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

摘要： 在美国，大约每500名男性中就有1人受到X连锁智力残疾(XLID)的影响。我们的 实验室之前已经鉴定了O-GlcNAc转移酶(OGT)基因的几个突变，这些突变是 一种综合征形式的XLID的病因和最近发现的三个新的催化错义突变 临床合作者的领域。OGT是一种必需的糖基转移酶，它单独负责 丝氨酸和苏氨酸上的翻译后修饰β-N-乙酰氨基葡萄糖(O-GlcNAc) 目标核蛋白和胞质蛋白。OGT和O-GlcNAc与多种细胞 过程和疾病，包括神经发育、转录调控和XLID。我们之前的工作 实验室生化表征了OGT四肽重复(TPR)结构域的突变，但统一的 XLID表型的下游对转录调控的影响尚不清楚。vt.给出 催化结构域和TPR结构域突变都是XLID的原因，我们的假设是两者都是新的 催化结构域变异体和先前描述的TPR结构域变异体导致基因失调 不能完全糖基化参与转录调控的关键靶蛋白的表达 OGT靶向缺失(TPR结构域)或OGT催化效率降低(催化域)。这一假设 有数据表明，与野生型相比，TPR结构域变体已经改变了转录 而催化结构域的变异可能是XLID的原因。为了验证我们的假设，我们将通过生物化学 表征新的催化结构域变体，我们将确定两者基因表达的变化 催化结构域变体和先前表征的TPR结构域变体。我们的实验室正准备独一无二地 由于我们在O-GlcNAc生物学方面的专业知识、之前对XLID变体的研究以及我们的 拥有表达OGT TPR结构域变体的Cas9工程男性胚胎干细胞。在……里面 目的1、采用体外检测和全细胞检测的方法，检测细胞生化特性的变化。 新的催化结构域变体包括热稳定性、动力学参数和对全球O-的影响 在纤维细胞中表达时的GlcNAc水平。在目标2中，我们将确定ALL之间基因表达的变化 我们将CRISPR/Cas9基因工程的人胚胎干细胞分化为神经干细胞时的变异特征 前体细胞。当结合CHIP-SEQ数据时，我们可以评估OGT变体对监管的影响 关于基因表达的。根据初步数据，我们还将研究TET2和HCF1作为潜在的OGT 相互作用因子/底物来解释基因表达的失调。这些方法将有助于阐明 不同功能缺陷的变异导致患者具有相同的XLID神经发育表型。 此外，这项研究将在加州大学复合碳水化合物研究中心进行 乔治亚州在兰斯·威尔斯博士的指导下，这将为学员提供一个良好的学习环境 在模范导师的指导下，掌握一般和专门的生化技能以及批判性思维技能。

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.