Utilizing Causal X-Linked Intellectual Disability Variants to Gain Insight into the O-GlcNAc Transferase Enzyme

Summary: Approximately 1 in 500 males in the United States are affected by X-Linked Intellectual Disability (XLID). Our laboratory has previously characterized several mutations in the O-GlcNAc Transferase (OGT) gene that are causal for a syndromal form of XLID and have recently discovered three novel missense mutations in the catalytic

domain with clinical collaborators. OGT is an essential glycosyltransferase that is solely responsible for the addition of the post-translational modification beta-N-acetylglucosamine (O-GlcNAc) onto serines and threonines of target nuclear and cytosolic proteins. OGT and O-GlcNAc have been implicated in a variety of cellular

processes and diseases including neurodevelopment, transcriptional regulation, and XLID. Previous work by our lab biochemically characterized mutations in the Tetratricopeptide Repeat (TPR) domain of OGT, but a unifying downstream effect on transcription regulation responsible for the XLID phenotype has yet to be elucidated. Given

both catalytic domain and TPR domain mutations are causal for XLID, our hypothesis is that both the novel catalytic domain variants and previously described TPR domain variants cause a dysregulation of gene expression by an inability to fully glycosylate key target proteins involved in transcriptional regulation due to a

loss of OGT targeting (TPR domain) or reduction in OGT catalytic efficiency (catalytic domain). This hypothesis is supported by data demonstrating that TPR domain variants have altered transcription compared to wildtype and that catalytic domain variants can be causal for XLID. To test our hypothesis, we will biochemically

characterize the novel catalytic domain variants, and we will determine changes to gene expression for both catalytic domain variants and previously characterized TPR domain variants. Our lab is uniquely poised to address this hypothesis due to our expertise in O-GlcNAc biology, previous work with XLID variants, and our

possession of Cas9-engineered male human embryonic stem cells expressing TPR domain variants of OGT. In aim 1, we will use in vitro assays and whole cell assays to determine changes in the biochemical characteristics of the novel catalytic domain variants including thermal stability, kinetic parameters, and impact on global O-

GlcNAc levels when expressed in cellulo. In aim 2, we will determine changes in gene expression between all characterized variants as we differentiate CRISPR/Cas9-engineered human embryonic stem cells to neural precursor cells. When combined with ChIP-Seq data, we can evaluate the impact of OGT variants on regulation

of gene expression. Based on preliminary data, we will also investigate Tet2 and HCF1 as potential OGT interactors/substrates to explain the dysregulation of gene expression. These approaches will help elucidate how variants deficient in different functions result in the same XLID neurodevelopmental phenotype in the patient.

Furthermore, this research will take place at the Complex Carbohydrate Research Center at the University of Georgia under the direction of Dr. Lance Wells, that will provide the trainee an excellent environment to learn general and specialized biochemical skills as well as critical thinking skills under exemplary mentorship.