Date of Award
Doctor of Philosophy
Genetics, Development and Cell Biology
Maura M. McGrail
Retinoblastoma-Binding Protein 4 (RBBP4) is a chromatin adaptor protein that associates with numerous activating and repressive chromatin regulatory complexes. RBBP4 is overexpressed in human gliomas, and has been shown to promote survival of GBM cells and p300-dependent expression of DNA repair genes (Kitange et al., 2016). We previously established a zebrafish RB-defective neural progenitor-like brain tumor model (Solin et al., 2015) and recently found Rbbp4 is highly upregulated in the RB- brain tumors. Here we demonstrate Rbbp4 is essential for zebrafish brain development and show it has distinct requirements in neural stem and progenitor cells. CRISPR/Cas9 targeting was used to isolate a 4 base-pair frameshift mutation in the zebrafish homolog rbbp4. Homozygous mutant rbbp4Δ4/Δ4 are lethal at approximately 5-7 days post fertilization and show a dramatic reduction in the size of the brain and retina. Overexpression of rbbp4 cDNA in a Tg(Tol2) transgenic line rescues the rbbp4 mutant phenotype, demonstrating disruption of neurogenesis in rbbp4Δ4/Δ4 homozygotes is due to lack of functional Rbbp4. Beginning at 2 days post fertilization, γ-H2AX and activated caspase labeling was detected in post-mitotic regions in the larval midbrain and retina, suggesting RBBP4 is required to prevent DNA-damage and apoptosis in neural progenitors or newborn neurons. γ-H2AX and caspase were not detected in the stem cell niches at the brain ventricle or retinal ciliary marginal zone, however, stem cell morphology was abnormally large. Pulse chase BrdU labeling experiments confirmed a delay in stem cell proliferation and a lack of survival of post-mitotic neural progenitors. TP53 morpholino knockdown in rbbp4Δ4/Δ4 homozygotes transiently suppressed neural progenitor apoptosis, indicating the absence of Rbbp4 activates TP53-dependent programmed cell death. Together these results suggest distinct requirements for Rbbp4 in neural stem cell proliferation and neural progenitor survival. Overall, our results indicate RBBP4 may drive RB-defective brain tumor growth by promoting proliferation and preventing DNA damage induced apoptosis of tumor cells.
Schultz, Laura, "Using zebrafish to elucidate the epigenetic mechanisms controlling neurogenesis and brain tumorigenesis" (2018). Graduate Theses and Dissertations. 16458.