Analyzing histone H3.3 function in neural stem cells via H3F3a knockout

Project (M.A., Biology (Stem Cell))--California State University, Sacramento, 2015.The histone variant protein H3.3 plays necessary roles in embryogenesis, spermatogenesis, and transcription.H3.3 is encoded for by two genes: H3F3A and H3F3B in humans and H3f3a and H3f3b in mice. Both genes encode for an identical protein, yet have different promoters, untranslated regions, intron/exon boundaries, and expression patterns. Additionally, mutations in H3.3 or its deposition complex members ATRX/DAXX have been found in roughly 45% of all pediatric glioblastoma multiforme patients. Although a growing body of work exists investigating the role of H3.3 in development, these studies have primarily utilized non-mammalian model organisms. Understanding the role that H3.3 plays in development in mammalian species, as well as in neural stem cell lineages, will hopefully provide insight into the mechanism by which H3.3 is associated with tumorigenicity. Here, we characterize a novel H3f3a knockout first mouse via qPCR and Western blotting. We show that there appears to be no gene dosage compensation mechanism in place where H3f3b can compensate for H3f3a disruption. Additionally, we provide evidence illustrating a decrease in both activating and repressive H3 histone marks, as well as a decrease in the H3.3-specific phosphorylation of serine 31. Taken together, these data suggests that disruption of H3f3a perturbs chromatin remodeling.Biological Sciences (Stem Cell