To dissect the mechanism of ubiquitin-dependent regulation of ribosome biogenesis and translation during neural crest and craniofacial development We had previously identified the multi-subunit ubiquitin E3 ligase CUL3 RING Ligase (CRL3) with its vertebrate-specific substrate adaptor KBTBD8 (CRL3-KBTBD8) as an essential regulator of human and Xenopus tropicalis neural crest cell formation. We showed that CRL3-KBTBD8 ubiquitylates the ribosome biogenesis regulator NOLC1 and its paralog TCOF1, whose mutation underlies Treacher Collins Syndrome, a neurocristopathy characterized by loss of cranial neural crest cells and defects in craniofacial development. Intriguingly, CRL3-KBTBD8 controls neural crest cell formation and craniofacial development by regulating the function of newly synthesized ribosomes to alter the translational program of differentiating cells. However, the precise molecular mechanism of ribosome specification and the signaling networks that integrate this novel type of regulation into the differentiation program have remained elusive. During the last funding period, we have been able to show that CRL3-KBTBD8-dependent monoubiquitylation and neural crest specification require multisite substrate phosphorylation by CK2, a kinase whose levels gradually increase during development of the nervous system. The essential CRL3KBTBD8-substrates TCOF1 and NOLC1 contain ten or more motifs that, following their phosphorylation by CK2, can be independently recognized by a conserved surface on KBTBD8. We found that seven or more CK2 motifs need to be present in the same substrate to mediate both monoubiquitylation by CRL3KBTBD8 as well as neural crest specification. Multisite dependency allows cells to convert a gradual increase in kinase inputs, as seen for embryonic CK2, into decisive activation of signaling outputs. We therefore propose that multisite dependency of CRL3KBTBD8 provides an elegant mechanism for switch-like cell-fate decisions controlled by monoubiquitylation. Taken together, our findings uncover an important role for multisite phosphorylation in integrating ubiquitin-dependent regulation of ribosome biogenesis and function into the neural crest differentiation program (Werner et al, 2018, Elife). Our study further identifies an essential role for dimerization for KBTBD8s function in cell-fate determination. In a collaborative study led by the laboratory of Michael Rape at UC Berkeley, we could show that KBTBD8 dimerization is subject to a quality control pathway that ensures proper neural crest formation (Mena et al, 2018, Science). 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. We have previously identified many of these BTB proteins to be tightly regulated in their mRNA expression during differentiation, raising the intriguing possibility that these CRL3-BTB complexes have important function during hESC maintenance and differentiation. During the last funding period, we have identified a particular CRL3-BTB complex as an essential regulator of hESC actin dynamics and of neuronal differentiation from embryoid bodies. Our preliminary data suggests that this CRL3-based E3 ligase controls cell-fate decision during hESC differentiation through an actin cytoskeleton signaling pathway. Our current efforts are geared towards elucidating the substrates and underlying mechanism through which this CRL3-BTB complex determines cell fate. 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 Lys residues, ubiquitylation is able to regulate various substrate fates ranging from substrate degradation to control of intracellular signaling pathways. Deubiquitylases (DUBs), a class of enzymes critical for many cellular processes, remove ubiquitin modifications from proteins by either primarily recognizing the protein (substrate-specific DUBs) or the ubiquitin conjugate (linkage-specific DUBs). While the underlying biochemistry and the regulatory principles impinging on many DUBs have extensively been studied during the last years, their physiological functions have remained less well understood. In collaboration with the lab of Daniel Kastner at NHGRI, we have shown that loss of function mutations in a particular subclass of DUB genes lead to severe congenital anomalies in humans. Affected individuals have clinical manifestations including structural brain malformations, congenital heart disease, ambiguous genitalia, post-axial polydactyly, arthrogryposis, and craniofacial defects. We are currently combining hESC/iPSC culture with biochemical and proteomic approaches to dissect the mechanisms of how mutations in these DUB genes lead to these severe phenotypes.
|Mena, Elijah L; Kjolby, Rachel A S; Saxton, Robert A et al. (2018) Dimerization quality control ensures neuronal development and survival. Science 362:|
|Werner, Achim; Baur, Regina; Teerikorpi, Nia et al. (2018) Multisite dependency of an E3 ligase controls monoubiquitylation-dependent cell fate decisions. Elife 7:|