Impact of histone serotonylation domain organization on neurodevelopment

点击翻译按钮获取中文摘要

Project Summary Emerging evidence suggests chromatin mechanisms contribute to brain development, including organization of H3 lysine 4 tri-methylation (H3K4me3) domains. Broad H3K4me3 domains are linked with cell-specific transcriptional activation and are associated with synaptic signaling in neurons, where H3K4me3 spreading was disrupted in postmortem prefrontal cortex (PFC) neurons from patients with autism spectrum disorders (ASD). Thus, H3K4me3 breadth likely influences important neurodevelopmental processes, but how this occurs is unclear. Spreading of H3K4me3 broad peaks is regulated, in part, by the enzyme Lysine methyltransferase 2e (Kmt2e), where over 30 genetic variants - including a valine-to-isoleucine substitution at position 140 (V140I) in its chromatin-reading PHD finger - were observed in individuals with symptoms related to intellectual disability and developmental delay. Interestingly, H3K4me3 sits next to glutamine 5 that can be serotonylated, producing the combinatorial histone post-translational modification H3K4me3Q5ser that further enhances permissive transcription compared to H3K4me3 alone. Our preliminary data show that H3Q5ser enhances binding of Kmt2e to H3K4me3 and ChIP-sequencing of H3K4me3Q5ser in embryonic forebrain identified broad H3K4me3Q5ser domains that associate with neurodevelopment-associated processes. Thus, I hypothesize that Kmt2e regulates brain development via organization of H3K4me3 broad domains, and that the neighboring H3Q5ser influences such interactions. To specifically interrogate Kmt2e and H3K4me3Q5ser binding as a regulator of broad peak organization, the mutant Kmt2eV140I, that contains a mutation at the site where H3Q5ser would extend during H3K4me3 and Kmt2e PHD finger binding, was selected as a translationally relevant genetic variant that can be used to assess the mechanistic impact of this interaction. In Aim 1, I will quantitatively assess the binding affinity between H3K4me3Q5ser and Kmt2eWT vs. Kmt2eV140I PHD fingers as a crucial interaction in the developing brain that may be disrupted in some pathologies, using peptide immunoprecipitation followed by western blotting and isothermal titration calorimetry. In Aim 2, I will use CRISPR/Cas9 technology in diploid RPE1 cells to assess the impact of tagged Kmt2eWT vs. Kmt2eV140I knock-in vs. Kmt2e knockout on broad peak distribution using H3K4me3Q5ser ChIP-seq, on Kmt2e recruitment using Kmt2e ChIP-seq, and on downstream transcription using RNA-seq. In Aim 3, I will assess the impact of Kmt2e in developing brain by using in utero electroporation to transfect artificial miRNA designed to specifically knockdown Kmt2e expression in PFC progenitor cells, with simultaneous ‘rescue’ by adding back tagged Kmt2eWT vs. Kmt2eV140I transgenes, using H3K4me3Q5ser and Kmt2e ChIP-seq as readouts of epigenetic normalization, RNA-seq to evaluate developmental gene expression programs, and neuronal morphology analyses to assess Kmt2e impact on synapse development. Together, these studies will provide valuable insight into a novel epigenetic mechanism regulating neurodevelopment that may be perturbed in syndromes such as ASD.