A reciprocal regulatory loop between TAZ/YAP and G-protein Gαs regulates Schwann cell proliferation and myelination

ARTICLE Received 24 Oct 2016 | Accepted 3 Mar 2017 | Published 26 Apr 2017 DOI: 10.1038/ncomms15161 OPEN A reciprocal regulatory loop between TAZ/YAP and G-protein Gas regulates Schwann cell proliferation and myelination Yaqi Deng1,*, Lai Man Natalie Wu1,*, Shujun Bai2,*, Chuntao Zhao1, Haibo Wang1, Jincheng Wang1,3, Lingli Xu4, Masahide Sakabe1, Wenhao Zhou4, Mei Xin1 & Q. Richard Lu1,2,4 Schwann cell (SC) myelination in the peripheral nervous system is essential for motor function, and uncontrolled SC proliferation occurs in cancer. Here, we show that a dual role for Hippo effectors TAZ and YAP in SC proliferation and myelination through modulating G-protein expression and interacting with SOX10, respectively. Developmentally regulated mutagenesis indicates that TAZ/YAP are critical for SC proliferation and differentiation in a stage-dependent manner. Genome-wide occupancy mapping and transcriptome profiling reveal that nuclear TAZ/YAP promote SC proliferation by activating cell cycle regulators, while targeting critical differentiation regulators in cooperation with SOX10 for myelination. We further identify that TAZ targets and represses Gnas, encoding Gas-protein, which opposes TAZ/YAP activities to decelerate proliferation. Gnas deletion expands SC precursor pools and blocks peripheral myelination. Thus, the Hippo/TAZ/YAP and Gas-protein feedback circuit functions as a fulcrum balancing SC proliferation and differentiation, providing insights into molecular programming of SC lineage progression and homeostasis. 1 Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229, USA. 2 Key Laboratory of Birth Defects and Related Diseases of Women and Children (Ministry of Education), State Key Laboratory of Biotherapy, West China Second Hospital, Sichuan University, Chengdu 610041, China. 3 Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China. 4 Key Laboratory of Birth Defects, Children’s Hospital of Fudan University, Shanghai 201102, China. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to M.X. (email: ) or to Q.R.L. (email: ). NATURE COMMUNICATIONS | 8:15161 | DOI: 10.1038/ncomms15161 | www.nature.com/naturecommunications 1 ARTICLE S NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15161 chwann cells (SCs) produce multilamellar myelin sheaths that are essential for saltatory conduction of action potentials and axonal integrity in the vertebrate peripheral nervous system (PNS)1–3. The mutually exclusive aspects of proliferation and differentiation must be tightly regulated to allow both processes to operate properly to generate sufficient SCs for subsequent differentiation and myelination in developing peripheral nerves. Defects in SC generation and differentiation during development and regeneration may cause a failure in myelinogenesis, contributing to acquired or hereditary peripheral neuropathies associated with motor and sensory disabilities4. In contrast, SC over-proliferation, caused by mutations in tumour suppressor genes NF1 and NF2, results in peripheral nerve sheath tumours such as neurofibromas5 and Schwannomas6, respectively. SC development is a molecularly and ultrastructurally well-defined, multi-stage process that occurs over a protracted period of time. After specification from neural crest cells and transitioning to a SC precursor state, SC precursors and immature SCs undergo proliferation and expansion to establish a one-to-one relationship with axons during the radial sorting process. Shortly after birth, immature SCs differentiate into pro-myelinating cells and then further differentiate and form myelin sheaths around axons. SC lineage progression requires the coordinated activity of pro-myelinating factors (for example, SOX10, OCT6/POU3F1 and EGR2/KROX20) in promoting cell cycle exit and initiate differentiation, while suppressing negative regulatory factors of differentiation (for example, SOX2, NOTCH/HES and c-JUN)7,8. At present, the extrinsic and intrinsic signals that modulate and balance positive and negative factors to control SC proliferation and their transition to a differentiating state during peripheral myelination are not fully understood. Signalling by the tumour suppressor Hippo is an evolutionary conserved pathway that controls organogenesis by regulating cell proliferation, differentiation and, when dysregulated, tumorigenesis9–11. Yes-associated-protein (YAP) and transcriptional co-activator with PDZ-binding-motif (TAZ) are the major effectors of Hippo signalling12. YAP and TAZ associate with DNA-binding transcription factors, such as TEAD1–4, to regulate downstream gene expression11,13,14. Upstream Hippo kinase cascades phosphorylate and inactivate TAZ/YAP, thereby preventing their nuclear translocation and leading to their ubiquitin-mediated degradation15–18. TAZ/YAP activity can be regulated by multiple signalling pathways, including G-protein coupled receptors (GPCR), Transforming growth factor (TGF-b), wingless/integrated (WNT), and NOTCH, and by mechanical stimuli19–22. Activation of Gasprotein and cAMP-dependent protein kinase A pathways inhibits YAP activity through preventing its nuclear translocation, whereas elevation of G12/13 or Gai signalling, which antagonizes the Gasmediated cyclic adenosine monophosphate (cAMP) stimulation, enhances YAP expression19,23. Thus, the activity of TAZ/YAP is tightly regulated in response to context-specific signals9,10,24,25. Conversely, regulation of G-protein expression by YAP/TAZ remains elusive. Recent studies indicate that TAZ/YAP are required for SC radial sorting and peripheral myelination through regulating laminin receptors22 or KROX20 (ref. 26), and in control of myelin internodal length27. The nuclear localization of YAP/TAZ can be modulated by mechanical signals22. YAP/TAZ interact with TEAD1 to regulate expression of myelination-associated genes including Pmp22 (ref. 28), suggesting that YAP/TAZ may directly regulate the transcriptional programme necessary for SC differentiation. Given that the phenotype of Taz/Yap double mutants is much more severe than those observed in mice lacking laminin receptors29, it is conceivable that YAP/TAZ regulate 2 additional targets that are responsible for the severe peripheral dysmyelinating phenotype. Currently, the direct targets regulated by YAP/TAZ during SC lineage progression have not been fully defined. Whether YAP/TAZ have a direct role in the transition from SC proliferation-to-differentiation remains unresolved. Here we show that YAP/TAZ are expressed in SC nuclei in both culture and peripheral nerves through adulthood in mice and demonstrate that YAP/TAZ are crucial for SC proliferation in addition to myelin formation. We further map TAZ genome occupancy in SCs using chromatin immunoprecipitation and sequencing (ChIP-seq) and rev (...truncated)