Project Summary/Abstract The serotonergic (5HTergic) system is implicated in a wide range of neurodevelopmental and neuropsychiatric phenomena, including regulation of mood and stress reactivity. While 5HT actions have been assumed to be mediated exclusively through 5HT receptors, studies have established 5HT forms covalent bonds with histone H3—resulting in H3 glutamine 5 serotonin (H3Q5ser)–a process known as H3 serotonylation. H3 serotonylation plays key regulatory roles in establishing normal embryonic and adult patterns of brain transcriptional plasticity. However, functional roles for H3 serotonylation during postnatal brain development, have been unexplored, and the impact of environmental stimuli on this modification during early life remains unknown. Our laboratory implemented FANS coupled CUT&RUN (C&R)-seq to profile the cell type-specific epigenomic landscape of H3 serotonylation in mouse medial prefrontal cortex (mPFC), across critical periods of postnatal brain development. We identify cell-type-, sex-, developmental-, and early life stress- (ELS) induced alterations in H3 serotonylation genomic enrichment, with males showing more pronounced developmental and ELS-induced changes in both neuron and glia populations. In males, further analysis reveals, H3 serotonylation binding increases at oligodendrocyte genes in response to ELS. While these findings are insightful, they alone cannot elucidate the cell-type-specific functional roles of H3 serotonylation on gene expression or its potential influence on behavioral phenotypes. In the F99 phase, I will test the hypothesis that H3 serotonylation critically regulates neurodevelopmental gene expression, and perturbation by ELS results in altered gene expression and increased vulnerability to stress-related behavioral phenotypes as a function of this novel PTM. I will utilize multi-omic bioinformatic tools to integrate epigenomic and transcriptomic (ie. RNA and C&R-Seq) data to uncover the causal link between H3 serotonylation and gene expression during critical postnatal brain development and in response to ELS. Using a novel cell-type specific viral construct, I will attenuate ELS-induced H3 serotonylation changes, to elucidate the functional cell-type specific roles of H3 serotonylation in brain development and its connection to stress-related behaviors in adulthood. During this phase, I will receive technical training on large-scale data analysis, integration of cell-type specific “omic” data, and molecular cloning techniques that can be used to create gene editing tools to manipulate epigenetic modifications such as H3 serotonylation in vivo. In the K00 phase, I will focus on identifying laboratories for my postdoctoral work, prioritizing expertise in circuit-specific approaches (circuit characterization/manipulation), functional readouts of neuronal physiology (fiber photometry), and use of computer vision and machine learning to unbiasedly quantify animal behaviors. Together, this tra...