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Functional evolution of a cis-regulatory module.

Ludwig MZ, Palsson A, Alekseeva E, Bergman CM, Nathan J, Kreitman M - PLoS Biol. (2005)

Bottom Line: The evolution of this enhancer is non-clock-like, with important functional differences between closely related species and functional convergence between distantly related species.Functional divergence is attributable to differences in activation levels rather than spatiotemporal control of gene expression.Our findings have implications for understanding enhancer structure-function, mechanisms of speciation and computational identification of regulatory modules.

View Article: PubMed Central - PubMed

Affiliation: Department of Ecology and Evolution, University of Chicago, Illinois, USA. mludwig@midway.uchicago.edu <mludwig@midway.uchicago.edu>

ABSTRACT
Lack of knowledge about how regulatory regions evolve in relation to their structure-function may limit the utility of comparative sequence analysis in deciphering cis-regulatory sequences. To address this we applied reverse genetics to carry out a functional genetic complementation analysis of a eukaryotic cis-regulatory module-the even-skipped stripe 2 enhancer-from four Drosophila species. The evolution of this enhancer is non-clock-like, with important functional differences between closely related species and functional convergence between distantly related species. Functional divergence is attributable to differences in activation levels rather than spatiotemporal control of gene expression. Our findings have implications for understanding enhancer structure-function, mechanisms of speciation and computational identification of regulatory modules.

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Developmental Series of EVE Abundance(A–E) Immunofluorescence labeling of time-staged early EVEΔS2E homozygous embryos. This developmental sequence, which corresponds roughly from the initialization of cellularization (A) to its completion (E), takes approximately 45 min at 25 oC in wild-type flies [41].(F) Expression of en in same genotype at stage 10. Arrows mark third and fourth en stripes. Note the short interval between en stripes 3 and 4 (parasegment 3) and the reduced fourth stripe.(G) EVE expression in stripe 2 during the developmental series around cellularization, where times 1–5 correspond to pictures in A–E. Stage 1 is early cellularization, while the process has been completed for embryos in class 5. The series is comparable to time classes 4–8 on the FlyEx Web site (http://flyex.ams.sunysb.edu/flyex/) [34]. Estimated least square means (± SE) for EVEΔS2E/Cy stock and wild-type line w1118; note the Cy/Cy homozygote is essentially wild-type. Early eve pair-rule expression is not known to be autoregulated (as occurs in postcellularization stages), and we observe a 2-fold difference in early stripe expression, with an additive component (a) of 0.62 and negligible dominance deviation (d/a) = 0.01, for the first two stages. This dosage dependency is lost after the cellularization stage (3), presumably because all embryos carry two copies of the autoregulatory element.
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pbio-0030093-g003: Developmental Series of EVE Abundance(A–E) Immunofluorescence labeling of time-staged early EVEΔS2E homozygous embryos. This developmental sequence, which corresponds roughly from the initialization of cellularization (A) to its completion (E), takes approximately 45 min at 25 oC in wild-type flies [41].(F) Expression of en in same genotype at stage 10. Arrows mark third and fourth en stripes. Note the short interval between en stripes 3 and 4 (parasegment 3) and the reduced fourth stripe.(G) EVE expression in stripe 2 during the developmental series around cellularization, where times 1–5 correspond to pictures in A–E. Stage 1 is early cellularization, while the process has been completed for embryos in class 5. The series is comparable to time classes 4–8 on the FlyEx Web site (http://flyex.ams.sunysb.edu/flyex/) [34]. Estimated least square means (± SE) for EVEΔS2E/Cy stock and wild-type line w1118; note the Cy/Cy homozygote is essentially wild-type. Early eve pair-rule expression is not known to be autoregulated (as occurs in postcellularization stages), and we observe a 2-fold difference in early stripe expression, with an additive component (a) of 0.62 and negligible dominance deviation (d/a) = 0.01, for the first two stages. This dosage dependency is lost after the cellularization stage (3), presumably because all embryos carry two copies of the autoregulatory element.

Mentions: We created the eve S2E deficiency mutant by removing a 480-bp fragment corresponding to the minimal stripe 2 element (MSE; see Figure S1) from a 15-kb cloned copy of the eve locus [12]. A transgene containing the complete fragment is capable of rescuing eve mutant flies to fertile adulthood [12]. EVEΔS2E is functionally a allele for stripe 2, as evidenced by the expression of the segment polarity gene, engrailed (en). Establishment of en 14-stripe pattern is a complex process that includes involvement by eve early stripes [13,14]. Eve stripe 2 corresponds to parasegment 3, which is bordered by en stripes 3 and 4. We hypothesized that these en stripes might be developmental indicators of early eve stripe 2 expression. Indeed EVEΔS2E embryos lacking a functional S2E (Figure 3A–3F) produce a short parasegment 3 and vestigial en stripe 4 (Figure 3F). This defect alone is almost certainly a lethal condition.


