Quantitative mass spectrometry for comprehending epigenetic mechanisms in a new underlying neurological developmental disorder

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Project Summary Epigenetic mechanisms have been linked to many human disorders and diseases, most notably cancer. However, in recent years more and more human diseases have been found with possible epigenetic insults. In this regard, our collaborators at the Children’s Hospital of Philadelphia have through exome sequencing discovered germline-line mutations to the H3FA and H3FB genes in pediatric patients that suffer from similar neurological disorders and craniofacial abnormalities. These two genes encode for the histone variant H3.3, and thus represent the first germ-line mutations found on histone H3 in humans (neurohistone H3.3 mutations). Histones are small basic proteins that bind DNA to give rise to our chromatin structure. Along with DNA methylation and long non-coding RNA, post-translational modifications to histones regulate gene expression patterns (epigenetic mechanisms) and chromatin organization. Histone H3.3 is a specialized histone variant linked to active genes, and somatic mutations to this histone have been found in different brain cancers exclusively on the N-terminal tail. Our preliminary data have found that these neurohistone H3.3 mutations are spread across the entire protein from the N- to C-terminus. Therefore, we hypothesize that these mutations while resulting in similar phenotypes, do this by disrupting different epigenetic mechanisms involving histone H3.3. In this proposal, we aim to understand how these neurohistone mutations lead to neurodevelopmental problems. First, we aim to determine if these neurohistone mutations affect global or local histone modification patterns on wild-type or mutant histone variants using novel mass spectrometry (MS) approaches in cellular models. Next, we look to determine if these neurohistone mutants affect chromatin structure or conformation, or cause misincorporation of the H3.3 variant in the genome. Lastly, we will characterize proteome expression in a mouse model of one of the mutations using a novel in utero stable isotope labeling approach. It is our goal to determine how these neurohistone H3.3 mutations affect epigenetic and cellular signaling mechanisms to disrupt neurodevelopment and maintenance leading to neurological disorders in these pediatric patients.