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

项目摘要 CRL4泛素连接酶复合体调节许多重要的生物过程，如DNA DNA甲基化的复制、DNA修复、转录调控和表观遗传。我们 新近发现CRL4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SOX2(SRY-box2)。SOX2是一种剂量依赖的 在调节自我更新和多能性方面起关键作用的主要转录因子 ESCs、IPSCs以及许多胎儿和成人干细胞的多能性。在ESCs中，SOX2的增加 促进外胚层和中胚层的发育，同时SOX2的丢失或减少 诱导分化为内胚层和滋养外胚层。即使在4-细胞胚胎阶段， SOX2与靶基因的异质结合决定了第一个谱系决定。在人类身上， 单个Sox2等位基因上的功能丧失突变足以导致家族性 无眼球/小眼球综合症与癫痫、脑异常、生长缓慢、 运动技能迟缓和学习障碍。相反，基因扩增和过度表达 SOX2经常与许多低分化癌有关，包括肺癌、食道癌、 脑癌和乳腺癌。然而，目前还不清楚SOX2蛋白水平是如何在 发育过程中的各种干/祖细胞和组织动态平衡。我们最近发现， SOX2蛋白稳定性受一种新的基于CRL4的赖氨酸蛋白分解机制的调节 甲基化在胚胎干细胞和其他相关细胞中作为蛋白质分解的触发物。这种特殊的基因突变 CRL4功能损害胚胎发育。我们建议解开它的功能和调节。 这种新的和重要的基于CRL4的蛋白分解机制调节自我更新和 胚胎干细胞和胚胎发育的多能性。我们的具体目标1是定义 以CRL4为基础的泛素E3连接酶，针对甲基化的SOX2蛋白进行降解。我们的特定 目的2是确定甲基化的SOX2蛋白水平是如何动态调节的 胚胎干细胞的自我更新。在我们的具体目标3中，我们建议研究SOX2的功能是如何 在动物发育中使用甲基化缺陷的特定基因突变来调节- 依赖的蛋白分解途径。赛曼莎·劳埃德是一名大四的本科生 进行2023年夏季RO1拨款GM140185中提议的研究。她目前 考虑成为UNLV张博士实验室的一名博士研究生。

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.