Stefán Pétursson, Juan Ouyang, Kaan Okay, Katrín Möller and Hans T. Björnsson
Introduction
Mendelian disorders of the epigenetic machinery (MDEM) result from mutations in genes encoding proteins that regulate the chromatin state of the genome. Intellectual disabilities and growth abnormalities are prevalent in these disorders, however, the underlying molecular mechanisms are poorly understood. A potential mechanism involves disruption of neurodevelopment, which begins with neural progenitor cells (NPCs). Initially, these cells primarily produce neurons, but later differentiate into glial cells in a process called the neuro-gliogenic switch. This study aims to identify whether MDEM lead to abnormalities in this switch.
Methods
NPCs were isolated from mice, and knock-outs (KOs) of epigenetic machinery (EM) genes were created using CRISPR/Cas9. NPCs were differentiated into neural lineages using our neurodevelopmental model to observe the effects of EM-KOs on differentiation. Western Blots and immunofluorescence (IF) were used to analyse neural and glial-specific markers.
Results
We established an assay to estimate developmental stages and cell compositions after EM disruption using Western Blots and IF. Disruption of several EM genes, including Kmt2d, Kmt2a, and Dnmt1, affects NPC differentiation capacity, often resulting in precocious differentiation in our neurodevelopmental model.
Conclusions
Our findings suggest that disruptions of several EM genes lead to precocious differentiation of NPCs. The results will be used to identify candidate genes to create induced pluripotent stem cell lines with patient-specific mutations to further investigate whether precocious differentiation of NPCs occurs in MDEM patients. Understanding how EM disruption affects brain development will significantly improve our understanding of MDEMs and potentially lead to future treatment options.
