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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

Visualization of FAIRE-seq data in the VB genome browser. FAIRE-seq reads [36] mapped to version three of the A. aegypti scaffolds reference v.4 [20, 21] are accessible through the VB Genome Browser. FPs in the supercontig 1.551: 476,900–630,100 bp region are shown. The peak marked with an asterisk corresponds to reporter A in Table 2. The gene accession numbers noted here and in all subsequent tables and supplementary materials correspond to VB [20, 21]
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Fig1: Visualization of FAIRE-seq data in the VB genome browser. FAIRE-seq reads [36] mapped to version three of the A. aegypti scaffolds reference v.4 [20, 21] are accessible through the VB Genome Browser. FPs in the supercontig 1.551: 476,900–630,100 bp region are shown. The peak marked with an asterisk corresponds to reporter A in Table 2. The gene accession numbers noted here and in all subsequent tables and supplementary materials correspond to VB [20, 21]

Mentions: FAIRE-seq open chromatin profiling was performed to identify regulatory DNA in the A. aegypti genome. For these studies, embryos were collected 50 +/− 1 h after eggs were laid, a time point that coincides with the onset of axon pathfinding [33], a topic of interest to our laboratory [33–35], and a time period for which transcriptome data are available [29]. The FAIRE procedure was optimized using these A. aegypti embryos as input tissue until a 1 % FAIRE DNA yield (with respect to the total input DNA) was obtained, an amount that is within the optimal range noted by Simon et al. [7]. FAIRE-seq replicate experiments were highly reproducible, as evidenced by IDR [24, 25] analysis (Additional file 1) which detected no significant differences between three replicate experiments, the data from which were subsequently combined for downstream analyses. FAIRE-seq reads [36] were mapped to version three of the A. aegypti scaffolds reference v.4 [20, 21] and have been made accessible through the VB Genome Browser (Fig. 1). In total, FAIRE-seq open chromatin profiling resulted in the identification of ~121,600 FPs (Additional file 2) [36], a number which is consistent with results reported in other systems [7].Fig. 1


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)

Visualization of FAIRE-seq data in the VB genome browser. FAIRE-seq reads [36] mapped to version three of the A. aegypti scaffolds reference v.4 [20, 21] are accessible through the VB Genome Browser. FPs in the supercontig 1.551: 476,900–630,100 bp region are shown. The peak marked with an asterisk corresponds to reporter A in Table 2. The gene accession numbers noted here and in all subsequent tables and supplementary materials correspond to VB [20, 21]
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Visualization of FAIRE-seq data in the VB genome browser. FAIRE-seq reads [36] mapped to version three of the A. aegypti scaffolds reference v.4 [20, 21] are accessible through the VB Genome Browser. FPs in the supercontig 1.551: 476,900–630,100 bp region are shown. The peak marked with an asterisk corresponds to reporter A in Table 2. The gene accession numbers noted here and in all subsequent tables and supplementary materials correspond to VB [20, 21]
Mentions: FAIRE-seq open chromatin profiling was performed to identify regulatory DNA in the A. aegypti genome. For these studies, embryos were collected 50 +/− 1 h after eggs were laid, a time point that coincides with the onset of axon pathfinding [33], a topic of interest to our laboratory [33–35], and a time period for which transcriptome data are available [29]. The FAIRE procedure was optimized using these A. aegypti embryos as input tissue until a 1 % FAIRE DNA yield (with respect to the total input DNA) was obtained, an amount that is within the optimal range noted by Simon et al. [7]. FAIRE-seq replicate experiments were highly reproducible, as evidenced by IDR [24, 25] analysis (Additional file 1) which detected no significant differences between three replicate experiments, the data from which were subsequently combined for downstream analyses. FAIRE-seq reads [36] were mapped to version three of the A. aegypti scaffolds reference v.4 [20, 21] and have been made accessible through the VB Genome Browser (Fig. 1). In total, FAIRE-seq open chromatin profiling resulted in the identification of ~121,600 FPs (Additional file 2) [36], a number which is consistent with results reported in other systems [7].Fig. 1

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