IntroductionDuring the cell cycle, ubiquitin-mediated proteolysis provides aswift and precise means to regulate the abundance of cell cycleregulatory proteins, including cyclins and cyclin-dependent kinase(Cdk) inhibitors. This mechanism for regulating the turnover ofproteins is mediated through ubiquitin ligases, which transferubiquitin to target proteins, enabling their timely destruction(Hershko, 1997; Hershko and Ciechanover, 1998; Nakayama andNakayama, 2006; Reed, 2006; Reed, 2003). E3 ubiquitin-ligasespromote the specific attachment of poly-ubiquitin chains, whichthen triggers proteolysis by the 26S proteasome. SCF-type (Skp1–Cullin–F-box) E3 ubiquitin ligases are multi-subunit complexesthat consisting of Skp1, Cullin, Rbx1 and an F-box protein (FBP)(Ang and Wade, 2005; Deshaies, 1999; Jackson and Eldridge,2002; Nakayama and Nakayama, 2005; Vodermaier, 2004). It isthe FBP, which has a crucial role in specifically recruiting thetarget substrate, usually directed by post-translational modificationof the substrate (Cenciarelli et al., 1999; Hermand, 2006; Ho et al.,2008; Jin et al., 2004; Winston et al., 1999). In cell cycle regulation,several FBPs that regulate G1–S phase regulators have beenintensively studied. These include the prototypical S-phase kinase-associated protein 2 (Skp2, also known as Fbxl1) and the F-boxand WD repeat domain containing 7 (Fbxw7) that, respectively,regulate the levels of the Cdk inhibitors cyclin E and cyclin-dependent kinase inhibitor 1B (CDKN1B; also known as andhereafter referred to as p27). In addition, three FBPs – Fbxo4,Fbxw8, Fbxo31 – promote the ubiquitin-mediated degradation ofcyclin D1 (Lin et al., 2006; Santra et al., 2009; Okabe et al., 2006).
Another FBP that interacts with G1–S regulatory proteins is F-box protein 7 (Fbxo7). In contrast to the destabilising effects ofother FBPs, Fbxo7 acts as a specific assembly factor for cyclin D–Cdk6 complexes. Fbxo7 interacts directly with both Cdk6 and p27,and cooperatively increases cyclin D3 interactions with Cdk6 invitro (Laman et al., 2005). In vivo, Fbxo7 overexpression inimmortalised murine fibroblasts leads to their Cdk6-dependenttransformation. These cells had increased levels of cyclin D–Cdk6complexes and E2F activity, and formed tumours in athymic nudemice (Laman et al., 2005). Many mitogen and cytokine signallingpathways converge on cyclin D–Cdk activity, which – whenoverstimulated – promotes oncogenesis. Because of its Cdk6-dependent transforming activity, we propose that Fbxo7 functionsas an oncogene.
