Department or Program
From fertilization onwards, development of a viable organism is a highly regulated process requiring coordinated cell-signaling at all stages of development. Aryl-hydrocarbon receptor (Ahr) and nuclear factor-erythroid-2 (nfe2) related factors (nrfs) are transcription factors that are involved in developmental regulatory processes during periods of oxidative stress. Using 24 hours-post-fertilization (hpf) zebrafish embryos in their pharyngula period, the interactions between Ahr1B and nrfs are being assessed. Morpholino knockdown of Ahr1B in the presence of a strong Ahr ligand, TCDD, suggests that Ahr1B regulates most nrf genes. While these data suggest an interaction between the two pathways, it cannot distinguish whether the interaction is directly or indirectly influenced by Ahr1B. Using chromatin immunoprecipitation (ChIP) followed by qPCR, it is hypothesized that there is direct regulation of particular nrf genes by Ahr1B binding on cis-promoter xenobiotic response elements (XRE). Because ChIP procedures have never been used to study transcription factor binding in whole zebrafish embryos as late as 24 hpf, a ChIP procedure has been extensively optimized. Specifically, optimization has been focused on sonication parameters for DNA fragmentation, DNA-protein crosslinking conditions, and non-specific binding-removal techniques to minimize background noise in the results. Additionally, a negative control for immunoprecipitation has been added and qPCR primers have been designed for each of the XREs on the nrf genes to allow for quantification. Using a well-known histone protein binding-interaction as a positive control and a known absent protein as a negative control, the efficiency of the ChIP procedure for use in whole 24 hpf zebrafish embryos has been demonstrated. Once optimization of the Ahr1B antibody is complete, collection of results will be possible. The results of this study will allow us to understand specific regulatory mechanisms and further understand the functions of the nrf family transcription factors during Ahr1B-mediated oxidative stress responses in early embryological development.
Level of Access
Restricted: Embargoed [Open Access After Expiration]
Date of Graduation
Bachelor of Arts
Ulin, Alexandra Sharon Wyss, "Use of Chromatin Immunoprecipitation and qPCR in 24 Hours-post-fertilization Zebrafish (Danio rerio) Embryos to Show Direct Regulation of nrf genes by Ahr1B" (2017). Honors Theses. 223.
Number of Pages