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High-throughput cis-regulatory element discovery in the vector mosquito Aedes aegypti.

Behura SK, Sarro J, Li P, Mysore K, Severson DW, Emrich SJ, Duman-Scheel M - BMC Genomics (2016)

Bottom Line: FAIRE results in the preferential recovery of open chromatin DNA fragments that are not bound by nucleosomes, an evolutionarily conserved indicator of regulatory activity, which are then sequenced.All of the elements tested in vivo were confirmed to drive gene expression in transgenic Drosophila reporter assays.The results of this investigation indicate that FAIRE-seq is a powerful tool for identification of regulatory DNA in the genomes of non-model organisms, including human disease vector mosquitoes.

View Article: PubMed Central - PubMed

Affiliation: Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, 46556, USA.

ABSTRACT

Background: Despite substantial progress in mosquito genomic and genetic research, few cis-regulatory elements (CREs), DNA sequences that control gene expression, have been identified in mosquitoes or other non-model insects. Formaldehyde-assisted isolation of regulatory elements paired with DNA sequencing, FAIRE-seq, is emerging as a powerful new high-throughput tool for global CRE discovery. FAIRE results in the preferential recovery of open chromatin DNA fragments that are not bound by nucleosomes, an evolutionarily conserved indicator of regulatory activity, which are then sequenced. Despite the power of the approach, FAIRE-seq has not yet been applied to the study of non-model insects. In this investigation, we utilized FAIRE-seq to profile open chromatin and identify likely regulatory elements throughout the genome of the human disease vector mosquito Aedes aegypti. We then assessed genetic variation in the regulatory elements of dengue virus susceptible (Moyo-S) and refractory (Moyo-R) mosquito strains.

Results: Analysis of sequence data obtained through next generation sequencing of FAIRE DNA isolated from A. aegypti embryos revealed >121,000 FAIRE peaks (FPs), many of which clustered in the 1 kb 5' upstream flanking regions of genes known to be expressed at this stage. As expected, known transcription factor consensus binding sites were enriched in the FPs, and of these FoxA1, Hunchback, Gfi, Klf4, MYB/ph3 and Sox9 are most predominant. All of the elements tested in vivo were confirmed to drive gene expression in transgenic Drosophila reporter assays. Of the >13,000 single nucleotide polymorphisms (SNPs) recently identified in dengue virus-susceptible and refractory mosquito strains, 3365 were found to map to FPs.

Conclusion: FAIRE-seq analysis of open chromatin in A. aegypti permitted genome-wide discovery of CREs. The results of this investigation indicate that FAIRE-seq is a powerful tool for identification of regulatory DNA in the genomes of non-model organisms, including human disease vector mosquitoes.

No MeSH data available.


Related in: MedlinePlus

FP DNA sequences promote gene expression in vivo. GFP reporter expression (from the reporters indicated in Table 2) was detected in the following D. melanogaster tissues: embryo (reporter A in a; whole-mount is shown), embryonic midgut (reporter B in b), third instar larval salivary gland (reporter C in c), pupal eyes (reporter D in d), third instar larval gut (reporter E in e), third instar larval fat body (reporter F in f), adult antenna (reporter G in g, reporter H in h), third instar larval denticle belts (reporter I in i), third instar larval leg discs (reporter J in j), and the third instar larval brain (reporter K in k)
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Fig4: FP DNA sequences promote gene expression in vivo. GFP reporter expression (from the reporters indicated in Table 2) was detected in the following D. melanogaster tissues: embryo (reporter A in a; whole-mount is shown), embryonic midgut (reporter B in b), third instar larval salivary gland (reporter C in c), pupal eyes (reporter D in d), third instar larval gut (reporter E in e), third instar larval fat body (reporter F in f), adult antenna (reporter G in g, reporter H in h), third instar larval denticle belts (reporter I in i), third instar larval leg discs (reporter J in j), and the third instar larval brain (reporter K in k)

Mentions: To further assess whether FAIRE-seq identified regulatory regions in the A. aegypti genome, a number of FP DNA sequences were tested for their ability to drive gene expression in vivo. Although transgenic technology is available in A. aegypti, transgenic generation in D. melanogaster is mature, quick, and inexpensive, and it is relatively easier and cheaper to maintain fruit fly stocks. Given the merits of Drosophila transgenics, 11 A. aegypti FP DNA sequences (Table 2) were cloned into a Drosophila transformation vector containing EGFP under the control of a minimal hsp70 promoter. 100 % of these elements were confirmed to drive EGFP reporter expression in D. melanogaster (Fig. 4). Although the FAIRE-seq study was performed in A. aegypti embryos, in addition to embryos (Fig. 4a, b), a number of the FP sequences identified were found to be capable of driving gene expression at later stages of the Drosophila life cycle, including the larval (Fig. 4c, e, f, i, j, k), pupal (Fig. 4d), and adult (Fig. 4g, h) stages. These findings suggest that some of the regulatory sequences that function in embryos are also active at later stages of the life cycle, including adults. However, a recent FAIRE-seq study in D. melanogaster showed that following cell-type specification in the appendages, open chromatin profiles changed as the appendages progressed toward terminal differentiation, suggesting that stage-specific functions also require opening of new regulatory sites or the closing of existing sites [17]. In the future, it will be interesting to generate A. aegypti transgenic reporters to examine the activity of the regulatory elements assessed here in Drosophila (Table 2, Fig. 4) at multiple stages of the mosquito life cycle.Table 2


High-throughput cis-regulatory element discovery in the vector mosquito Aedes aegypti.

