PROJECT SUMMARY Chromatin-associated proteins are a major class of genes mutated in autism spectrum disorder (ASD) and intellectual disability (ID). These genetic data indicate the importance of gene regulation for brain development, however a significant challenge for the field is to determine how disruptions of chromatin regulators may converge on specific biological processes in developing neurons. To address this challenge, here we focus in detail on the biochemical and cellular mechanisms by which ID-associated frameshift mutations in the linker histone H1.4 result in impaired neuronal development. As a histone, H1.4 is a direct component of chromatin; thus, studying how mutations in this gene impair neural development offers the opportunity to gain specific biochemical insight into mechanisms of chromatin regulation in neurons. Rahman Syndrome (RMNS) is a rare, genetic form of ID caused by de novo heterozygous mutations in H1-4, which encodes histone H1.4. All RMNS-associated mutations in H1-4 are small insertions or deletions that create a shared C-terminal frameshift. Genetic data indicate that the mutant protein likely functions in a dominant negative or neomorphic manner to lead to RMNS phenotypes, but the biochemical and cellular consequences of expressing RMNS mutant histone H1.4 are poorly understood. We have found that expressing RMNS mutant histone H1.4 in rat hippocampal neurons leads to the disruption of synaptic gene expression and neuronal firing. We hypothesize that RMNS mutant H1.4 disrupts chromatin architecture in differentiating neurons to impair gene expression programs that are required for synapse development and neuronal function. To determine how the RMNS mutation of histone H1.4 leads to aberrant transcription and to evaluate the consequences for brain development, we will use both biochemical and molecular genetic approaches in the developing mouse brain and in human neurons. In Aim 1, we will build a foundation for these studies by using leading edge proteomic and molecular genetic methods to characterize the expression, regulation, and chromatin distribution of histone H1.4 over the course of neuronal differentiation in the mouse. To determine how RMNS mutations disrupt synaptic gene expression in brain development, in Aim 2 we will use a novel in vivo protein tagging strategy to identify proteins that interact with wildtype versus RMNS mutant H1.4. Finally, to determine how RMNS mutations disrupt chromatin regulation, in Aim 3 we will study generate RMNS mutant H1.4 expressing iPSC- derived neurons for biochemical histone H1 proteomics and gene expression analyses. We will then use a novel method for low-input three-dimensional chromatin conformation capture to test the hypothesis that RMNS mutant histone H1.4 disrupts higher level chromatin architecture. These studies will advance knowledge of the causes of brain developmental abnormalities in RMNS, and they will contribute to understanding of the specific mecha...