The E3 ubiquitin ligase c-Cbl restricts development and functions of hematopoietic stem cells

Abstract Abstract 1335 Fanconi anemia (FA) is associated with a hereditary predisposition to bone marrow failure. The proteins encoded by the FANC genes are primarily involved in DNA repair responses through the formation of a large, multisubunit complex that has E3 ubiquitin ligase activity (Annual Review of Genetics 2009;43:223). FA hematopoietic stem cells display defective stem cell properties and limited replicative potential. However, the molecular basis for how a FA genetic background contributes to those defects remains poorly understood. Here we provide evidence that FANCL, which has E3 ubiquitin ligase activity, enhances beta-catenin activity (Figure A) and protein expression. Beta-catenin is a nuclear effector of canonical Wnt signaling. The Wnt/beta-catenin pathway is active in normal hematopoietic stem cells in the native bone marrow environment and disruption of this signaling pathway results in defective hematopoietic stem cells (Nature 2003;423:409). To test whether FANCL positively regulates beta-catenin through its ubiquitination activity, we performed cell-based ubiquitination assays. We show that FANCL functionally ubiquitinates beta-catenin (Figure B) and that ubiquitin chain extension can occur via non-lysine-48 ubiquitin linkages. Accumulating evidence reveal diverse, non-proteolytic biological roles for proteins modified by atypical ubiquitin chains (EMBO Reports 2008;9:536). Our data suggests that FANCL may enhance the protein function of beta-catenin via ubiquitination with atypical ubiquitin chains. Importantly, we demonstrate that suppression of FANCL expression in human CD34+ cord blood stem cells reduces beta-catenin expression (Figure C) and multilineage progenitor expansion. These results demonstrate a role for the FA pathway in regulating Wnt/beta-catenin signaling. Therefore, diminished Wnt/beta-catenin signaling may be an important underlying molecular defect in FA hematopoietic stem cells leading to their accelerated loss. A, LEF-TCF-luciferase reporter assay showing increasing beta-catenin activity in 293FT cells with increasing FANCL expression compared with vector-control (VC) (n=4). B, Immunoprecipitation of beta-catenin in cells transfected with vector-control or FANCL and probed for hemagglutinin (HA)-tagged ubiquitin shows increased ubiquitinated forms of beta-catenin with FANCL expression (n=4). C, shRNA suppression of FANCL expression in CD34+ cord blood stem cells results in decreased beta-catenin expression compared with a scramble control (Scr) by immunofluorescence analysis (three different shRNA constructs, n=3 for each construct). Disclosures: No relevant conflicts of interest to declare.

Download Full-text

FANCL ubiquitinates β-catenin and enhances its nuclear function

Blood ◽

10.1182/blood-2011-11-388355 ◽

2012 ◽

Vol 120 (2) ◽

pp. 323-334 ◽

Cited By ~ 23

Author(s):

Kim-Hien T. Dao ◽

Michael D. Rotelli ◽

Curtis L. Petersen ◽

Stefanie Kaech ◽

Whitney D. Nelson ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ubiquitin Ligase ◽

Fanconi Anemia ◽

E3 Ubiquitin Ligase ◽

Chain Extension ◽

Molecular Defect ◽

Ubiquitin Chain ◽

Hematopoietic Stem ◽

Ubiquitin Ligase Activity

Abstract Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates β-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, β-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate β-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34+ stem and progenitor cells results in fewer β-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/β-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss.

Download Full-text

Ubiquitin Ligase Component Fbw7 Regulates the Quiescence of Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v108.11.79.79 ◽

2006 ◽

Vol 108 (11) ◽

pp. 79-79 ◽

Cited By ~ 1

Author(s):

Sahoko Matsuoka ◽

Yuichi Oike ◽

Ichiro Onoyama ◽

Keiyo Takubo ◽

Keisuke Ito ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ubiquitin Ligase ◽

Mononuclear Cells ◽

Human Cancer ◽

Self Renewal ◽

Hematopoietic Stem ◽

Human Cancer Cell Lines ◽

Adult Mice ◽

Deficient Mice

Abstract Fbw7 is a SCF ubiquitin ligase component that catalyzes the ubiquitination of c-Myc, Cyclin E, and Notch. In several human cancer cell lines and primary cancer cells, Fbw7 is mutated and functions as a tumor suppressor gene. Previously we have reported that Fbw7-deficient mice died at embryonic day 10.5–11.5 with deficiencies in hematopoietic and vascular development, indicating that Fbw7 has a pivotal role in hematopoiesis (Tsunematsu R et al. J Biol Chem. 2004). Fbw7 is widely expressed in various hematopoietic lineages in BM of adult mice, but little has been known about the function of Fbw7 in hematopoiesis. To assess the requirement of Fbw7 in adult hematopoietic cells, we generated Fbw7-deficient mice by conditional gene targeting. Fbw7 was conditionally deleted from Mx-1-Cre;Fbw7fl/− adult mice by injection of pIpC over 1 week to induce Cre expression. We examined Fbw7fl/+ littermates as a control. We found progressive pancytopenia in Fbw7-deficient mice. Furthermore, most Fbw7-deficient mice developed leukemia (mainly ALL) within 3 months after pIpC treatment, suggesting that Fbw7 is essential to maintain normal hematopoiesis and loss of Fbw7 accelerates leukemogenesis. The portion of Fbw7-deficient Lin−Sca-1+c-Kit+CD34− hematopoietic stem cells (HSCs) in the G0 phase was 2.5-fold decreased and the frequency of cell division of Fbw7-deficient HSCs markedly increased in culture. These data suggest that Fbw7 promotes quiescence of HSCs. To examine the function of Fbw7-deficient HSCs, we transplanted 1500 Lin−Sca-1+cKit+ BM cells from Fbw7-dificient mice or littermate controls into lethally irradiated recipient mice with 4×105 normal BM mononuclear cells. In the result, Fbw7-deficient HSCs are impaired in long-term repopulating activity and multipotency. It has been reported that c-Myc controls the self-renewal activity of HSCs through the cell adhesion to the osteoblastic niche (Wilson A et al. Genes Dev. 2004). We found that c-Myc is significantly accumulated in Fbw7-deficient Lin−Sca-1+cKit+ BM cells, suggesting that HSCs leave the niche and show the active cell cycling. We propose that a ubiquitin ligase, Fbw7 is a key mediator of HSC quiescence and self renewal capacity.

Download Full-text