In U2OS and NIH3T3 cells, Fbxo7 has shown selectivity forCdk6 over Cdk2 and Cdk4. Although the biochemical propertiesof Cdk4 and Cdk6 are similar, more-recent studies have indicatedthat differences can be discerned in their selective binding to co-factors (Sugimoto et al., 1999; Laman et al., 2005), preference forphosphorylation sites in pRb in vitro (Takaki et al., 2005),sensitivities to INK4 family members (Tourigny et al., 2002; Joneset al., 2007) and in vivo partnering with D-type cyclins (Ely et al.,2005). Also, in studies of knockout mice, tissue-specific defectsare seen: Cdk4-knockout mice have impaired development ofpancreatic -islet cells (Rane et al., 1999), whereas Cdk6-null miceshow deficiencies in haematopoiesis (Malumbres et al., 2004; Huet al., 2009). In addition, whereas Cdk4–Cdk6 or Cdk2–Cdk4double-knockout mice are embryonic lethal, Cdk2–Cdk4 double-
SummaryFbxo7 is an unusual F-box protein because most of its interacting proteins are not substrates for ubiquitin-mediated degradation. Fbxo7directly binds p27 and Cdk6, enhances the level of cyclin D–Cdk6 complexes, and its overexpression causes Cdk6-dependenttransformation of immortalised fibroblasts. Here, we test the ability of Fbxo7 to transform haematopoietic pro-B (Ba/F3) cells which,unexpectedly, it was unable to do despite high levels of Cdk6. Instead, reduction of Fbxo7 expression increased proliferation, decreasedcell size and shortened G1 phase. Analysis of cell cycle regulators showed that cells had decreased levels of p27, and increased levelsof S phase cyclins and Cdk2 activity. Also, Fbxo7 protein levels correlated inversely with those of CD43, suggesting direct regulationof its expression and, therefore, of B cell maturation. Alterations to Cdk6 protein levels did not affect the cell cycle, indicating thatCdk6 is neither rate-limiting nor essential in Ba/F3 cells; however, decreased expression of Cdk6 also enhanced levels of CD43,indicating that expression of CD43 is independent of cell cycle regulation. The physiological effect of reduced levels of Fbxo7 wasassessed by creating a transgenic mouse with a LacZ insertion into the Fbxo7 locus. Homozygous Fbxo7LacZ mice showed significantlyincreased pro-B cell and pro-erythroblast populations, consistent with Fbxo7 having an anti-proliferative function and/or a role inpromoting maturation of precursor cells.
Knockdown of Fbxo7 reveals its regulatory role inproliferation and differentiation of haematopoieticprecursor cellsEl Kahina Meziane*, Suzanne J. Randle*, David E. Nelson, Mikhail Lomonosov and Heike Laman‡
University of Cambridge, Department of Pathology, Division of Cellular and Genetic Pathology, Tennis Court Road, Cambridge CB2 1QP, UK*These authors contributed equally to this work‡Author for correspondence ([email protected])
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knockout mice are viable (Barriere et al., 2007; Malumbres et al.,2004; Berthet et al., 2006). Moreover, there is mounting evidencefor a specific role for Cdk6 in differentiation, especially in neuronaland haematopoietic cells (Ericson et al., 2003; Slomiany et al.,2006; Fujimoto et al., 2007; Grossel and Hinds, 2006; Matushanskyet al., 2003). These studies support the idea that the G1 Cdks haveoverlapping, yet distinct, activities.
Fbxo7 also directly interacts with and stabilizes p27, althoughFbxo7 does not require p27 to increase levels of cyclin D–Cdk6(Laman et al., 2005). p27 was first identified as an inhibitor ofcyclin-dependent kinases but it also acts as an assembly factor forcyclin D–Cdk complexes. More recently, p27 has been shown tohave roles that extend beyond direct cell cycle regulation to functionsin differentiation, motility and migration, and cytoskeletal signalling(reviewed in Borriello et al., 2007). As such, p27 has both tumoursuppressor and oncogenic activities – dependent on its subcellularlocalization and cell type (Besson et al., 2007; Chu et al., 2008).
Because Fbxo7 exhibited a preference for Cdk6, we reasonedthat it might have different functions in haematopoietic cells whereCdk6 activity predominates (Laman, 2006). In this study, we testedthe activity of Fbxo7 in B cell lines. In contrast to our findings infibroblasts and osteosarcoma cells, data presented here indicatethat Fbxo7 is not a transforming gene in pro-B cells. Instead, thereduction of Fbxo7 protein levels led to an increased rate ofproliferation and changed the expression of B cell markers toresemble a less-mature state. In addition, a mouse model presentingreduced Fbxo7 protein levels showed increased populations ofprecursor pro-B cells and pro-erythroblasts. These data areconsistent with a model, in which Fbxo7 has an anti-proliferativerole in precursor cells and also a cell-cycle-independent regulatoryrole in the maturation of haematopoietic cells.
