In eukaryotic cells, octamers of histone proteins intricately organize DNA, forming a macromolecule known as nucleosomes. A nucleosome is comprised of two copies of histone H2A, H2B, H4, and H3. Histone H3.3 (H3.3), a histone variant, is often found at actively transcribed loci. H3.3 plays a role in cellular inheritance as ablation of H3.3 expression leads to loss of active gene states and dysfunction of heterochromatin telomeric structures. H3F3A and H3F3B, the two genes are known to encode H3.3, are expressed in all human cells with higher expression in the gonads and brain. A recent publication detailed H3.3 as the causative gene in a neurodevelopmental disorder with craniofacial abnormalities. The underlying cause of the disease is unknown; however, H3.3 is involved in a wide range of central nervous system functions. This dissertation highlights work that aimed to profile the effects of these germline mutations. We performed a comprehensive screen on pathogenic H3.3 variants utilizing a combination of techniques and methodology, including tissue culture, bottom-up proteomics, and neurodevelopmental assays in Xenopus laevis. We found global alterations to histone post-translational modifications with significant alterations to histone acetylation on histone H2a, H3, and H4 peptides. In addition, performing quantitative proteomics in the 293T cells led us to determine that these mutations affect several cellular processes, such as RNA splicing, cell motility, neurofilament maintenance, folic acid metabolism, and post-synaptic density, all processes known to be dysregulated in other neurological syndromes. Utilizing the model organism Xenopus, we introduced two of the pathogenic variants into embryos. We observed reduced craniofacial cartilage, abnormal head shape, and impaired motility due to kinked tails in the mutant tadpoles. We performed quantitative proteomics on these tadpoles and found pathways related to carbon metabolism and amino acid degradation upregulated. Lastly, our transcriptomic analyses corroborated some of these finds and an upregulation of the TGF-beta signaling pathway in one of the mutants. This work provides the first mechanistic study of these germline H3.3 mutations, introduces pathways dysregulated that can be further studied. Understanding the basic biology of these mutations will shed light on the molecular mechanisms of H3.3 in neurodevelopment
