuclear localization signal, sites 52–60 and 134–180 for FOXO1a, sites 152–154 and 173,174 for FOXO3a, sites 144–183 for FOXO4 [42,45,46] Yellow: transactivation domain, sites 389–428 for FOXO1a, sites 221–326 and 378–428 for FOXO4 [38,39] Blue Bar: phosphorylation site, sites 20, 157 and 216 for FOXO1a, FOXO3a and FOXO4, additionally site 218 for FOXO1a, sites 379 and 383 for FOXO4 [44,48-52,55,56]E. FoxP cluster of 10 sequences. Purple: glutamine rich region, sites 6–182 [58,61,79-81] Blue: zinc finger, sites 288–311 [58,60,61,81] Yellow: leucine zipper, sites 324–349 [58-61] Green: forkhead domain, sites 434–506 Grey Bar: region involved in repression, sites 1–505 [59,80] Red Bar: EH1 motif, sites 398–408 and 501–511 for FOXP1, sites 501–511 for FOXP2.

Mentions: Codon site models M0, M1a, M2a, M3, M7 and M8 were implemented in codeml for each of the six clusters and compared using likelihood ratio tests. For each cluster the M3 vs. M0 LRT was significant (Table 3), indicating that one category of ω was insufficient to describe the variability in selection pressure across amino acid sites. LRTs testing for positive selection, M2a vs M1a and M8 vs M7, were also insignificant for each cluster (Table 3), therefore the amino acid changes within each cluster are neutral or under negative selection. Table 4 reports the parameter estimates for the least parameter rich model, M1a, which best describes the variation in selection pressures across sites. Graphs were constructed showing the posterior weighted ω, the mean of ω over the site classes weighted by the posterior probability of each class, of each residue analyzed (Figure 2). Since ambiguous sites were removed, the residue numbers along the bottom of the graphs do not correspond to residue numbers of the analyzed sequences. Underneath each graph is a cartoon of the important regions contained in human forkhead gene(s) within that cluster. Few functional regions have been examined in human FoxA and FoxP proteins therefore functional information identified in rat and mouse protein studies has been included in the FoxA and FoxP figures respectively. The location of the forkhead domain for each human sequence was taken from the NCBI Entrez Protein [11] database record for that sequence.


Identification and analysis of evolutionary selection pressures acting at the molecular level in five forkhead subfamilies.

Fetterman CD, Rannala B, Walter MA - BMC Evol. Biol. (2008)

Selection pressures on amino acids in each of the six clusters analyzed. In each cluster the forkhead domain position was obtained from the NCBI Entrez Protein [11] database record for that sequence (see Additional file 1 for accession numbers). A. FoxA cluster of 31 sequences. Peach: conserved domain IV, site 1 [36,74] Blue: conserved domain V, sites 2–7 [36,74] Green: forkhead domain, sites 25–101 Yellow: conserved domain II, sites 147–160 [72] Pink: conserved domain III, sites 174–177 [72] Black Bar: nuclear localization signal, sites 19–32 and 88–101 [74] Red Bar: EH1 motif, sites 151–160 [33,34]B. FoxD cluster of 24 sequences. Green: forkhead domain, sites 42–119 Acidic and alanine/proline rich regions described by Ernstsson et al. 1996; Sutton et al. 1996; Ernstsson et al. 1997; Freyaldenhoven, Fried, and Wielckens 2002. Red Bar: EH1 motif, sites 163–169 for all of the FOXD4s, sites 176–186 for FOXD1 and FOXD3 [33,34]C. FoxI cluster of 10 sequences. Green: forkhead domain, sites 123–200 Yellow: transactivation domain, sites 196–282 [78]D. FoxO cluster of 12 sequences. Green: forkhead domain, sites 61–141 Orange: nuclear export signal, sites 1–43 and 235–264 for FOXO1a, sites 244–252 and 258–266 for FOXO3a, sites 250–256 for FOXO4 [42,46,47,76] Black Bar: nuclear localization signal, sites 52–60 and 134–180 for FOXO1a, sites 152–154 and 173,174 for FOXO3a, sites 144–183 for FOXO4 [42,45,46] Yellow: transactivation domain, sites 389–428 for FOXO1a, sites 221–326 and 378–428 for FOXO4 [38,39] Blue Bar: phosphorylation site, sites 20, 157 and 216 for FOXO1a, FOXO3a and FOXO4, additionally site 218 for FOXO1a, sites 379 and 383 for FOXO4 [44,48-52,55,56]E. FoxP cluster of 10 sequences. Purple: glutamine rich region, sites 6–182 [58,61,79-81] Blue: zinc finger, sites 288–311 [58,60,61,81] Yellow: leucine zipper, sites 324–349 [58-61] Green: forkhead domain, sites 434–506 Grey Bar: region involved in repression, sites 1–505 [59,80] Red Bar: EH1 motif, sites 398–408 and 501–511 for FOXP1, sites 501–511 for FOXP2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: Selection pressures on amino acids in each of the six clusters analyzed. In each cluster the forkhead domain position was obtained from the NCBI Entrez Protein [11] database record for that sequence (see Additional file 1 for accession numbers). A. FoxA cluster of 31 sequences. Peach: conserved domain IV, site 1 [36,74] Blue: conserved domain V, sites 2–7 [36,74] Green: forkhead domain, sites 25–101 Yellow: conserved domain II, sites 147–160 [72] Pink: conserved domain III, sites 174–177 [72] Black Bar: nuclear localization signal, sites 19–32 and 88–101 [74] Red Bar: EH1 motif, sites 151–160 [33,34]B. FoxD cluster of 24 sequences. Green: forkhead domain, sites 42–119 Acidic and alanine/proline rich regions described by Ernstsson et al. 1996; Sutton et al. 1996; Ernstsson et al. 1997; Freyaldenhoven, Fried, and Wielckens 2002. Red Bar: EH1 motif, sites 163–169 for all of the FOXD4s, sites 176–186 for FOXD1 and FOXD3 [33,34]C. FoxI cluster of 10 sequences. Green: forkhead domain, sites 123–200 Yellow: transactivation domain, sites 196–282 [78]D. FoxO cluster of 12 sequences. Green: forkhead domain, sites 61–141 Orange: nuclear export signal, sites 1–43 and 235–264 for FOXO1a, sites 244–252 and 258–266 for FOXO3a, sites 250–256 for FOXO4 [42,46,47,76] Black Bar: nuclear localization signal, sites 52–60 and 134–180 for FOXO1a, sites 152–154 and 173,174 for FOXO3a, sites 144–183 for FOXO4 [42,45,46] Yellow: transactivation domain, sites 389–428 for FOXO1a, sites 221–326 and 378–428 for FOXO4 [38,39] Blue Bar: phosphorylation site, sites 20, 157 and 216 for FOXO1a, FOXO3a and FOXO4, additionally site 218 for FOXO1a, sites 379 and 383 for FOXO4 [44,48-52,55,56]E. FoxP cluster of 10 sequences. Purple: glutamine rich region, sites 6–182 [58,61,79-81] Blue: zinc finger, sites 288–311 [58,60,61,81] Yellow: leucine zipper, sites 324–349 [58-61] Green: forkhead domain, sites 434–506 Grey Bar: region involved in repression, sites 1–505 [59,80] Red Bar: EH1 motif, sites 398–408 and 501–511 for FOXP1, sites 501–511 for FOXP2.
Mentions: Codon site models M0, M1a, M2a, M3, M7 and M8 were implemented in codeml for each of the six clusters and compared using likelihood ratio tests. For each cluster the M3 vs. M0 LRT was significant (Table 3), indicating that one category of ω was insufficient to describe the variability in selection pressure across amino acid sites. LRTs testing for positive selection, M2a vs M1a and M8 vs M7, were also insignificant for each cluster (Table 3), therefore the amino acid changes within each cluster are neutral or under negative selection. Table 4 reports the parameter estimates for the least parameter rich model, M1a, which best describes the variation in selection pressures across sites. Graphs were constructed showing the posterior weighted ω, the mean of ω over the site classes weighted by the posterior probability of each class, of each residue analyzed (Figure 2). Since ambiguous sites were removed, the residue numbers along the bottom of the graphs do not correspond to residue numbers of the analyzed sequences. Underneath each graph is a cartoon of the important regions contained in human forkhead gene(s) within that cluster. Few functional regions have been examined in human FoxA and FoxP proteins therefore functional information identified in rat and mouse protein studies has been included in the FoxA and FoxP figures respectively. The location of the forkhead domain for each human sequence was taken from the NCBI Entrez Protein [11] database record for that sequence.