ResultsIncreased Fbxo7 levels do not transform Ba/F3 cellsTo address whether Fbxo7 is capable of transforminghaematopoietic cells, we used the pro-B cell line Ba/F3, whichdepends on IL-3 for survival, and tested whether exogenous
expression of Fbxo7 would alter the proliferation rate or substitutefor IL-3 signalling. Ba/F3 cells were infected either with a retrovirusexpressing human FBXO7–IRES–GFP or the empty vector, andfluorescence-activated cell sorting (FACS) sorted for GFP+ cells.Immunoblotting of lysates from polyclonal cell lines demonstratedthe presence of exogenous Fbxo7 expression, and the increasedmobility on SDS-PAGE of human Fbxo7 compared with theendogenous murine Fbxo7 (Fig. 1A). We first tested whetherincreased Fbxo7 levels would enhance the proliferation rate whichwas assessed by seeding cells at equal densities and counting livecells at periodic intervals. However, both cell lines proliferated atthe same rate (Fig. 1B). In addition, the proportion of live and deadcells was measured, and no significant differences were observedbetween control and Fbxo7-expressing Ba/F3 cells (Fig. 1C). Next,the ability of Fbxo7 to substitute for IL-3 signalling was tested bywithdrawing it from the growth medium and measuring cellviability. As seen in Fig. 1D, vector control and Fbxo7-expressingcells lost viability with essentially the same kinetics. We noticedthat 48 hours post withdrawal ~5% more Fbxo7-expressing cellswere alive compared with those expressing the vector control;however, this small effect, although statistically significant(P0.027), was transient, and suggests that Fbxo7 has only a weakability to provide survival signals to Ba/F3 cells. Thus Fbxo7expression did not relieve IL-3 dependence in Ba/F3 cells nor wasit rate-limiting for their proliferation.
Decreasing Fbxo7 levels enhanced Ba/F3 cell proliferationWe next determined whether Fbxo7 is required for proliferation orviability. To achieve this, Ba/F3 cells were infected with retrovirusesthat encoded miR-30-based short-hairpin RNA targeting theexpression of Fbxo7 mRNA or an empty vector. Cell lines werecloned by limiting dilution under antibiotic selection and screenedby immunoblotting lysates to test for Fbxo7 expression. Data fortwo independent clonal lines are shown in Fig. 2A. Expression ofshort hairpin RNA achieved a significant reduction in Fbxo7 levels,arguing against the idea that high levels of Fbxo7 are required forproliferation or viability. In fact, during cloning, we noticed that
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Fig. 1. Increased Fbxo7 expression in Ba/F3 cells does notaffect growth or proliferation. (A)Immunoblot of cell lysatesfrom Ba/F3 cells infected with retroviruses that express eitherhuman FBXO7 or the empty vector. Actin was used as a control forequivalent loading. (B)Time course of the proliferation rate ofcells seeded at equal densities and counted on the day indicated.(C)Percentages of viable and non-viable cells in an asynchronousculture assessed by PI staining of live cells and by FACS (mean ±s.d., n3). (D)Kinetics of cell survival upon withdrawal of IL-3from the culture medium. Cells were seeded at equal densities inmedium without IL-3 and the number of viable cells wasdetermined as indicated; *P<0.05.
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cultures with reduced Fbxo7 protein levels proliferated more rapidlyand were smaller than control cells. By measuring the rate ofproliferation (P<0.01 on day 3 and P<0.001 on day 17) and the cellvolume (P<0.001), we confirmed the statistical significance ofthese observations (Fig. 2B,C).
To investigate the molecular basis for the Ba/F3 cell cyclephenotypes, protein levels of other cell cycle regulators were alsodetermined. Immunoblotting the lysates from control and Fbxo7-knockdown cells showed that the total levels of subunits (cyclinD2, cyclin D3 and Cdk4, Cdk6) were not significantly changed;however, protein expression of the S phase cyclins E and A wasmarkedly increased, whereas p27 protein levels were reduced (Fig.2A). These alterations suggested that Cdk2 activity waspredominantly affected in these cells. To test this, Cdk2 kinaseassays were performed. In independent experiments endogenousCdk2 activity was quantified as being 25% (data not shown) and52% (Fig. 2D) increased in cells with reduced Fbxo7 as compared
with vector control cells. In addition, although we have previouslyshown in fibroblasts and osteosarcoma cells that Fbxo7 is requiredfor Cdk6 association with D-type cyclins (Laman et al., 2005), inBa/F3 cells, the amount of Cdk6 associated with endogenous cyclinD2 and D3 was unaffected by the reduction of Fbxo7 levels astested by in vivo co-immunoprecipitation assays (Fig. 2E). Weconclude, therefore, that Cdk6 association with D-type cyclinsdoes not depend on Fbxo7 in this cell type. Together, these dataindicate that reducing Fbxo7 protein levels augmented Cdk2activity, and promoted a rapid G1 to S phase transition and asmaller cell size.
