DOT1L-mediated H3K79 methylation as a regulator of histone variant H3.3 and neuronal development

The National Institute of Neurological Disorders and Stroke awarded The Trustees of The University of Pennsylvania $100,228 on December 1, 2026, under the Extramural Research Programs in the Neurosciences and Neurological Disorders program (CFDA 93.853) to conduct research defining how DOT1L-mediated H3K79 methylation regulates histone variant H3.3 and impacts neuronal development.

The research addresses neurodevelopmental disorders caused by genetic variants in the histone methyltransferase DOT1L. The project uses multiple isogenic mouse embryonic stem cell lines that reduce H3K79 methylation through distinct mechanisms, differentiating them into neural progenitor cells and post-mitotic neurons. Research Aim 1 will define the mechanism by which H3K79 methylation regulates H3.3. Aim 2 will determine how H3K79 methylation loss impacts neuronal transcription, maturation, and function. The work is performed in Philadelphia, Pennsylvania, with a period of performance extending from December 1, 2026, through June 30, 2029.

Abstract

Neurodevelopmental disorders (NDDS) affect approximately 1 in 12 children in the united states and many are caused by genetic variants in chromatin regulators. Recent work identified a new NDD caused by missense variants in the histone methyltransferase DOT1L, the sole enzyme that catalyzes histone 3 lysine 79 methylation (H3K79ME). Nearly all DOT1L NDD patient variants are located within the catalytic domain of DOT1L and those tested disrupt its methyltransferase activity, implicating disrupted H3K79ME as the driver of the disorder, as opposed to DOT1L's non-catalytic functions. However, while H3K79ME is strongly associated with active transcription, its downstream effects on chromatin remain unclear, especially in neurons, as no specific readers or effectors of the mark have been identified. Preliminary data suggest that H3K79ME may regulate chromatin by modulating the histone variant H3.3, a critical and well-established regulator of neuronal chromatin and neurodevelopment whose disruption causes a similar NDD. H3.3 is the dominant H3 variant in neurons and is dynamically deposited and evicted at active chromatin regions to support transcriptional plasticity required for neuronal differentiation and function. Loss of H3K79ME leads to global depletion of H3.3, raising the possibility that H3K79ME contributes to DOT1L-associated NDDS by disrupting H3.3 chromatin dynamics. The goal of the proposed research is to define the mechanism by which H3K79ME regulates H3.3 (aim 1) and determine how H3K79ME loss impacts neuronal transcription, maturation, and function (aim 2). To isolate the specific role of H3K79ME, the project will use multiple isogenic mouse embryonic stem cell lines that reduce H3K79ME through distinct mechanisms and differentiate them into neural progenitor cells and post-mitotic neurons. These lines include 1) a DOT1L catalytic inactivation mutation, 2) an H3.3K79A mutation that blocks methylation of the dominant neuronal H3 variant, and 3) a DOT1L D157N variant identified in NDD patients that causes partial H3K79ME loss. Aim 1 will test the hypothesis that H3K79ME promotes H3.3 retention post- deposition to maintain its occupancy at active chromatin regions. Stable isotope labeling and mass spectrometry will be used to quantify the rate of H3.3 incorporation into and eviction from nucleosomes, and proteasome inhibition will test whether evicted H3.3 is degraded. Cut & run will identify genomic regions where H3K79ME loss depletes H3.3 during neuronal differentiation. Aim 2 will test the hypothesis that H3K79ME regulates synaptic gene expression and is required for the maturation and function of neurons. RNA-SEQ will define the genes regulated by H3K79ME during neuronal differentiation, and neuronal morphology and activity analyses will define the role of H3K79ME in neuronal maturation and function. This work will define the role of H3K79ME in neurons and uncover the mechanism of its previously unrecognized regulation of H3.3. The findings will provide mechanistic insight into the molecular basis of DOT1L-associated NDDS and may identify chromatin pathways amenable to therapeutic interventions.