Supplement: Regulation of SOX Proteins by Methylation-dependent Proteolysis in Stem Cells and Development

项目摘要 CRL 4泛素连接酶复合物调节许多重要的生物过程，如DNA 复制，DNA修复，转录调控和DNA甲基化的表观遗传。我们 最近发现，CRL 4还调节胚胎干细胞的自我更新和多能性 （ESCs）通过重要的干细胞蛋白如SOX 2（SRY-box 2）。SOX 2是一种剂量依赖性 在调节自我更新和多能性中起关键作用的主转录因子，或 干细胞是胚胎干细胞、诱导多能干细胞以及许多胎儿和成体干细胞的多能性。在ESC中，SOX 2的增加 促进发育成外胚层和中胚层谱系，而SOX 2的丢失或减少 诱导分化成内胚层和滋养外胚层谱系。即使在4细胞胚胎阶段， SOX 2与靶基因的异质结合决定了第一谱系决定。在人类中， 单个Sox 2等位基因上的功能缺失突变足以引起家族性 无眼/小眼综合征与癫痫发作、脑异常、生长缓慢， 运动技能延迟和学习障碍。相反，基因扩增和过度表达 SOX 2通常与许多低分化癌症相关，包括肺癌、食道癌、 脑癌和乳腺癌。然而，目前还不清楚SOX 2蛋白水平是如何调节的， 各种干/祖细胞在发展和组织稳态。我们最近发现， SOX 2的蛋白质稳定性由一种新的基于CRL 4的蛋白水解机制调节， 甲基化作为ESC和其他相关细胞中的蛋白水解触发剂。这种特殊的基因突变 CRL 4功能损害胚胎发育。我们建议解开的功能和调节 这种新的和重要的基于CRL 4的蛋白水解机制在调节自我更新和 胚胎干细胞和胚胎发育期间的多能性。我们的具体目标1是确定 基于CRL 4的泛素E3连接酶，靶向甲基化SOX 2蛋白进行降解。我们的具体 目的2是确定甲基化SOX 2蛋白的水平如何动态调节， ESCs的自我更新。在我们的具体目标3中，我们建议检查SOX 2的功能是如何被激活的。 在动物发育中使用甲基化缺陷的特定遗传突变体进行调节- 依赖的蛋白水解途径。Symantha Lloyd是一名高年级本科生， 进行研究中提出的RO 1赠款GM 140185为2023年夏天。她目前正在 我正在考虑去UNLV的张博士实验室读博士研究生。

Project Summary The CRL4 ubiquitin ligase complexes regulate many important biological processes such as DNA replication, DNA repair, transcriptional regulation, and epigenetic inheritance of DNA methylation. We recently found that CRL4 also regulates the self-renewal and pluripotency of embryonic stem cells (ESCs) through important stem cell proteins such as SOX2 (SRY-box2). SOX2 is a dose-dependent master transcriptional factor that plays a key role in regulating the self-renewal and pluripotency or multipotency of ESCs, iPSCs, and many fetal and adult stem cells. In ESCs, an increase of SOX2 promotes development into ectoderm and mesoderm lineages, while loss or reduction of SOX2 induces differentiation into endoderm and trophectoderm lineages. Even at the 4-cell embryonic stage, the heterogeneous binding of SOX2 to target genes determines the first lineage decision. In human, loss-of-function mutations on a single Sox2 allele is sufficient to cause the familial anophthalmia/microphthalmia syndrome associated with seizures, brain abnormalities, slow growth, delayed motor skills and learning disability. Conversely, gene amplification and over-expression of SOX2 are frequently associated with many poorly differentiated cancers including lung, esophagus, brain, and breast cancers. However, it remains unclear how the SOX2 protein level is regulated in various stem/progenitor cells during development and tissue homeostasis. We recently found that the protein stability of SOX2 is regulated by a novel CRL4-based proteolytic mechanism using lysine methylation as a proteolytic trigger in ESCs and other related cells. Genetic mutation of this particular CRL4 function impairs embryonic development. We propose to unravel the function and regulation of this novel and important CRL4-based proteolytic mechanism in regulating self-renewal and pluripotency of ESCs and during embryonic development. Our specific aim 1 is to define the roles of CRL4-based ubiquitin E3 ligases that target the methylated SOX2 protein for degradation. Our specific aim 2 is to determine how the levels of methylated SOX2 proteins are dynamically regulated during the self-renewal of ESCs. In our specific aim 3, we propose to examine how the function of SOX2 is regulated in animal development using specific genetic mutants defective in the methylation- dependent proteolysis pathway. Symantha Lloyd is a senior undergraduate student that is scheduled to conduct research proposed in the RO1 grant GM140185 for the summer of 2023. She is currently considering to become a PhD graduate student in Dr. Zhang’s Laboratory at UNLV.