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

National Institute Of Dental & Craniofacial Research

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Abstract

To dissect the functions and mechanism of deubiquitylases during embryonic development The subfamily of ovarian tumor (OTU) DUBs have emerged as important regulators of ubiquitin chain architecture to control essential cellular processes and diverse aspects of human physiology and development. OTU DUBs elicit their functions by hydrolyzing specific linkage types within polyubiquitin to modulate the stability, activity, or interaction landscapes of their substrates. In particular, cleavage of K48-linked ubiquitin chains protects substrates from proteasomal degradation and cleavage of M1- and K63-linked ubiquitin chains limits intracellular signaling. Five out of the sixteen OTU DUBs have been linked to human disease: mutations in OTULIN and TNFAIP3 (encoding A20) cause autoinflammatory disorders, mutations in OTUD7A and ALG13 are associated with neurodevelopmental disorders, and mutations in OTUD6B cause multiple congenital anomalies. However, with the exception of OTULIN and A20, the underlying mechanisms and cognate substrates for these disease-associated enzymes are ill-defined and the physiological functions for other OTU DUB members have remained largely unknown. We have identified a cohort of patients with mutations in several OTU DUBs, all with developmental anomalies. Thus, we hypothesize that linkage-specific deubiquitylation activity of OTU DUBs is essential to cleave ubiquitin modifications to ensure proper ubiquitin signaling during embryonic development. The major goal of this project to mechanistically dissect how alterations in activity of particular OTU DUBs lead to developmental disease. 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 (Beck*, Basar* et al, 2021, Sci Adv). 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. To determine the functions of spatially regulated E1 activity and ubiquitin activation during hematopoietic cell-fate decisions Together with the labs of Dr. Daniel Kastner and Peter Grayson, we have employed a genotype-first approach focusing on ubiquitylation genes to identify a novel autoinflammatory syndrome caused by mutations in the major ubiquitin E1 activating enzyme (Beck et al, 2020, N Eng J Med)(Poulter*, Collins* et al, 2021, Blood). We found 25 male patients with somatic variants at or around codon 41 in X-linked UBA1 (most commonly p.Met41Val, p.Met41Thr, p.Met41Leu). These individuals all present with severe late-onset autoinflammatory disease characterized by fevers, cytopenias, and unique, pathognomonic vacuoles in myeloid and erythroid precursors cells. These patients fulfilled clinical criteria for inflammatory (relapsing polychondritis, Sweet syndrome, polyarteritis nodosa, giant cell arteritis) and hematologic (myelodysplastic syndrome or multiple myeloma) conditions. We have named this disease VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. VEXAS syndrome is defined by somatic mosaicism: in affected patients the recurrent UBA1p.Met41 mutation is present in more than half of hematopoietic stem cells in the bone marrow, yet in the peripheral blood, it is found exclusively in myeloid cells but not in lymphocytes, suggesting genetic selection. These findings highlight a novel paradigm for rheumatic diseases in which, similar to malignancy, somatic variants drive inflammation. To determine the molecular basis of VEXAS, we employed patient-cell based and biochemical assays. As expected from loss of the initiator methionine at Met41, these studies revealed that VEXAS mutations resulted in loss of the canonical cytoplasmic isoform UBA1b in mutant myeloid but not lymphoid wildtype lineages. Surprisingly, this was accompanied by expression of a novel, catalytically impaired isoform initiated at Met67. Accordingly, we detected decreases in ubiquitylation specifically in mutant peripheral blood myeloid cells, which also underwent necrotic cell death and exhibited activated innate immune pathways. Together, these findings suggest that loss of cytoplasmic UBA1 function in myeloid lineages is a major driver of autoinflammation in VEXAS syndrome. While all analyzed patient monocytes showed loss of cytoplasmic UBA1 function through reduction in UBA1b and emergence of UBA1c, for some individuals we also noted a reduction in nuclear UBA1a protein. To better define the contribution of nuclear and cytoplasmic UBA1 isoforms to inflammatory disease in vivo, we established CRISPR/ Cas9-edited zebrafish models. Zebrafish and human UBA1 genes are highly homologous. As uba1 is essential for viability, we assessed inflammation during early development. Homozygous loss of either all isoforms of uba1 or loss of uba1b alone, but not uba1a in mpx:EGFP transgenic zebrafish led to decreased numbers of neutrophils compared to sibling controls. All three zebrafish lines deficient in uba1 also showed growth abnormalities and early lethality, a finding that may in part be due to the germline nature of these mutations as compared to the somatic variants found in patients with VEXAS. Finally, loss of uba1 or uba1b alone, but not uba1a in zebrafish led to upregulation of similar inflammatory pathway genes as in patients. Thus, we conclude that loss of cytoplasmic UBA1 function causes inflammation in vivo and is the major molecular cause of VEXAS. Taken together, our ubiquitylation-focused genotype-first approach allowed the discovery of VEXAS syndrome, a novel autoinflammatory disorder caused by somatic, recurrent, missense mutations in UBA1. Through studying VEXAS mutations, we have identified loss of cytoplasmic ubiquitin activation through aberrant translation of UBA1 isoforms as a major cause of this disease. In addition, our findings elucidate an unexpected regulatory layer of ubiquitin signaling at its apex, which we predict to contribute to developmental cell-fate decisions.