Characterization of the Hookworm FOXO Transcription Factor DAF-16 and its Target Genes
Open AccessAbstractHookworm disease is an intestinal parasitic disease of humans and animals. It continues to rank among the most important neglected tropical diseases. Blood loss resulting from heavy hookworm infections has devastating impacts for children, pregnant women, and the elderly. As do many parasitic nematodes, hookworms undergo developmental arrest as free-living infective larvae (L3) prior to host entry, and their post infection development as a parasite is re-activated by host-specific signals. The transition to parasitism and subsequent development is poorly understood. Accumulating evidence suggests that the insulin-like signaling (ILS) pathway is involved in hookworm infection and development. A key output of ILS pathway is the forkhead transcription factor DAF-16/FOXO, which has been implicated in diverse biological processes. In this study, an ortholog of DAF-16 from hookworm A. caninum, Ac-DAF-16, was characterized. The amino acid sequence of Ac-DAF-16/FOXO DNA binding domain (DBD) shared high identity (94%) with the b and c isoforms of C. elegans DAF-16. Other functional motifs including Akt/PKB phosphorylation sites and 14-3-3 protein binding sites common to DAF-16/FOXO, are also conserved in Ac-DAF-16/FOXO. We demonstrated that Ac-DAF-16/FOXO was transcriptionally active through a canonical DBE (DAF-16 family protein binding element) and negatively regulated by growth factor stimulation.To better understand the roles of Ac-DAF-16/FOXO in hookworm parasitism, we combined molecular techniques and bioinformatics to identify Ac-DAF-16/FOXO binding sites in the hookworm genome and a group of its target genes. The DBD of Ac-DAF-16 was used to select genomic fragments by in vitro genomic selection. Twenty four bound genomic fragments were analyzed for the presence of the DBE and putative alternative Ac-DAF-16/FOXO binding motifs. The 22 genes linked to those genomic fragments were identified as direct gene targets of Ac-DAF-16/FOXO and their developmental stage-specific expression patterns were examined. The results suggested that Ac-DAF-16/FOXO was involved in many biological processes throughout hookworm development. Comprehensive characterization of Ac-DAF-16/FOXO gene targets will provide insights into the molecular mechanisms of its regulatory functions. For this reason, we selected an identified Ac-DAF-16/FOXO target gene in our genome wide screening studies, Ac-snr-3, for further investigation, since it was linked to a genomic fragment with strong binding affinity to Ac-DAF-16/FOXO. Ac-snr-3 encodes a core Sm protein, Sm-D1, in snRNP (small nuclear ribonucleoprotein) particles. It spans at least 5.6 kb in genome and contains 3 exons and 2 introns. The transcriptional profile of hookworm snr-3 for post infection developmental stages suggested a function in regulating hookworm maturation into the adulthood. Its 3-end genomic fragment (Fragment 2.23) was able to drive transcription of a reporter gene in the presence of Ac-DAF-16/FOXO, regardless of the location relative to reporter gene, indicating that it might act as cis-regulatory element mediating the effects of Ac-DAF-16/FOXO in hookworm. The unresponsiveness of this fragment to serum stimulation suggested that co-acting protein factors were involved in its interaction with Ac-DAF-16/FOXO.In overview, the findings presented in this dissertation identified a key transcription factor in hookworm ILS, Acitalic>-DAF-16/FOXO, and demonstrated that it regulated gene expression through the specific cis-regulatory elements in hookworm development.
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