Epigenetic regulation of neural stem cell biology by Tet DNA dioxygenases

ABSTRACT The ten-eleven translocation family (TET1/2/3) of enzymes are epigenetic regulators of gene expression that are highly expressed in neural stem cells (NSCs) and during mammalian nervous system development. TET enzymes are dioxygenases that promote active and passive DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethycytosine (5hmC) and higher-order oxidized derivatives. In addition to its role as a demethylation intermediate, 5hmC can function as a stable epigenetic mark and is highly enriched and dynamic in the developing nervous system. TET enzymes and 5hmC dysregulation have been implicated in human neurodevelopmental syndromes, intellectual disability, craniofacial abnormalities, and neurodegeneration. These observations suggest a critical role for TET enzymes in the developing nervous system and has led to interest in their roles in the biology of NSCs. However, the functions of TET enzymes in NSCs and neurodevelopment remain poorly understood. Preliminary data from our lab demonstrates that Tet triple- knockout NSCs (T123–/–) derived from embryonic stem cells exhibit severe defects in self-renewal, multipotency, and expression of neurodevelopmental genes. We therefore hypothesize that TET enzymes have essential functions in epigenetic regulation of gene expression programs critical for NSC maintenance and multipotency and in embryonic neurodevelopment. To test this hypothesis, we have derived embryonic forebrain NSC lines containing floxed alleles of Tet1/2/3 and a tamoxifen-inducible Cre recombinase transgene expressed from the constitutive Rosa26 locus (T123F/F; +/R26-CreER) for conditional, combined deletion of all three Tet genes. We have also established a colony of Tet1/2/3 triple-floxed mice expressing a tamoxifen-inducible Cre recombinase transgene under control of the neural-specific Nestin promoter (T123F/F; +/Nestin-CreERT2). Using these models, we will (1) define the role of TET enzymes in the maintenance and multipotency of NSCs , (2) establish the requirement of TETs in embryonic neurodevelopment, and (3) identify TET-mediated epigenetic and transcriptional regulatory mechanisms in NSCs. Findings from these studies will define novel roles played by TET enzymes in NSC biology and neurodevelopment, provide insights into how dysregulation of TETs contribute to human neurodevelopmental disorders, and identify novel targets for therapy. Under the joint mentorship of Drs. Meelad Dawlaty and Jean Hébert, I will successfully execute the proposed research and training plan. This research program will further my knowledge of the epigenetic regulation of neural stem cell biology and facilitate my scientific and professional development by equipping me with the necessary skills to become a physician- scientist.