Johanson, Michael (2013-08). An Investigation of Insulator Proteins in Mosquito Genomes. Master's Thesis.
Transgenic mosquitoes are beneficial for the design and implementation of various pathogen control programs. However, low and variable expression of transgenes caused by position effects is a hindrance to the characterization and effective use of transgenes in mosquito species. The use of insulator sequences to flank transgenes may have the ability to overcome position effects caused by the genomic environment surrounding the insertion site. CTCF is a multifunctional protein, conserved from humans to Drosophila. Its role as an enhancer blocker in the Drosophila bithorax complex and its proximal binding to other insulator proteins on Drosophila chromosomes makes it a good candidate for identifying insulator sequences throughout the mosquito genome that may be used to improve mosquito transgenesis. Its multi-functionality as a transcription factor and genome organizer also makes CTCF worthy of investigation for an improved understanding of the regulation of the mosquito genome. This study uses chromatin immunoprecipitation with an An. gambiae CTCF antibody followed by Illumina deep sequencing (ChIP-Seq) to identify regions of CTCF binding throughout the An. gambiae genome. A subset of the CTCF binding site peaks was validated using ChIP-PCR. Another subset of this data set, including the ChIP-PCR validated peaks, was input into the motif finding tool, AlignACE, in order to identify a CTCF binding site consensus. Four motifs were identified, none of which were found in more than 11.9% of the ChIP-Seq data set. These results lead us to conclude that An. gambiae CTCF binds to a wider variety of sequences compared to Drosophila CTCF. This work also includes a comparison of the expression profiles of the dipteran insulator proteins, Su(Hw) and CP190, with that of CTCF across multiple life stages in Ae. aegypti. The results of this study suggest the possibility of genomic colocalization, as has been recently discovered in Drosophila. The identification of CTCF binding site peaks throughout the An. gambiae genome provides a large data set of potential insulator sequences that may be used to improve mosquito transgenesis, and provide a new model for the study of CTCF function in a species with medical significance.