As cells acquire much of their mass during G1 phase, cell cycleprofiling was performed. Asynchronous populations of cells werefixed, stained with propidium iodide (PI) and assayed by FACS todetermine DNA content (n10). Representative plots shown inFig. 2F demonstrate that, compared with vector control, Fbxo7-knockdown cells had fewer cells with 2N DNA content in G1
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Fig. 2. Stable reduction of Fbxo7 levels causes an increase in proliferation and a decrease in the size of Ba/F3 cells. (A)Immunoblot of various cell cycleproteins in lysates from independent clones of Ba/F3 cells infected with retroviruses expressing a miR-30-based hairpin RNA which targets Fbxo7 expression.Actin is used as a loading control. (B)Time course of the proliferation rate of an empty vector control cell line and two independent clonal cell lines that havereduced Fbxo7 protein expression. Cells were seeded at equal densities and counted on the day indicated. (C)Size measurements of cell lines described in B,seeded at equal densities. (D)Kinase assays on immunoprecipitations with antibodies against Cdk2 from cell lysates of Ba/F3 cells with reduced or endogenouslevels of Fbxo7. pRb was used as a substrate and the amount of incorporated [-33P]ATP was quantified on a phosphoimager (Cyclone). (E)Immunoblotting ofendogenous Cdk6 associated with D cyclins in immunoprecipitations from cell lysates of Ba/F3 cells with reduced or endogenous levels of Fbxo7.(F)Representative FACS plots (from n10) of an asynchronous population of cells, that had been fixed and whose DNA content had been quantified by PI staining,to determine the percentages of cells in the cell cycle phase indicated. (G)Percentages of viable and non-viable cells in an asynchronous culture assessed by PIstaining of live cells and by FACS (mean ± s.d., n3). (H)Immunoblotting for pocket proteins in cell lysates of Ba/F3 cells with reduced and endogenous levels ofFbxo7. **P<0.01, ***P<0.001.
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phase and more cells with >2N DNA content in S and G2–Mphases. These data indicate that the more-rapid proliferation andsmaller size of the Fbxo7-knockdown cells are due to a shorter G1phase. The possibility that cells with less Fbxo7 protein resist celldeath during culturing, which would account for the increased cellnumber, was investigated by assaying live cell exclusion of PI;however, the percentages of viable cells were similar for both celllines (Fig. 2G). The inactivation of pocket proteins (pRb, p107 andp130) is rate-limiting for transition through G1 and passage throughthe restriction point. Consistent with the observed shortened G1phase, immunoblotting the pocket proteins in lysates from cellswith reduced Fbxo7 levels also showed their levels decreasedcompared with vector control cells (Fig. 2H).
