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

阐明CUL3环泛素连接酶在hESC维持和分化中的新作用 在600个人类E3中，CUL3环连接酶(CRL3)是一个多亚基E3家族，它使用90个含BTB结构域的蛋白质作为底物接头。虽然已知特定的CRL3-BTB复合体调节人类发育和生理的关键方面，但大多数CRL3 E3的生物学功能仍有待发现。这一目标的主要目标是确定CRL3-BTB复合体在发育中的新角色，并剖析其作用机制的分子基础。 我们以前已经发现了一个特殊的CUL3-BTB复合体，它是hESC肌动蛋白动力学和拟胚体向神经元分化的重要调节因子。在上一个资助阶段，通过结合蛋白质组学和生化方法以及hESC分化实验，我们已经确定了一个肌动蛋白细胞骨架信号模块，通过该模块，CUL3-BTB复合体调控基于胚体的神经前体和神经元的形成。由于已知该信号模块许多组件的突变会导致神经发育疾病，我们假设CUL3-BTB复合体本身的突变也可能导致人类神经发育缺陷。因此，我们与Daniel Kastners实验室(NHGRI)合作，系统地查询了未诊断的发育性疾病患者的外显子组数据库中的BTB变体，重点是那些罕见的等位基因频率和位于功能蛋白结构域的疾病。事实上，我们在两个表型重叠的患者中发现了突变，两人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，我们可以证明这些患者变体要么减少与催化亚单位CUL3的结合，要么减少与肌动蛋白细胞骨架信号模块的结合。这强烈表明，CUL3-BTB复合体的泛素化活性是通过肌动蛋白细胞骨架信号模块传递信号所必需的，如果减少，就会导致神经发育疾病。我们目前的努力是机械地剖析这些过程。 探讨脱泛素酶在胚胎发育过程中的作用和机制 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，协调人类发育的许多方面。泛素化通过连接一个泛素分子或通过不同K残基连接的泛素链，能够调节从底物降解到细胞内信号通路的各种底物命运。卵巢肿瘤脱泛素酶家族(OTU DUBS)是泛素编码的重要调节者，控制着人类生理的关键方面。OTU DUB通过靶向多聚泛素内不同的连接类型来调节其底物的稳定性、活性或相互作用环境，从而诱导其功能。虽然一些OTU DUB具有很好的特征，并与单基因疾病有关，但大多数OTU DUB的生理功能和潜在机制在很大程度上仍然难以捉摸。 在过去的资助期间，我们发现了一种新的多发性先天性畸形综合征，即连锁(LINKage-specific-deubiquitylation-deficiency-induced胚胎缺陷综合征，由脱泛素酶OTUD5/DUBA的低形态半合子错义变异引起。受影响的个体有临床表现，包括结构性脑畸形、先天性心脏病、轴心后多指畸形和头面部缺陷。在研究体外、小鼠和人类多能干细胞神经外胚层分化模型中的连锁突变时，我们发现了一种新的调节电路，它协调了早期分化过程中的染色质重塑途径。我们发现OTUD5的K48链特异的去泛素化活性对小鼠和人类的发育是必不可少的，如果降低，会导致异常的细胞命运指定。OTUD5通过阻止包括ARID1A/B和HDAC2在内的多种染色质调节因子的降解来控制分化，这些染色质调节因子的突变是与相关患者表现出表型重叠的发育综合征的基础。因此，在早期分化过程中，OTUD5的缺失导致神经和神经脊增强子的染色质更难获得，从而导致基因表达网络的异常重新布线。我们的工作在表型相关的发育障碍和在早期细胞命运决定过程中对底物组(即染色质重塑复合体)的连接特异性泛素编辑的基本功能之间发现了一种新的机制联系，我们预测这一调控概念将成为胚胎发育的一般特征。

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.