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Evolution acts on enhancer organization to fine-tune gradient threshold readouts.

Crocker J, Tamori Y, Erives A - PLoS Biol. (2008)

Bottom Line: Furthermore, by precisely altering the organization of NEEs with different morphogen gradient threshold readouts, we show that CRM organizational evolution is sufficient for explaining changes in enhancer activity.Thus, evolution can act on CRM organization to fine-tune morphogen gradient threshold readouts over a wide dynamic range.Our study demonstrates that equivalence classes of CRMs are powerful tools for detecting lineage-specific adaptations by gene regulatory sequences.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Dartmouth College, Hanover, NH, USA.

ABSTRACT
The elucidation of principles governing evolution of gene regulatory sequence is critical to the study of metazoan diversification. We are therefore exploring the structure and organizational constraints of regulatory sequences by studying functionally equivalent cis-regulatory modules (CRMs) that have been evolving in parallel across several loci. Such an independent dataset allows a multi-locus study that is not hampered by nonfunctional or constrained homology. The neurogenic ectoderm enhancers (NEEs) of Drosophila melanogaster are one such class of coordinately regulated CRMs. The NEEs share a common organization of binding sites and as a set would be useful to study the relationship between CRM organization and CRM activity across evolving lineages. We used the D. melanogaster transgenic system to screen for functional adaptations in the NEEs from divergent drosophilid species. We show that the individual NEE modules across a genome in any one lineage have independently evolved adaptations to compensate for lineage-specific developmental and/or genomic changes. Specifically, we show that both the site composition and the site organization of NEEs have been finely tuned by distinct, lineage-specific selection pressures in each of the three divergent species that we have examined: D. melanogaster, D. pseudoobscura, and D. virilis. Furthermore, by precisely altering the organization of NEEs with different morphogen gradient threshold readouts, we show that CRM organizational evolution is sufficient for explaining changes in enhancer activity. Thus, evolution can act on CRM organization to fine-tune morphogen gradient threshold readouts over a wide dynamic range. Our study demonstrates that equivalence classes of CRMs are powerful tools for detecting lineage-specific adaptations by gene regulatory sequences.

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Precise Changes in NEE Organization Determine Lineage-Specific Threshold Readouts of Morphogen Gradient(A–E) Minimal modification of the D. melanogaster brk NEE configuration so that it resembles the D. virilis spacing (A) is sufficient to expand expression to levels seen for the D. virilis brk NEE-driven transgene (B–E). Asterisk (*) indicates that the spacing has been mutated in an otherwise wild-type D. melanogaster brk NEE.(F–J) A series of minimal modifications to the D. melanogaster vein (vn) NEE configuration (F) so that it differs by −1 bp, 0 bp (wild-type), +1 bp, and +2 bp, which is similar to the broadly expressed D. melanogaster sog NEE configuration, yields a series of monotonically increasing widths for lateral stripes of expression (G–J). The in situ staining experiments in this figure were conducted in parallel and with the same anti-sense lacZ probe to facilitate comparisons.
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pbio-0060263-g006: Precise Changes in NEE Organization Determine Lineage-Specific Threshold Readouts of Morphogen Gradient(A–E) Minimal modification of the D. melanogaster brk NEE configuration so that it resembles the D. virilis spacing (A) is sufficient to expand expression to levels seen for the D. virilis brk NEE-driven transgene (B–E). Asterisk (*) indicates that the spacing has been mutated in an otherwise wild-type D. melanogaster brk NEE.(F–J) A series of minimal modifications to the D. melanogaster vein (vn) NEE configuration (F) so that it differs by −1 bp, 0 bp (wild-type), +1 bp, and +2 bp, which is similar to the broadly expressed D. melanogaster sog NEE configuration, yields a series of monotonically increasing widths for lateral stripes of expression (G–J). The in situ staining experiments in this figure were conducted in parallel and with the same anti-sense lacZ probe to facilitate comparisons.

Mentions: For example, the Drosophila brk NEEs have a conserved organization consisting of a central invariant Dorsal site flanked on either side by invariant CA-core E-box motifs (5′-CACATGT) (Figure 2 details 17–19, and Figure 6A). However, the D. virilis Dorsal to E-box spacer is shorter by exactly 3 bp on either side of the Dorsal motif relative to the D. melanogaster NEE (Figures 2 and 6A), in addition to many other substitutions and insertions and deletions (indels) throughout these enhancers. Recall that while the D. melanogaster brk NEE drives a lateral stripe of about eight or nine nuclei wide, the D. virilis brk NEE drives a lateral stripe of ∼13 nuclei in D. melanogaster stage 5(2) embryos (Figure 6B–6D). We therefore reduced the D. melanogaster NEE Dorsal site to E-box spacers by 3 bp on each side, mimicking the D. virilis configuration. This precise adjustment in spacing is sufficient to broaden the expression of the D. melanogaster brk NEE driven transgene to D. virilis brk NEE levels (Figure 6B and 6E). These in situ detection experiments were conducted in parallel to aid comparison. Furthermore, double labeling with probes to the mesodermal marker snail and the lacZ transgene shows that this functional change extends to both intensity of expression as well as expansion of the dorsal border of expression (Figure 7). However, even after normalizing the peak concentrations, a measurable difference in width is still evident (compare Figure 7D and 7E). These in situ detection experiments were also conducted in parallel to aid comparison.


