Browsing by Author "Sarro, Joseph"

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    High-throughput cis-regulatory element discovery in the vector mosquito Aedes aegypti
    (BioMed Central, 2016-05-10) Behura, Susanta K.; Sarro, Joseph; Li, Ping; Mysore, Keshava; Severson, David W.; Emrich, Scott J.; Duman-Scheel, Molly; Department of Medical & Molecular Genetics, IU School of Medicine
    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.
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    Requirement for commissureless2 function during dipteran insect nerve cord development
    (Wiley, 2013-12) Sarro, Joseph; Andrews, Emily; Sun, Longhua; Behura, Susanta K.; Tan, John C.; Zeng, Erliang; Severson, David W.; Duman-Scheel, Molly; Medical & Molecular Genetics, School of Medicine
    BACKGROUND: In Drosophila melanogaster, commissureless (comm) function is required for proper nerve cord development. Although comm orthologs have not been identified outside of Drosophila species, some insects possess orthologs of Drosophila comm2, which may also regulate embryonic nerve cord development. Here, this hypothesis is explored through characterization of comm2 genes in two disease vector mosquitoes. RESULTS: Culex quinquefasciatus (West Nile and lymphatic filiariasis vector) has three comm2 genes that are expressed in the developing nerve cord. Aedes aegypti (dengue and yellow fever vector) has a single comm2 gene that is expressed in commissural neurons projecting axons toward the midline. Loss of comm2 function in both A. aegypti and D. melanogaster was found to result in loss of commissure defects that phenocopy the frazzled (fra) loss of function phenotypes observed in both species. Loss of fra function in either insect was found to result in decreased comm2 transcript levels during nerve cord development. CONCLUSIONS: The results of this investigation suggest that Fra down-regulates repulsion in precrossing commissural axons by regulating comm2 levels in both A. aegypti and D. melanogaster, both of which require Comm2 function for proper nerve cord development.