Ubiquitin-dependent cell-fate decisions during human development and disease

人类发育和疾病过程中泛素依赖性细胞命运决定

• 批准号: 10248894
• 负责人: Achim Werner
• 金额: $ 143.94万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10248894
• 关键词: Actins; Affect; BTB/POZ Domain; Binding; Biochemical; Biogenesis; Biological Assay; Biological Process; CUL3 gene; Catalytic Domain; Cell Differentiation process; Cell Fate Control; Cells; Chromatin; Chromatin Remodeling Factor; Clinical; Co-Immunoprecipitations; Code; Collaborations; Complex; Craniofacial Abnormalities; Cytoskeleton; Databases; Defect; Deposition; Development; Disease; Embryo; Embryonic Development; Enhancers; Enzymes; Excision; Exhibits; Family; Funding; Gene Expression; Gene Frequency; Goals; HDAC2 gene; Human; Human Development; Human Genetics; Immunoblotting; In Vitro; Individual; Intellectual functioning disability; Ligase; Link; Maintenance; Modeling; Molecular; Mus; Mutation; National Human Genome Research Institute; Neural Crest; Neuronal Differentiation; Neurons; Pathway interactions; Patients; Phenotype; Physiological; Physiology; Polydactyly; Polyubiquitin; Post-Translational Protein Processing; Process; Proteins; Proteomics; Regulation; Research; Ribosomes; Role; Signal Pathway; Signal Transduction; Structure; Syndrome; Tertiary Protein Structure; Translations; Ubiquitin; Variant; Work; base; brain malformation; cell fate specification; chromatin remodeling; congenital anomaly; congenital heart disorder; craniofacial development; developmental disease; exome; human disease; human embryonic stem cell; human pluripotent stem cell; insight; nerve stem cell; neurodevelopment; neuron development; novel; novel therapeutic intervention; ovarian neoplasm; prevent; programs; rare variant; self-renewal; stem cells; ubiquitin ligase; ubiquitin-protein ligase

To elucidate novel roles for specific CUL3-RING ubiquitin ligases in hESC maintenance and differentiation Amongst 600 human E3s, Cul3-RING Ligases (CRL3s) are a family of multi-subunit E3s that use 90 BTB domain-containing proteins as substrate adaptors. While particular CRL3-BTB complexes are known to regulate crucial aspects of human development and physiology, biological functions of the majority of the CRL3 E3s are still to be discovered. It is the major goal of this aim to identify novel roles for CRL3-BTB complexes in development and dissect the molecular underpinnings of their mechanism of action. We have previously identified a particular CUL3-BTB complex as an essential regulator of hESC actin dynamics and of neuronal differentiation from embryoid bodies. During the last funding period, through combining proteomic and biochemical approaches with hESC differentiation assays, we have identified an actin cytoskeleton signaling module through which the CUL3-BTB complex regulates embryoid-body based neural progenitor and neuron formation. Since mutations in many of the components of this signaling module are known to cause neurodevelopmental diseases, we hypothesized that also mutations in the CUL3-BTB complex itself could cause defects in human neurodevelopment. Thus, in collaboration with the lab of Daniel Kastners lab (NHGRI), we systematically queried exome databases for BTB variants in patients with undiagnosed developmental diseases, focusing on those with rare allele frequency and located in functional protein domains. Indeed, we found mutations in two phenotypically overlapping patients both exhibiting intellectually disability and structural brain malformations. Intriguingly, using immunoblotting and co-immunoprecipitation, we can show that these patient variants either reduce binding to the catalytic subunit CUL3 or to the actin cytoskeleton signaling module. This strongly suggests that ubiquitylation activity by the CUL3-BTB complex is required for signaling through the actin cytoskeleton signaling module for proper neuronal development and, if reduced, leads to neurodevelopmental disease. Our current efforts are geared towards mechanistically dissecting these processes. To dissect the functions and mechanism of deubiquitylases during embryonic development Ubiquitylation, the covalent attachment of ubiquitin to proteins, is an essential post-translational modification that orchestrates many aspects of human development. Through attachment of either one ubiquitin molecule or chains of ubiquitin typically linked through different K residues, ubiquitylation is able to regulate various substrate fates ranging from substrate degradation to control of intracellular signaling pathways. Deubiquitylases of the ovarian tumor family (OTU DUBs) are important regulators of the ubiquitin code and control crucial aspects of human physiology. OTU DUBs elicit their functions by targeting distinct linkage types within polyubiquitin to modulate the stability, activity, or interaction landscapes of their substrates. While some OTU DUBs are well characterized and have been linked to monogenetic diseases, the physiological functions and underlying mechanisms of the majority of OTU DUBs have remained largely elusive. During the last funding period, we have discovered LINKED (LINKage-specific-deubiquitylation-deficiency-induced Embryonic Defects) syndrome, a novel multiple congenital anomaly disorder caused by hypomorphic hemizygous missense variants in the deubiquitylase OTUD5/DUBA. Affected individuals have clinical manifestations including structural brain malformations, congenital heart disease, post-axial polydactyly, and craniofacial defects. Studying LINKED mutations in vitro, in mouse, and in models of neuroectodermal differentiation of human pluripotent stem cells, we have uncovered a novel regulatory circuit that coordinates chromatin remodeling pathways during early differentiation. We show that the K48-linkage-specific deubiquitylation activity of OTUD5 is essential for murine and human development and, if reduced, leads to aberrant cell-fate specification. OTUD5 controls differentiation through preventing the degradation of multiple chromatin regulators including ARID1A/B and HDAC2, mutation of which underlie developmental syndromes that exhibit phenotypic overlap with LINKED patients. Accordingly, loss of OTUD5 during early differentiation leads to less accessible chromatin at neural and neural crest enhancers and thus aberrant rewiring of gene expression networks. Our work identifies a novel mechanistic link between phenotypically related developmental disorders and an essential function for linkage-specific ubiquitin editing of substrate groups (i.e. chromatin remodeling complexes) during early cell-fate decisions a regulatory concept, we predict to be a general feature of embryonic development.