Bottom Line: Branch-site models, which allow estimated selection pressures along specified lineages to vary as compared to the remaining phylogeny, identified positive selection along branches leading to the FoxA3 and Protostomia clades in the FoxA cluster and the branch leading to the FoxO3 clade in the FoxO cluster.Consideration of selection pressures observed in conjunction with known functional information allowed prediction of residue function and refinement of domain boundaries.Identification of residues that differentiate orthologs and paralogs provided insight into the development and functional consequences of paralogs and forkhead subfamily composition differences among species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada. christina.fetterman@ualberta.ca

ABSTRACT

Background: Members of the forkhead gene family act as transcription regulators in biological processes including development and metabolism. The evolution of forkhead genes has not been widely examined and selection pressures at the molecular level influencing subfamily evolution and differentiation have not been explored. Here, in silico methods were used to examine selection pressures acting on the coding sequence of five multi-species FOX protein subfamily clusters; FoxA, FoxD, FoxI, FoxO and FoxP.

Results: Application of site models, which estimate overall selection pressures on individual codons throughout the phylogeny, showed that the amino acid changes observed were either neutral or under negative selection. Branch-site models, which allow estimated selection pressures along specified lineages to vary as compared to the remaining phylogeny, identified positive selection along branches leading to the FoxA3 and Protostomia clades in the FoxA cluster and the branch leading to the FoxO3 clade in the FoxO cluster. Residues that may differentiate paralogs were identified in the FoxA and FoxO clusters and residues that differentiate orthologs were identified in the FoxA cluster. Neutral amino acid changes were identified in the forkhead domain of the FoxA, FoxD and FoxP clusters while positive selection was identified in the forkhead domain of the Protostomia lineage of the FoxA cluster. A series of residues under strong negative selection adjacent to the N- and C-termini of the forkhead domain were identified in all clusters analyzed suggesting a new method for refinement of domain boundaries. Extrapolation of domains among cluster members in conjunction with selection pressure information allowed prediction of residue function in the FoxA, FoxO and FoxP clusters and exclusion of known domain function in residues of the FoxA and FoxI clusters.

Conclusion: Consideration of selection pressures observed in conjunction with known functional information allowed prediction of residue function and refinement of domain boundaries. Identification of residues that differentiate orthologs and paralogs provided insight into the development and functional consequences of paralogs and forkhead subfamily composition differences among species. Overall we found that after gene duplication of forkhead family members, rapid differentiation and subsequent fixation of amino acid changes through negative selection has occurred.

Show MeSH