Epigenetic Maintenance of Neural Cell Identity

Neurodevelopmental disorders (NDDs) comprise a group of genetically and phenotypically heterogeneous pathologies commonly characterized by psychiatric impairment. The molecular basis of these neuropathologies remains poorly understood. Recent whole-genome-sequencing studies revealed that mutations in genes encoding heterochromatin modifiers are significantly associated with NDDs. This class of transcriptional regulators is thought to stabilize neural cell identity and function by enforcing heritable silencing of lineage non-specific genes through epigenetic chromatin modifications. However, since most heterochromatin modifiers are ubiquitously expressed and lack sequence-specificity, (1) how precise targeting of repressive chromatin is controlled and (2) how mutations in general heterochromatin modifiers contribute to NDD- associated neuronal defects remains unclear. To gain experimental traction on these questions, we will examine the mechanism by which a high-confidence NDD risk gene, ZNF462, recruits the heterochromatin modifiers EHMT1/2. We will test whether and how ZNF462 restricts lineage non-specific gene expression and maintains neural cell identity. ZNF462 haploinsufficiency causes Weiss-Kruszka syndrome, a complex NDD characterized by neurodevelopmental defects including developmental delay and autism. However, the neurodevelopmental role of the C2H2 zinc finger protein is unknown. We previously discovered that mouse Zfp462, is required for endodermal gene repression, directing Ehmt1/2-dependent heterochromatin to transposable element (TE)-derived enhancers in neural progenitor cells. We hypothesize that human ZNF462 controls facultative heterochromatin formation, by specifically restricting non-neural gene expression during neurogenesis. However, we predict that due to rapid species-specific evolution of TEs, ZNF462 will have novel human targets and control a distinct gene regulatory network. W e will therefore: (Aim 1) employ neural differentiation of human embryonic stem cells (hESCs) coupled to epigenome and transcriptome profiling to investigate the impact of ZNF462 heterozygosity on maintenance of neural gene expression, (Aim 2) perform structure-function analysis and functional complementation in mESCs to identify ZNF462 protein domains responsible for homodimerization, DNA binding and transcriptional repression and (Aim 3) profile CTCF binding and three-dimensional chromosome conformation in neuroepithelial stem cells (NESCs) to investigate the impact of ZNF462 heterozygosity on neuro-specific genome architecture. Our proposal provides a path to novel insight into the molecular mechanism of ZNF462-dependent gene silencing, and enhance our understanding of the etiology of Weiss-Kruszka syndrome. The following strategy will reveal new concepts in gene regulation and neurobiology and elucidate the link between mutations in heterochromatin modifiers and NDDs. Overall, our work will inform novel strategies to prevent and treat NDDs arising from epigenetic dysregulation.