CTCF-dependent mechanisms of ATRX in neuronal differentiation

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PROJECT SUMMARY ATRX is an epigenetic factor that is mutated in ATRX syndrome. ATRX is required for the maintenance of multipotent neuroprogenitor cells (NPCs), particularly as these cells initiate differentiation programs in the brain. Mutations that cause ATRX syndrome cluster within two domains: the ATRX PHD finger domain, and the helicase domain. These mediate interactions with histones, and remodel chromatin, respectively. Interestingly, mutations in the PHD finger are associated with severe intellectual disability and psychomotor impairment, while mutations in the helicase domain often manifest with milder neurodevelopmental delays but more severe genital abnormalities13. The basis for this genotype-phenotype correlation has never been investigated. ATRX has well- established roles in molecular processes that are crucial for normal brain development including histone variant H3.3 deposition and Polycomb repressive complex 2 targeting for epigenetic silencing. It also has a poorly understood role in regulating CTCF, a critical genome architectural protein that is essential for development. Our preliminary data indicate PHD finger and helicase mutations of ATRX differentially regulate CTCF localization and may underlie genotype-phenotype correlations. Our preliminary data has uncovered a genome-wide role for ATRX in CTCF localization. We found that ATRX knock down (KD) in mouse embryonic stem cells (mESCs) results in CTCF accumulation at many genomic sites, including both imprinted and non-imprinted loci. We also discovered that ATRX interacts with ADNP and DNMT3L, both of which can prevent CTCF binding. Using CRISPR/Cas9, we have generated isogenic ESCs where the endogenous Atrx allele has been replaced by point mutants found in ATRX syndrome patients. We show that mutations in the PHD finger cause significant impairment of NPC differentiation, while mutations in the helicase domain cause more subtle differentiation delays. Our preliminary findings and novel reagents uniquely position us to interrogate the consequence of ATRX mutations, both for genome organization through CTCF, and for the ability of ESCs to differentiate into NPCs. Based on our preliminary data, we propose to test the hypothesis that distinct ATRX domains regulate specific chromatin processes that impinge to different extents on the function of CTCF. In Aim 1, we will investigate the consequence of ATRX syndrome mutations to gene expression and genome organization during neuronal differentiation. In Aim 2, we will decipher the mechanisms by which ATRX regulates CTCF localization.