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The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans.

Camino EM, Butts JC, Ordway A, Vellky JE, Rebeiz M, Williams TM - PLoS Genet. (2015)

Bottom Line: Morphological traits result from gene regulatory networks (GRNs) that form a web of transcription factors, which regulate multiple cis-regulatory element (CRE) sequences to control the coordinated expression of differentiation genes.By elucidating how yellow and tan are connected to the web of abdominal trans-regulators, we discovered that the yellow and tan abdominal CREs are composed of distinct regulatory inputs that exhibit contrasting responses to the same Hox proteins and Hox cofactors.These results provide an example in which CRE origination underlies a recently evolved novel trait, and highlights how coordinated expression patterns can evolve in parallel through the generation of unique regulatory linkages.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Dayton, Dayton, Ohio, United States of America.

ABSTRACT
The origination and diversification of morphological characteristics represents a key problem in understanding the evolution of development. Morphological traits result from gene regulatory networks (GRNs) that form a web of transcription factors, which regulate multiple cis-regulatory element (CRE) sequences to control the coordinated expression of differentiation genes. The formation and modification of GRNs must ultimately be understood at the level of individual regulatory linkages (i.e., transcription factor binding sites within CREs) that constitute the network. Here, we investigate how elements within a network originated and diversified to generate a broad range of abdominal pigmentation phenotypes among Sophophora fruit flies. Our data indicates that the coordinated expression of two melanin synthesis enzymes, Yellow and Tan, recently evolved through novel CRE activities that respond to the spatial patterning inputs of Hox proteins and the sex-specific input of Bric-à-brac transcription factors. Once established, it seems that these newly evolved activities were repeatedly modified by evolutionary changes in the network's trans-regulators to generate large-scale changes in pigment pattern. By elucidating how yellow and tan are connected to the web of abdominal trans-regulators, we discovered that the yellow and tan abdominal CREs are composed of distinct regulatory inputs that exhibit contrasting responses to the same Hox proteins and Hox cofactors. These results provide an example in which CRE origination underlies a recently evolved novel trait, and highlights how coordinated expression patterns can evolve in parallel through the generation of unique regulatory linkages.

No MeSH data available.


Related in: MedlinePlus

Characterization of the direct Hox inputs shaping tan expression.(A) The SM6 region of the t_MSE possesses five sites, S1-S5, with sequences characteristic of Abd-B (TTAT) and Abd-A (TAAT) binding sites. This CRE region also possesses sites resembling sequences bound by Exd and Hth, though the functionality of the Exd site was not studied here. (B-F) EGFP reporter transgene expression in male pupae at ~95 hours after puparium formation. (B) The t_MSE2 sequence drives robust expression in the male A5 and A6 segments. When all of the (C) TTAT sites and (D) TTAT and TAAT sites depicted in (A) were mutated, regulatory activity in the male A5 segment was reduced to 89±6% and 88±3% respectively. (F) When the entire SM6 region was mutated, regulatory activity decreased to 31±1%. (C) The TTAT site mutations resulted in activity increasing in the A4 and A3 segments respectively to 169±4% and 261±6%. (D) The TTAT and TAAT site mutations resulted in activity increasing in the A4 and A3 segments respectively to 207±2% and 281±2%. (E) When the Hth site was mutated, regulatory activity in the male A5, A4, and A3 segments respectively increased to 122±7%, 236±6%, and 276±4%.(F) The entire SM6 region mutation resulted in activity decreasing in the A4 and A3 segments respectively to 64±1% and 73±1%. Blue arrowheads indicate segments where activity was notably increased and Red arrowheads indicate segments with notably decreased activity compared to the wild type sequence. Gel shift assays for sequences possessing wild type and mutant site (G) 3, (H) 4, and (I) 5 and the DNA-binding domains for Abd-A and Abd-B. Binding reactions used increasing amounts of the GST-DNA binding domain fusion protein (from left to right: 0 ng, 111 ng, 333 ng, 1000 ng, and 3000 ng). Binding correlated with the amount of input protein for the probes with the non-mutant sequence, whereas binding was dramatically reduced for the probes with a mutant Hox site.
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pgen.1005136.g006: Characterization of the direct Hox inputs shaping tan expression.(A) The SM6 region of the t_MSE possesses five sites, S1-S5, with sequences characteristic of Abd-B (TTAT) and Abd-A (TAAT) binding sites. This CRE region also possesses sites resembling sequences bound by Exd and Hth, though the functionality of the Exd site was not studied here. (B-F) EGFP reporter transgene expression in male pupae at ~95 hours after puparium formation. (B) The t_MSE2 sequence drives robust expression in the male A5 and A6 segments. When all of the (C) TTAT sites and (D) TTAT and TAAT sites depicted in (A) were mutated, regulatory activity in the male A5 segment was reduced to 89±6% and 88±3% respectively. (F) When the entire SM6 region was mutated, regulatory activity decreased to 31±1%. (C) The TTAT site mutations resulted in activity increasing in the A4 and A3 segments respectively to 169±4% and 261±6%. (D) The TTAT and TAAT site mutations resulted in activity increasing in the A4 and A3 segments respectively to 207±2% and 281±2%. (E) When the Hth site was mutated, regulatory activity in the male A5, A4, and A3 segments respectively increased to 122±7%, 236±6%, and 276±4%.(F) The entire SM6 region mutation resulted in activity decreasing in the A4 and A3 segments respectively to 64±1% and 73±1%. Blue arrowheads indicate segments where activity was notably increased and Red arrowheads indicate segments with notably decreased activity compared to the wild type sequence. Gel shift assays for sequences possessing wild type and mutant site (G) 3, (H) 4, and (I) 5 and the DNA-binding domains for Abd-A and Abd-B. Binding reactions used increasing amounts of the GST-DNA binding domain fusion protein (from left to right: 0 ng, 111 ng, 333 ng, 1000 ng, and 3000 ng). Binding correlated with the amount of input protein for the probes with the non-mutant sequence, whereas binding was dramatically reduced for the probes with a mutant Hox site.