Functional evolution of a cis-regulatory module.

Ludwig MZ, Palsson A, Alekseeva E, Bergman CM, Nathan J, Kreitman M - PLoS Biol. (2005)

Developmental Series of EVE Abundance(A–E) Immunofluorescence labeling of time-staged early EVEΔS2E homozygous embryos. This developmental sequence, which corresponds roughly from the initialization of cellularization (A) to its completion (E), takes approximately 45 min at 25 oC in wild-type flies [41].(F) Expression of en in same genotype at stage 10. Arrows mark third and fourth en stripes. Note the short interval between en stripes 3 and 4 (parasegment 3) and the reduced fourth stripe.(G) EVE expression in stripe 2 during the developmental series around cellularization, where times 1–5 correspond to pictures in A–E. Stage 1 is early cellularization, while the process has been completed for embryos in class 5. The series is comparable to time classes 4–8 on the FlyEx Web site (http://flyex.ams.sunysb.edu/flyex/) [34]. Estimated least square means (± SE) for EVEΔS2E/Cy stock and wild-type line w1118; note the Cy/Cy homozygote is essentially wild-type. Early eve pair-rule expression is not known to be autoregulated (as occurs in postcellularization stages), and we observe a 2-fold difference in early stripe expression, with an additive component (a) of 0.62 and negligible dominance deviation (d/a) = 0.01, for the first two stages. This dosage dependency is lost after the cellularization stage (3), presumably because all embryos carry two copies of the autoregulatory element.
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Related In: Results  -  Collection

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pbio-0030093-g003: Developmental Series of EVE Abundance(A–E) Immunofluorescence labeling of time-staged early EVEΔS2E homozygous embryos. This developmental sequence, which corresponds roughly from the initialization of cellularization (A) to its completion (E), takes approximately 45 min at 25 oC in wild-type flies [41].(F) Expression of en in same genotype at stage 10. Arrows mark third and fourth en stripes. Note the short interval between en stripes 3 and 4 (parasegment 3) and the reduced fourth stripe.(G) EVE expression in stripe 2 during the developmental series around cellularization, where times 1–5 correspond to pictures in A–E. Stage 1 is early cellularization, while the process has been completed for embryos in class 5. The series is comparable to time classes 4–8 on the FlyEx Web site (http://flyex.ams.sunysb.edu/flyex/) [34]. Estimated least square means (± SE) for EVEΔS2E/Cy stock and wild-type line w1118; note the Cy/Cy homozygote is essentially wild-type. Early eve pair-rule expression is not known to be autoregulated (as occurs in postcellularization stages), and we observe a 2-fold difference in early stripe expression, with an additive component (a) of 0.62 and negligible dominance deviation (d/a) = 0.01, for the first two stages. This dosage dependency is lost after the cellularization stage (3), presumably because all embryos carry two copies of the autoregulatory element.
Mentions: We created the eve S2E deficiency mutant by removing a 480-bp fragment corresponding to the minimal stripe 2 element (MSE; see Figure S1) from a 15-kb cloned copy of the eve locus [12]. A transgene containing the complete fragment is capable of rescuing eve mutant flies to fertile adulthood [12]. EVEΔS2E is functionally a allele for stripe 2, as evidenced by the expression of the segment polarity gene, engrailed (en). Establishment of en 14-stripe pattern is a complex process that includes involvement by eve early stripes [13,14]. Eve stripe 2 corresponds to parasegment 3, which is bordered by en stripes 3 and 4. We hypothesized that these en stripes might be developmental indicators of early eve stripe 2 expression. Indeed EVEΔS2E embryos lacking a functional S2E (Figure 3A–3F) produce a short parasegment 3 and vestigial en stripe 4 (Figure 3F). This defect alone is almost certainly a lethal condition.

Bottom Line: The evolution of this enhancer is non-clock-like, with important functional differences between closely related species and functional convergence between distantly related species.Functional divergence is attributable to differences in activation levels rather than spatiotemporal control of gene expression.Our findings have implications for understanding enhancer structure-function, mechanisms of speciation and computational identification of regulatory modules.

View Article: PubMed Central - PubMed

Affiliation: Department of Ecology and Evolution, University of Chicago, Illinois, USA. mludwig@midway.uchicago.edu <mludwig@midway.uchicago.edu>

ABSTRACT
Lack of knowledge about how regulatory regions evolve in relation to their structure-function may limit the utility of comparative sequence analysis in deciphering cis-regulatory sequences. To address this we applied reverse genetics to carry out a functional genetic complementation analysis of a eukaryotic cis-regulatory module-the even-skipped stripe 2 enhancer-from four Drosophila species. The evolution of this enhancer is non-clock-like, with important functional differences between closely related species and functional convergence between distantly related species. Functional divergence is attributable to differences in activation levels rather than spatiotemporal control of gene expression. Our findings have implications for understanding enhancer structure-function, mechanisms of speciation and computational identification of regulatory modules.

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