WNT-mediated signaling relay in stem cells and oncogenesis - from basic biology to applications

Elucidating the role of stem-cell specfic ubiquitin ligases

Early stage researcher 12 (ESR12) project

Supervision: Bon-Kyoung Koo

Lab webpage:  http://www.stemcells.cam.ac.uk/researchers/principal-investigators/bon-kyoung-koo

Aim:
Understanding the role of PA-RING E3s ubiquitin ligases in tumourigenesis

Methodology:
ESR12 will clone all members of mouse PA-RING cDNAs for mammalian expression. Using a luciferase-based assay, ESR12 will screen for PA-RING family members that modify responses to WNT. Mass spectrometry, interaction studies, ubiquitination assays, and endocytosis tests with candidate WNT (co)receptors will be employed to identify the substrate(s) (international secondment: Bryja, collaboration  Maurice, private sector secondment: Attana). To validate substrate(s), ESR12 will use intestinal organoid cultures to overexpress PA-RING ligases using retrovirus and assess effects on stem cell proliferation and viability. ESR12 will generate conditional knockout line(s) to evaluate their role in vivo. These lines will be tested as conventional knockout by germline exon deletion and conditionally through tissue- and stage-specific deletion.

Collaborators:
Maurice, Bryja, Attana

Project goals:
ESR12 will identify FZD E3 ubiquitin ligases of the PA-RING family and characterize the physiological role of novel FZD E3s for a better understanding of FZD receptor and RNF43-related E3 interactions.

Risk assessment and contingency plans:
All required cellular and biochemical assays are established. We have expertise in mouse genetics and primary 3D organoid culture to carry out the suggested experiments.

Key publications:

  1. Koo BK, Spit M, Jordens I, Low TY, Stange DE, van de Wetering M, van Es JH, Mohammed S, Heck AJR, Maurice MM, Clevers H (2012) Tumour suppressor RNF43 is a stem cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488(7413):665-9.
  2. Koo BK*, Stange DE*, Sato T, Karthaus W, Farin H, Huch M, van Es JH, Clevers H (2011) *equal contribution. Controlled gene expression in primary Lgr5 organoid cultures Nature Methods 4;9(1):81-3.
  3. de Lau W, Barker N, Low TY, Koo BK, Li VS, Teunissen H, Kujala P, Haegebarth A, Peters PJ, van de Wetering M, Stange DE, van Es JE, Guardavaccaro D, Schasfoort RB, Mohri Y, Nishimori K, Mohammed S, Heck AJ, Clevers H (2011) Lgr5 homologues associate with Wnt receptors and mediate Rspondin signalling. Nature 4;476(7360):293-7.