These data demonstrated that Fbxo7 has an unexpected anti-proliferative role in Ba/F3 cells. To test whether this is specific toBa/F3 cells, cell size and proliferation rates were tested in othertypes of cell that have reduced Fbxo7 levels. The human pre-B cellleukemic cell line Nalm-6 was infected with a retrovirus encodinga miR-30-based short hairpin RNA that targets FBXO7 expression.Immunoblotting lysates from FBXO7-knockdown cells showedthat levels of Cdk2, Cdk4, Cdk6 and cyclin D3 were unaffected,whereas levels of cyclin A were elevated and those of p27 werereduced when compared with vector control (Fig. 3A). In agreementwith the results for the Ba/F3 cells, the reduction of Fbxo7 proteinlevels also caused a statistically significant increase in the rate ofproliferation and a decrease in the volume of these cells (P<0.001)(Fig. 3B,C). Similarly, in the human cervical carcinoma cell lineC-33A, reduction of Fbxo7 protein levels by using a different miR-30 short hairpin RNA construct resulted in cells with a fasterproliferation rate than in vector control cells (P value <0.05) (Fig.3D). Cells with less Fbxo7 were also smaller than vector controlcells, although the reduction in cell size was more variable and notstatistically significant (P0.052) (Fig. 3E). Immunoblottingshowed a reduction in Fbxo7 and a small decrease in p27, but noreduction in the levels of cyclin E in this cell line (Fig. 3F). Thus,in human and mouse B cell lines, and in an adherent humanepithelial cell line, reduction of Fbxo7 caused cells to proliferatefaster and become smaller in size, indicating that the proliferationof several cell types is regulated by Fbxo7.
Changes to Cdk6 expression did not alter the size orproliferation of Ba/F3 cellsWe next wanted to investigate the role of Cdk6 in the cell cycle ofBa/F3 cells. We first tested whether overexpression of Cdk6 affectsproliferation rates and cell size. A polyclonal population of cellsthat overexpress Cdk6 was generated by retroviral infection ofviruses that encode CDK6-IRES-GFP, and by using FACS sortingfor GFP+ cells. Immunoblotting of lysates from these cells showedthat levels of Cdk6 were approximately fivefold increased, whereasthe levels of the cell cycle regulators Fbxo7, p27 and cyclin E werethe same as for the vector control cells (Fig. 4A). In addition, weused in vivo co-immunoprecipitation assays to test the amount ofCdk6 associated with cyclin D2. Overexpression of Cdk6 alonedid not change its association with cyclin D2, arguing that Cdk6levels are in excess and not limiting its association with cyclin D2(Fig. 4B). We found that proliferation rate and cell size for cellsthat overexpress Cdk6 are virtually identical to those of vectorcontrol (Fig. 4C,D).
Cdk6 requirement for cell proliferation was tested by usingretroviruses that encode short-hairpin RNAs targeting Cdk6 or theempty vector to infect Ba/F3 cells to create stable cell lines. Two
independent clonal cell lines were assayed for Cdk6 proteinexpression and, in both cases, its levels were substantially reducedwhile levels of Fbxo7 and p27 were unchanged (Fig. 4E). Inaddition, in vivo co-immunoprecipitation assays showed the lackof Cdk6 associated with cyclin D2 (Fig. 4F). However, when therate of proliferation and the cell volume were measured, thesecharacteristics were similar between control cells and those withreduced Cdk6 (Fig. 4G,H). Taken together, these data indicate thatCdk6 levels do not influence cell growth or proliferation or impacton the amount of Fbxo7 or p27. Thus Cdk6 is neither rate-limitingnor essential for the proliferation of Ba/F3 cells.
Reduced Fbxo7 levels cannot bypass cell cycle inhibitioncaused by increased levels of p27 in Ba/F3 cellsFbxo7 interacted directly with the N-terminus of p27 and stabilizedits levels in U2OS cells (Laman et al., 2005) and also in the cell
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Fig. 3. Reduced Fbxo7 levels in Nalm-6 and C-33A cells increases theirproliferation rate. Immunoblot of various cell cycle proteins in lysates fromNalm-6 cells (A) or C-33A cells (F) infected with retroviruses expressingdifferent miR-30-based hairpin RNAs that target FBXO7 expression. Timecourse of the proliferation rate of an empty vector control cell line and eitherNalm-6 (B) or C-33A (D) cell lines with reduced Fbxo7 protein expression.Cells were seeded at equal densities and counted on the indicated day. Cellsize measurements of Nalm-6 (C) or C-33A (E) cells seeded at equal densities.*P<0.05, ***P<0.001.