Behura SK, Sarro J, Li P, Mysore K, Severson DW, Emrich SJ, Duman-Scheel M - BMC Genomics (2016)

FP DNA sequences promote gene expression in vivo. GFP reporter expression (from the reporters indicated in Table 2) was detected in the following D. melanogaster tissues: embryo (reporter A in a; whole-mount is shown), embryonic midgut (reporter B in b), third instar larval salivary gland (reporter C in c), pupal eyes (reporter D in d), third instar larval gut (reporter E in e), third instar larval fat body (reporter F in f), adult antenna (reporter G in g, reporter H in h), third instar larval denticle belts (reporter I in i), third instar larval leg discs (reporter J in j), and the third instar larval brain (reporter K in k)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4862039&req=5

Fig4: FP DNA sequences promote gene expression in vivo. GFP reporter expression (from the reporters indicated in Table 2) was detected in the following D. melanogaster tissues: embryo (reporter A in a; whole-mount is shown), embryonic midgut (reporter B in b), third instar larval salivary gland (reporter C in c), pupal eyes (reporter D in d), third instar larval gut (reporter E in e), third instar larval fat body (reporter F in f), adult antenna (reporter G in g, reporter H in h), third instar larval denticle belts (reporter I in i), third instar larval leg discs (reporter J in j), and the third instar larval brain (reporter K in k)
Mentions: To further assess whether FAIRE-seq identified regulatory regions in the A. aegypti genome, a number of FP DNA sequences were tested for their ability to drive gene expression in vivo. Although transgenic technology is available in A. aegypti, transgenic generation in D. melanogaster is mature, quick, and inexpensive, and it is relatively easier and cheaper to maintain fruit fly stocks. Given the merits of Drosophila transgenics, 11 A. aegypti FP DNA sequences (Table 2) were cloned into a Drosophila transformation vector containing EGFP under the control of a minimal hsp70 promoter. 100 % of these elements were confirmed to drive EGFP reporter expression in D. melanogaster (Fig. 4). Although the FAIRE-seq study was performed in A. aegypti embryos, in addition to embryos (Fig. 4a, b), a number of the FP sequences identified were found to be capable of driving gene expression at later stages of the Drosophila life cycle, including the larval (Fig. 4c, e, f, i, j, k), pupal (Fig. 4d), and adult (Fig. 4g, h) stages. These findings suggest that some of the regulatory sequences that function in embryos are also active at later stages of the life cycle, including adults. However, a recent FAIRE-seq study in D. melanogaster showed that following cell-type specification in the appendages, open chromatin profiles changed as the appendages progressed toward terminal differentiation, suggesting that stage-specific functions also require opening of new regulatory sites or the closing of existing sites [17]. In the future, it will be interesting to generate A. aegypti transgenic reporters to examine the activity of the regulatory elements assessed here in Drosophila (Table 2, Fig. 4) at multiple stages of the mosquito life cycle.Table 2

Bottom Line: FAIRE results in the preferential recovery of open chromatin DNA fragments that are not bound by nucleosomes, an evolutionarily conserved indicator of regulatory activity, which are then sequenced.All of the elements tested in vivo were confirmed to drive gene expression in transgenic Drosophila reporter assays.The results of this investigation indicate that FAIRE-seq is a powerful tool for identification of regulatory DNA in the genomes of non-model organisms, including human disease vector mosquitoes.

View Article: PubMed Central - PubMed

Affiliation: Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN, 46556, USA.

ABSTRACT

Background: Despite substantial progress in mosquito genomic and genetic research, few cis-regulatory elements (CREs), DNA sequences that control gene expression, have been identified in mosquitoes or other non-model insects. Formaldehyde-assisted isolation of regulatory elements paired with DNA sequencing, FAIRE-seq, is emerging as a powerful new high-throughput tool for global CRE discovery. FAIRE results in the preferential recovery of open chromatin DNA fragments that are not bound by nucleosomes, an evolutionarily conserved indicator of regulatory activity, which are then sequenced. Despite the power of the approach, FAIRE-seq has not yet been applied to the study of non-model insects. In this investigation, we utilized FAIRE-seq to profile open chromatin and identify likely regulatory elements throughout the genome of the human disease vector mosquito Aedes aegypti. We then assessed genetic variation in the regulatory elements of dengue virus susceptible (Moyo-S) and refractory (Moyo-R) mosquito strains.

Results: Analysis of sequence data obtained through next generation sequencing of FAIRE DNA isolated from A. aegypti embryos revealed >121,000 FAIRE peaks (FPs), many of which clustered in the 1 kb 5' upstream flanking regions of genes known to be expressed at this stage. As expected, known transcription factor consensus binding sites were enriched in the FPs, and of these FoxA1, Hunchback, Gfi, Klf4, MYB/ph3 and Sox9 are most predominant. All of the elements tested in vivo were confirmed to drive gene expression in transgenic Drosophila reporter assays. Of the >13,000 single nucleotide polymorphisms (SNPs) recently identified in dengue virus-susceptible and refractory mosquito strains, 3365 were found to map to FPs.

Conclusion: FAIRE-seq analysis of open chromatin in A. aegypti permitted genome-wide discovery of CREs. The results of this investigation indicate that FAIRE-seq is a powerful tool for identification of regulatory DNA in the genomes of non-model organisms, including human disease vector mosquitoes.

No MeSH data available.


Related in: MedlinePlus