阐明特异性CUL 3-RING泛素连接酶在hESC维持和分化中的新作用 在600种人E3中，Cul 3-RING连接酶（CRL 3）是使用90种含有BTB结构域的蛋白作为底物衔接子的多亚基E3家族。虽然已知特定的CRL 3-BTB复合物调节人类发育和生理学的关键方面，但大多数CRL 3 E3的生物学功能仍有待发现。这一目标的主要目标是确定CRL 3-BTB复合物在发育中的新作用，并剖析其作用机制的分子基础。 我们以前已经确定了一个特定的CUL 3-BTB复合物作为hESC肌动蛋白动力学和胚状体神经元分化的重要调节因子。在上一个资助期间，通过将蛋白质组学和生物化学方法与hESC分化测定相结合，我们已经鉴定了肌动蛋白细胞骨架信号传导模块，CUL 3-BTB复合物通过该模块调节基于胚体的神经祖细胞和神经元形成。由于已知该信号传导模块的许多组分的突变会导致神经发育疾病，因此我们假设CUL 3-BTB复合物本身的突变也可能导致人类神经发育缺陷。因此，我们与丹尼尔卡斯特纳斯实验室（NHGRI）的实验室合作，系统地查询了未诊断发育疾病患者中BTB变体的外显子组数据库，重点关注那些具有罕见等位基因频率并位于功能蛋白质结构域的基因。事实上，我们在两个表型重叠的患者中发现了突变，这两个患者都表现出智力残疾和结构性脑畸形。有趣的是，使用免疫印迹和免疫共沉淀，我们可以表明，这些患者的变体减少结合到催化亚基CUL 3或肌动蛋白细胞骨架信号模块。这强烈地表明，通过CUL 3-BTB复合物的泛素化活性是通过肌动蛋白细胞骨架信号传导模块进行信号传导所需的，用于适当的神经元发育，并且如果降低，则导致神经发育疾病。我们目前的努力是面向机械解剖这些过程。 探讨去泛素化酶在胚胎发育过程中的作用及机制 泛素化是泛素与蛋白质的共价连接，是一种重要的翻译后修饰，它协调了人类发育的许多方面。通过连接一个泛素分子或通常通过不同K残基连接的泛素链，泛素化能够调节各种底物命运，从底物降解到控制细胞内信号传导途径。卵巢肿瘤家族的去泛素化酶（OTU DUBs）是泛素编码的重要调节剂，并且控制人类生理学的关键方面。OTU DUB通过靶向多聚泛素内的不同连接类型来调节其底物的稳定性、活性或相互作用景观来引发其功能。虽然一些OTU DUB被很好地表征并且已经与单基因疾病相关联，但是大多数OTU DUB的生理功能和潜在机制在很大程度上仍然是难以捉摸的。 在上一个资助期内，我们发现了LINKED（LINKage-specific-deubiquitylation-deficiency-induced Embryonic Defects）综合征，这是一种由去泛素化酶OTUD 5/杜巴中的半合子错义变体引起的新型多发性先天性异常疾病。受影响的个体具有临床表现，包括结构性脑畸形、先天性心脏病、轴后多指畸形和颅面缺陷。在体外，在小鼠和人类多能干细胞的神经外胚层分化模型中研究LINKED突变，我们发现了一种新的调节电路，在早期分化过程中协调染色质重塑途径。我们发现OTUD 5的K48-连接特异性去泛素化活性对于小鼠和人类发育是必不可少的，如果减少，会导致异常的细胞命运特化。OTUD 5通过防止包括ARID 1A/B和HDAC 2在内的多种染色质调节因子的降解来控制分化，ARID 1A/B和HDAC 2的突变是表现出与LINKED患者表型重叠的发育综合征的基础。因此，在早期分化过程中OTUD 5的丢失导致神经和神经嵴增强子处的染色质不易接近，从而导致基因表达网络的异常重新布线。我们的工作确定了表型相关的发育障碍之间的一种新的机制联系，以及在早期细胞命运决定过程中底物组（即染色质重塑复合物）的联系特异性泛素编辑的基本功能，我们预测这是胚胎发育的一般特征。