Evolution acts on enhancer organization to fine-tune gradient threshold readouts.

Crocker J, Tamori Y, Erives A - PLoS Biol. (2008)

Precise Changes in NEE Organization Determine Lineage-Specific Threshold Readouts of Morphogen Gradient(A–E) Minimal modification of the D. melanogaster brk NEE configuration so that it resembles the D. virilis spacing (A) is sufficient to expand expression to levels seen for the D. virilis brk NEE-driven transgene (B–E). Asterisk (*) indicates that the spacing has been mutated in an otherwise wild-type D. melanogaster brk NEE.(F–J) A series of minimal modifications to the D. melanogaster vein (vn) NEE configuration (F) so that it differs by −1 bp, 0 bp (wild-type), +1 bp, and +2 bp, which is similar to the broadly expressed D. melanogaster sog NEE configuration, yields a series of monotonically increasing widths for lateral stripes of expression (G–J). The in situ staining experiments in this figure were conducted in parallel and with the same anti-sense lacZ probe to facilitate comparisons.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2577699&req=5

pbio-0060263-g006: Precise Changes in NEE Organization Determine Lineage-Specific Threshold Readouts of Morphogen Gradient(A–E) Minimal modification of the D. melanogaster brk NEE configuration so that it resembles the D. virilis spacing (A) is sufficient to expand expression to levels seen for the D. virilis brk NEE-driven transgene (B–E). Asterisk (*) indicates that the spacing has been mutated in an otherwise wild-type D. melanogaster brk NEE.(F–J) A series of minimal modifications to the D. melanogaster vein (vn) NEE configuration (F) so that it differs by −1 bp, 0 bp (wild-type), +1 bp, and +2 bp, which is similar to the broadly expressed D. melanogaster sog NEE configuration, yields a series of monotonically increasing widths for lateral stripes of expression (G–J). The in situ staining experiments in this figure were conducted in parallel and with the same anti-sense lacZ probe to facilitate comparisons.
Mentions: For example, the Drosophila brk NEEs have a conserved organization consisting of a central invariant Dorsal site flanked on either side by invariant CA-core E-box motifs (5′-CACATGT) (Figure 2 details 17–19, and Figure 6A). However, the D. virilis Dorsal to E-box spacer is shorter by exactly 3 bp on either side of the Dorsal motif relative to the D. melanogaster NEE (Figures 2 and 6A), in addition to many other substitutions and insertions and deletions (indels) throughout these enhancers. Recall that while the D. melanogaster brk NEE drives a lateral stripe of about eight or nine nuclei wide, the D. virilis brk NEE drives a lateral stripe of ∼13 nuclei in D. melanogaster stage 5(2) embryos (Figure 6B–6D). We therefore reduced the D. melanogaster NEE Dorsal site to E-box spacers by 3 bp on each side, mimicking the D. virilis configuration. This precise adjustment in spacing is sufficient to broaden the expression of the D. melanogaster brk NEE driven transgene to D. virilis brk NEE levels (Figure 6B and 6E). These in situ detection experiments were conducted in parallel to aid comparison. Furthermore, double labeling with probes to the mesodermal marker snail and the lacZ transgene shows that this functional change extends to both intensity of expression as well as expansion of the dorsal border of expression (Figure 7). However, even after normalizing the peak concentrations, a measurable difference in width is still evident (compare Figure 7D and 7E). These in situ detection experiments were also conducted in parallel to aid comparison.

Bottom Line: Furthermore, by precisely altering the organization of NEEs with different morphogen gradient threshold readouts, we show that CRM organizational evolution is sufficient for explaining changes in enhancer activity.Thus, evolution can act on CRM organization to fine-tune morphogen gradient threshold readouts over a wide dynamic range.Our study demonstrates that equivalence classes of CRMs are powerful tools for detecting lineage-specific adaptations by gene regulatory sequences.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Dartmouth College, Hanover, NH, USA.

ABSTRACT
The elucidation of principles governing evolution of gene regulatory sequence is critical to the study of metazoan diversification. We are therefore exploring the structure and organizational constraints of regulatory sequences by studying functionally equivalent cis-regulatory modules (CRMs) that have been evolving in parallel across several loci. Such an independent dataset allows a multi-locus study that is not hampered by nonfunctional or constrained homology. The neurogenic ectoderm enhancers (NEEs) of Drosophila melanogaster are one such class of coordinately regulated CRMs. The NEEs share a common organization of binding sites and as a set would be useful to study the relationship between CRM organization and CRM activity across evolving lineages. We used the D. melanogaster transgenic system to screen for functional adaptations in the NEEs from divergent drosophilid species. We show that the individual NEE modules across a genome in any one lineage have independently evolved adaptations to compensate for lineage-specific developmental and/or genomic changes. Specifically, we show that both the site composition and the site organization of NEEs have been finely tuned by distinct, lineage-specific selection pressures in each of the three divergent species that we have examined: D. melanogaster, D. pseudoobscura, and D. virilis. Furthermore, by precisely altering the organization of NEEs with different morphogen gradient threshold readouts, we show that CRM organizational evolution is sufficient for explaining changes in enhancer activity. Thus, evolution can act on CRM organization to fine-tune morphogen gradient threshold readouts over a wide dynamic range. Our study demonstrates that equivalence classes of CRMs are powerful tools for detecting lineage-specific adaptations by gene regulatory sequences.

Show MeSH