Mentions: We next considered the SM6 region, for which the scanning mutation (SM6) resulted in a drastic reduction of male activity to 31±1% of the wild type CRE (compare Fig 6B and 6F). Within this 85 bp SM6 region resides 5 sequences matching either TTAT or TAAT sites, or both (Fig 6A, sites 1–5 or S1-S5). We generated and tested two mutant t_MSE2 reporter constructs, one in which the three TTAT sequences were mutated and the other for which the TTAT and TAAT sites were mutated (S11 Fig). To our surprise, the activity of the TTAT site mutant was only modestly reduced to 89±6% of the wild type CRE in the A5 segment (compare Fig 6B and 6C). Moreover, when the two TAAT sites were additionally mutated, regulatory activity was measured at 88±3% of wild type (Fig 6D). This lack of a pronounced regulatory effect contrasts with that caused by the 85 bp scanning mutation 6 (Fig 6F). Thus, with respect to the posterior male segment activity of the t_MSE2, it appears that Abd-B and Abd-A play little-to-no role as direct activators.


The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans.

Camino EM, Butts JC, Ordway A, Vellky JE, Rebeiz M, Williams TM - PLoS Genet. (2015)

Characterization of the direct Hox inputs shaping tan expression.(A) The SM6 region of the t_MSE possesses five sites, S1-S5, with sequences characteristic of Abd-B (TTAT) and Abd-A (TAAT) binding sites. This CRE region also possesses sites resembling sequences bound by Exd and Hth, though the functionality of the Exd site was not studied here. (B-F) EGFP reporter transgene expression in male pupae at ~95 hours after puparium formation. (B) The t_MSE2 sequence drives robust expression in the male A5 and A6 segments. When all of the (C) TTAT sites and (D) TTAT and TAAT sites depicted in (A) were mutated, regulatory activity in the male A5 segment was reduced to 89±6% and 88±3% respectively. (F) When the entire SM6 region was mutated, regulatory activity decreased to 31±1%. (C) The TTAT site mutations resulted in activity increasing in the A4 and A3 segments respectively to 169±4% and 261±6%. (D) The TTAT and TAAT site mutations resulted in activity increasing in the A4 and A3 segments respectively to 207±2% and 281±2%. (E) When the Hth site was mutated, regulatory activity in the male A5, A4, and A3 segments respectively increased to 122±7%, 236±6%, and 276±4%.(F) The entire SM6 region mutation resulted in activity decreasing in the A4 and A3 segments respectively to 64±1% and 73±1%. Blue arrowheads indicate segments where activity was notably increased and Red arrowheads indicate segments with notably decreased activity compared to the wild type sequence. Gel shift assays for sequences possessing wild type and mutant site (G) 3, (H) 4, and (I) 5 and the DNA-binding domains for Abd-A and Abd-B. Binding reactions used increasing amounts of the GST-DNA binding domain fusion protein (from left to right: 0 ng, 111 ng, 333 ng, 1000 ng, and 3000 ng). Binding correlated with the amount of input protein for the probes with the non-mutant sequence, whereas binding was dramatically reduced for the probes with a mutant Hox site.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4383587&req=5

