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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

FPs are enriched around TSSs. The aggregated FAIRE signal adjacent to transcription start sites (TSSs) was increased within 100–200 bp upstream of TSSs
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Fig2: FPs are enriched around TSSs. The aggregated FAIRE signal adjacent to transcription start sites (TSSs) was increased within 100–200 bp upstream of TSSs

Mentions: Several lines of evidence indicate that FAIRE reliably identified regulatory elements in A. aegypti. In total, 5 % of the FPs were found to be located at proximal promoters (Additional file 3), a figure that is consistent with reports in other systems [37, 38]. These sequences can include both core promoters as well as regulatory elements adjacent to the promoters. However, one limitation of FAIRE is that other assays, including DNAse-seq, may be better for identification of nucleosome-depleted promoters of highly expressed genes [7, 38]. The aggregated FAIRE signal around all transcription start sites (TSSs) showed higher frequency within 100–200 bp upstream of TSSs (Fig. 2), which is also expected [7]. Furthermore, transcription factor binding sites are often associated with regulatory sequences in eukaryotes [39]. Known transcription factor binding sites are enriched in the FPs (Table 1) with respect to the rest of the genome. Among them, the top 20 TF-binding sites that are significantly enriched in FPs are shown in Fig. 3. Of these, the following consensus sequences are most abundant: FoxA1, Gfi, Hunchback, Klf4, MYB/ph3 and Sox9. Some of these elements, particularly Hunchback and FoxA1, were also abundant in FPs localized to the UTRs and intragenic regions (these regions are discussed further below).Fig. 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)

FPs are enriched around TSSs. The aggregated FAIRE signal adjacent to transcription start sites (TSSs) was increased within 100–200 bp upstream of TSSs
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: FPs are enriched around TSSs. The aggregated FAIRE signal adjacent to transcription start sites (TSSs) was increased within 100–200 bp upstream of TSSs
Mentions: Several lines of evidence indicate that FAIRE reliably identified regulatory elements in A. aegypti. In total, 5 % of the FPs were found to be located at proximal promoters (Additional file 3), a figure that is consistent with reports in other systems [37, 38]. These sequences can include both core promoters as well as regulatory elements adjacent to the promoters. However, one limitation of FAIRE is that other assays, including DNAse-seq, may be better for identification of nucleosome-depleted promoters of highly expressed genes [7, 38]. The aggregated FAIRE signal around all transcription start sites (TSSs) showed higher frequency within 100–200 bp upstream of TSSs (Fig. 2), which is also expected [7]. Furthermore, transcription factor binding sites are often associated with regulatory sequences in eukaryotes [39]. Known transcription factor binding sites are enriched in the FPs (Table 1) with respect to the rest of the genome. Among them, the top 20 TF-binding sites that are significantly enriched in FPs are shown in Fig. 3. Of these, the following consensus sequences are most abundant: FoxA1, Gfi, Hunchback, Klf4, MYB/ph3 and Sox9. Some of these elements, particularly Hunchback and FoxA1, were also abundant in FPs localized to the UTRs and intragenic regions (these regions are discussed further below).Fig. 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