Functional characterization of clinically-identified coding variants in the transcription factor gene ZEB2

Project Summary/Abstract Mutations in the ZEB2 transcription factor (TF) gene are the cause of Mowat-Wilson syndrome (MOWS), an intellectual and developmental disability (IDD). While MOWS is due primarily to large deletions or premature stop codons in ZEB2, little is known about rare missense variants in the gene. To date, there has not been a

genome-wide identification of ZEB2 DNA binding sites and its target genes in IDD-relevant cells. This proposal aims to fill this knowledge gap using modified protocols developed in the Myers Lab for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). The proposal applies these methods

to identify the wild-type (WT) ZEB2 binding sites in induced pluripotent stem cells (iPSCs) as well as in neuronal cells differentiated from these iPSCs; these cell types are highly relevant for IDD research. The lab’s recently published method for assaying missense variants in TF genes will be used to perform the experiment

for five clinically-identified variants across domains of the ZEB2 gene. Differential binding will be assessed in these variant cells, as well as the genomic association sites for two essential cofactors for ZEB2: SMAD1 and CTBP1. This information will further clarify both WT and mutant ZEB2 functional activity. Finally, analytical

methods will be used to establish likely target genes for ZEB2 regulatory activity, and RNA-seq will be performed to measure gene expression, correlating differential expression in variant cell lines with differential binding. This work will be highly impactful for ZEB2 and MOWS research and future clinical diagnoses and will

lay the foundation for potential therapeutic interventions.