pgen.1005136.g006: Characterization of the direct Hox inputs shaping tan expression.(A) The SM6 region of the t_MSE possesses five sites, S1-S5, with sequences characteristic of Abd-B (TTAT) and Abd-A (TAAT) binding sites. This CRE region also possesses sites resembling sequences bound by Exd and Hth, though the functionality of the Exd site was not studied here. (B-F) EGFP reporter transgene expression in male pupae at ~95 hours after puparium formation. (B) The t_MSE2 sequence drives robust expression in the male A5 and A6 segments. When all of the (C) TTAT sites and (D) TTAT and TAAT sites depicted in (A) were mutated, regulatory activity in the male A5 segment was reduced to 89±6% and 88±3% respectively. (F) When the entire SM6 region was mutated, regulatory activity decreased to 31±1%. (C) The TTAT site mutations resulted in activity increasing in the A4 and A3 segments respectively to 169±4% and 261±6%. (D) The TTAT and TAAT site mutations resulted in activity increasing in the A4 and A3 segments respectively to 207±2% and 281±2%. (E) When the Hth site was mutated, regulatory activity in the male A5, A4, and A3 segments respectively increased to 122±7%, 236±6%, and 276±4%.(F) The entire SM6 region mutation resulted in activity decreasing in the A4 and A3 segments respectively to 64±1% and 73±1%. Blue arrowheads indicate segments where activity was notably increased and Red arrowheads indicate segments with notably decreased activity compared to the wild type sequence. Gel shift assays for sequences possessing wild type and mutant site (G) 3, (H) 4, and (I) 5 and the DNA-binding domains for Abd-A and Abd-B. Binding reactions used increasing amounts of the GST-DNA binding domain fusion protein (from left to right: 0 ng, 111 ng, 333 ng, 1000 ng, and 3000 ng). Binding correlated with the amount of input protein for the probes with the non-mutant sequence, whereas binding was dramatically reduced for the probes with a mutant Hox site.
Mentions: We next considered the SM6 region, for which the scanning mutation (SM6) resulted in a drastic reduction of male activity to 31±1% of the wild type CRE (compare Fig 6B and 6F). Within this 85 bp SM6 region resides 5 sequences matching either TTAT or TAAT sites, or both (Fig 6A, sites 1–5 or S1-S5). We generated and tested two mutant t_MSE2 reporter constructs, one in which the three TTAT sequences were mutated and the other for which the TTAT and TAAT sites were mutated (S11 Fig). To our surprise, the activity of the TTAT site mutant was only modestly reduced to 89±6% of the wild type CRE in the A5 segment (compare Fig 6B and 6C). Moreover, when the two TAAT sites were additionally mutated, regulatory activity was measured at 88±3% of wild type (Fig 6D). This lack of a pronounced regulatory effect contrasts with that caused by the 85 bp scanning mutation 6 (Fig 6F). Thus, with respect to the posterior male segment activity of the t_MSE2, it appears that Abd-B and Abd-A play little-to-no role as direct activators.

Bottom Line: Morphological traits result from gene regulatory networks (GRNs) that form a web of transcription factors, which regulate multiple cis-regulatory element (CRE) sequences to control the coordinated expression of differentiation genes.By elucidating how yellow and tan are connected to the web of abdominal trans-regulators, we discovered that the yellow and tan abdominal CREs are composed of distinct regulatory inputs that exhibit contrasting responses to the same Hox proteins and Hox cofactors.These results provide an example in which CRE origination underlies a recently evolved novel trait, and highlights how coordinated expression patterns can evolve in parallel through the generation of unique regulatory linkages.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Dayton, Dayton, Ohio, United States of America.

ABSTRACT
The origination and diversification of morphological characteristics represents a key problem in understanding the evolution of development. Morphological traits result from gene regulatory networks (GRNs) that form a web of transcription factors, which regulate multiple cis-regulatory element (CRE) sequences to control the coordinated expression of differentiation genes. The formation and modification of GRNs must ultimately be understood at the level of individual regulatory linkages (i.e., transcription factor binding sites within CREs) that constitute the network. Here, we investigate how elements within a network originated and diversified to generate a broad range of abdominal pigmentation phenotypes among Sophophora fruit flies. Our data indicates that the coordinated expression of two melanin synthesis enzymes, Yellow and Tan, recently evolved through novel CRE activities that respond to the spatial patterning inputs of Hox proteins and the sex-specific input of Bric-à-brac transcription factors. Once established, it seems that these newly evolved activities were repeatedly modified by evolutionary changes in the network's trans-regulators to generate large-scale changes in pigment pattern. By elucidating how yellow and tan are connected to the web of abdominal trans-regulators, we discovered that the yellow and tan abdominal CREs are composed of distinct regulatory inputs that exhibit contrasting responses to the same Hox proteins and Hox cofactors. These results provide an example in which CRE origination underlies a recently evolved novel trait, and highlights how coordinated expression patterns can evolve in parallel through the generation of unique regulatory linkages.

No MeSH data available.


Related in: MedlinePlus