Insights into EHMT1 Variants in the Neurodevelopmental Disorder Kleefstra Syndrome through Structural Dynamics and Functional Analysis

Euchromatic histone methyltransferase 1 (EHMT1, also known as GLP) is a key histone methylation writer intricately associated with Kleefstra Syndrome (KS), a multifaceted neurodevelopmental disorder characterized by intellectual disability, developmental delay, childhood hypotonia, distinctive facial features, and comorbidities,

including autism spectrum disorders. Its modular structure encompasses the ankyrin repeat scaffolding domain responsible for methyl-lysine recognition (histone reader function) and the SET catalytic domain governing histone methylation (writer function). Despite the identification of numerous mutations in patients, their precise

effects on EHMT1 function and direct links to pathogenicity remain elusive. As such, most are annotated as variants of uncertain significance (VUS). To enhance EHMT1 genomic variation interpretation and uncover novel insights into its structure-function relationship, we have harnessed a comprehensive computational

approach to perform deep variant phenotyping, which integrates mechanisms rooted in sequence, structure, and dynamics. Through this approach, we have identified domain-specific metrics and reclassified KS variants within the SET domain. In this proposal, we aim to perform the first in-depth comprehensive deep variant

phenotyping of KS-associated variants within the ankyrin repeat domain and further elucidate the structure- function relationship of variants within both the SET and ankyrin repeat domains to better understand pathophysiological mechanisms underlying the disease. Our CENTRAL HYPOTHESIS is that KS-associated

genomic variants within EHMT1 alter structural dynamics as well as biochemical and biophysical properties to impact domain-specific functions. The rationale is that comprehensive investigations into the biochemical, biophysical, and structural dynamics of KS-associated genomic variants within the EHMT1 SET and ankyrin

domains will reveal detailed mechanisms underlying the disruption of domain-specific functions, providing novel insight into the molecular basis of KS. To test our central hypothesis, we propose two interrelated, yet independent AIMS: (1) to define the impact of KS-associated genomic variants within the ankyrin domain on

the structural dynamics of EHMT1 using deep variant phenotyping and (2) to elucidate the underlying biochemical and biophysical mechanisms of EHMT1 dysfunction caused by KS-associated genomic variants. These findings will establish crucial foundations for subsequent investigations involving cell-based and animal

model studies. Ultimately, our discoveries will have the potential to guide future therapeutic strategies aimed at ameliorating the impacts of KS-associated variants on EHMT1 function and, by extension, on chromatin regulation and epigenetic processes. Collectively, these studies will enhance the annotation of genomic

variants and facilitate the development of more precise, personalized approaches to disease management and treatment, ultimately improving the well-being of affected children and offering support to their families.