Genetic dissection of the transcriptional hypoxia response and genomic regional capture for massively parallel sequencing
Turnbull, Douglas William, 1979-
MetadataShow full item record
Turnbull, Douglas William, 1979-
When cells are faced with the stress of oxygen deprivation (hypoxia), they must alter their physiology in order to survive. One adaptation cells make during hypoxia entails the transcriptional activation of specific groups of genes as well as the concurrent repression of other groups. This modulation is achieved through the actions of transcription factors, proteins that are directly involved in this transcriptional activation and repression. I studied the transcriptional response to hypoxia in the model organism Drosophila melanogaster utilizing DNA microarrays to examine the transcriptomes of five different mutant Drosophila strains deficient in the hypoxia-responsive transcription factors HIF-1, FOXO, NFkB, p53, and MTF-1. By comparing hypoxia responsive gene expression in these mutants to that of wild type flies and subsequently identifying binding sites for each transcription factor near putative target genes, I was able to identify the transcripts regulated by each transcription factor during hypoxia. I discovered that FOXO plays an unexpectedly large role in hypoxic gene regulation, regulating a greater number of genes than any other transcription factor. I also identified multiple interesting targets of other transcription factors and uncovered a potential regulatory link between HIF-1 and FOXO. This study is the most in-depth examination of the transcriptional hypoxia response to date. I was also involved in additional research on transcriptional stress responses in Drosophila. Also included in this dissertation are two papers on which I was the second author. One paper identified a regulatory link between the transcriptional responses to hypoxia and heat-shock. The other examined elevated CO 2 stress (hypercapnia) in Drosophila, showing that this stress causes the down-regulation of NFkB-dependent antimicrobial peptide gene expression. My studies of stress responses would not have been possible without well-described mutant fly strains. Another part of my dissertation research involved the creation of a method for characterizing new mutants for future studies. When researchers seek to identify the molecular nature of a mutation that causes an interesting phenotype, they must ultimately determine the specific responsible genomic sequence change. While classical genetic methods and other techniques can easily be used to roughly map the location of a mutation in a genome, regions identified by these means are usually so large that sequencing them to precisely identify the polymorphism is laborious and slow. I have developed a technique that makes sequencing genomic regions of this size much easier. My technique involves capturing genomic regions by hybridization of fragmented genomic target DNA to biotinylated probes generated from fosmid DNA, which are subsequently immobilized and washed on streptavidin beads. Genomic DNA fragments are then eluted by denaturation and sequenced using the latest generation of massively parallel sequencing technology. I have demonstrated the effectiveness of this approach by sequencing a mutation-containing 336-kilobase genomic region from a Caenorhabditis elegans strain. My entire protocol can be completed in two days, is relatively inexpensive, and is broadly applicable to any situation in which one wants to sequence a specific genomic region using massively parallel sequencing. This dissertation includes both my previously published and my coauthored materials.