Metazoan development requires the precise execution of highly coordinated differentiation programs that allow pluripotent stem cells to adopt specific fates. Differentiation is frequently brought about by global changes to chromatin architecture or transcriptional networks, and, as only recently discovered, can also be driven by regulated translation via remodeling of ribosome composition and function. However, mechanisms underlying the formation of these specialized ribosomes and their roles in regulating the translational output of differentiating cells have remained elusive. Dr. Werner has identified the multi-subunit ubiquitin E3 ligase CRL3 with its vertebrate-specific substrate adaptor KBTBD8 (CRL3KBTBD8) as an important regulator of cell fate. Specifically, CRL3KBTBD8 is essential for the formation of neural crest cells in a human embryonic stem cell (hESC) model. During neural induction of hESCs, CRL3KBTBD8 monoubiquitylates ribosome biogenesis regulator NOLC1 and its paralog TCOF1, a protein whose mutation underlies Treacher Collins Syndrome, a disorder of craniofacial development characterized by loss of cranial neural crest cells. Ubiquitylation of NOLC1 and TCOF1 promotes interaction of the rDNA synthesis and rRNA modification machinery, thereby altering the translational program of newly produced ribosomes to direct differentiation from a central nervous system fate towards a neural crest stem cell program. Thus, the mechanism of action of CRL3KBTBD8 provides compelling evidence that hESCs employ a ubiquitin-dependent mechanism to regulate ribosome function to guide differentiation towards a specific cell fate. In this project, Dr. Werner will build on thes results and proposes (Aim 1) to determine the molecular mechanisms of ubiquitin-dependent ribosome specification, (Aim 2) to dissect signaling networks controlling this dynamic regulation, and (Aim 3) to determine the role of ubiquitin-dependent ribosome regulation in craniofacial development in a mouse model with deleted KBTBD8 alleles. The results of these studies will uncover fundamental principles of ribosome regulation during cell fate determination, thus providing the molecular basis for the development of therapeutic approaches for treatment of Treacher Collins Syndrome. Under the guidance of his mentor, Prof. Rape, Dr. Werner has gained extensive knowledge in biochemistry, mass spectrometry-based approaches, and human embryonic stem cell techniques, which will be crucial for the successful conduct of the proposed research. At the same time, Dr. Werner will require additional training in performing and analyzing RNA sequencing and ribosomal profiling experiments, CRISPR-mediated genome editing, and mouse genetics during the mentored phase of his application. He will receive this training from advisors and collaborators, who are experts in these techniques and leaders of their respective research fields. In addition to his primary mentor, these advisors and collaborators will also participate in Dr. Werner's mentorship committee, which he has created to receive feedback on his experimentation and guidance for his career development. Further training activities during the mentored phase will be geared at Dr. Werner's professional education (i.e. international conferences and workshops, participation in institutional journal clubs and seminars) and development of his mentorship skills (i.e. continued supervision of rotation students and technical assistants, participation in workshops). In conclusion, Dr. Werner's strong research application as well as training and career development plan will enable him to launch a vigorous, technically diverse, and innovative research direction with focus on ribosomal and craniofacial biology. This will provide him with a strong foundation to successfully transition into an independent investigator at an academic institution in the US.

Public Health Relevance

Metazoan development is accomplished through tightly controlled cellular differentiation mechanisms that if misregulated can lead to devastating developmental disorders. The proposed research aims to understand the role of regulated ribosome function during differentiation processes, focusing on those involved in craniofacial development. Results obtained from these studies will help uncover fundamental principles of ribosome regulation during cell fate determination and provide the molecular basis for launching novel therapeutic approaches for the treatment of Treacher Collins Syndrome, a craniofacial development disorder.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Career Transition Award (K99)
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NIDCR Special Grants Review Committee (DSR)
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Frieden, Leslie A
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University of California Berkeley
Schools of Arts and Sciences
United States
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Werner, Achim; Baur, Regina; Teerikorpi, Nia et al. (2018) Multisite dependency of an E3 ligase controls monoubiquitylation-dependent cell fate decisions. Elife 7:
Werner, Achim; Rape, Michael (2017) Powering stem cell decisions with ubiquitin. Cell Death Differ 24:1823-1824
Werner, Achim; Manford, Andrew G; Rape, Michael (2017) Ubiquitin-Dependent Regulation of Stem Cell Biology. Trends Cell Biol 27:568-579
Yau, Richard G; Doerner, Kerstin; Castellanos, Erick R et al. (2017) Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control. Cell 171:918-933.e20
Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen A et al. (2015) Cell-fate determination by ubiquitin-dependent regulation of translation. Nature 525:523-7