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types tested here (Fig. 2A, Fig. 3A,F). To test directly whetherFbxo7 is required to stabilize levels of p27, we investigated whetherFbxo7-knockdown cells become resistant to the cell cycle inhibitoryeffects of exogenous p27 expression. For this, a plasmid containingthe coding region of p27 fused to dsRED was transfected intoBa/F3 cells with reduced levels of Fbxo7. The dsRED-positivecells were collected by FACS sorting, and DNA content and cellvolume were measured. Exogenous expression of p27approximately doubled the percentage of cells containing 2N DNA(G1 phase) (38% compared with 19.5% in untransfected cells),cells expressing p27-dsRED also showed a 13% increase in cellsize (data not shown). These data argue against the idea that Fbxo7is required for p27 to function as a cell cycle inhibitor, becausereduction in Fbxo7 levels did not overcome a p27-induced blockto G1–S phase progression.
Decreased p27 levels in Ba/F3 cells increased theirproliferation rate and reduced cell sizeBecause a reduction of Fbxo7 levels led to a decrease in p27 inBa/F3 cells (Fig. 2A), we next investigated the phenotypic effectsof decreasing p27 levels specifically in Ba/F3 cells. Cell lines werecreated that stably express a miR-30-based short-hairpin RNAtargeting p27 expression. Two independent clonal cell lines withreduced p27 protein levels were created (Fig. 5A). When rate ofproliferation and cell size were assayed, cells with less p27 proteinshowed statistically significant increased rates of proliferation(P<0.001 from day 4 onwards) (Fig. 5B) and a smaller size(P0.005) (Fig. 5C). The DNA content was also measured byFACS analysis (n3); representative plots demonstrating the
decreased percentage of cells in G1 phase and a commensurateincreased percentage of cells in S phase are shown in Fig. 5D.Furthermore, when the expression of other cell cycle regulatorswas assayed, no changes were seen in the expression levels ofcyclin D2, Cdk4, Cdk6, Fbxo7 or cyclin E (Fig. 5A). These datademonstrate that, in Ba/F3 cells, reduction of p27 protein aloneaffects the distribution of cell cycle phases, proliferation rate andcell size, but not the total levels of cell cycle regulators. Thus p27also has an anti-proliferative role in Ba/F3 cells.
Ba/F3 cells with reduced levels of Fbxo7 showedincreased levels of CD43It has been reported that lengthening the G1 phase can increasethe probability of differentiation (Johnson et al., 1993; Carroll etal., 1995), whereas shortening the G1 phase decreases theprobability of differentiation and favours proliferation (Kato andSherr, 1993). Reducing the cellular levels of Fbxo7 shortened theduration of G1 phase, so we wanted to investigate any effectsFbxo7 might have on differentiation. Stages of B celldifferentiation can be distinguished by the expression of cellsurface antigens, so a subset of these common surface markerswas assayed, including B220, B7.1, B7.2, CD43, CD25, MHCII(Ia) (Zola et al., 1991). Ba/F3 empty vector control cells andcells with reduced levels of Fbxo7 were stained with fluorescentantibody conjugates and analysed by FACS. Expression of mostof the above markers was not changed; however, staining forCD43 showed differences between cells with endogenous orreduced levels of Fbxo7 (Fig. 6A). In separate experiments, Ba/F3cells with stable Fbxo7 knockdown expressed high levels of CD43
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Fig. 4. Alterations to Cdk6 levels do not affect growth or proliferation of Ba/F3 cells. (A)Immunoblot of cell lysates from polyclonal cell lines generated byinfecting Ba/F3 cells with retroviruses expressing human CDK6–IRES–GFP and sorted for GFP expression. (B)Immunoblot of endogenous Cdk6 associated withcyclin D2 in immunoprecipitations from cell lysates of Ba/F3 cells with overexpression of Cdk6. (C,D,G,H) Effect of overexpression or reduction of Cdk6 levelson proliferation rate (C,G) and cell size (D,H). Cells were seeded at equal densities and counted on the indicated day. (E)Immunoblot (as in A) of cell cycleregulatory proteins in lysates from independent clones of Ba/F3 cells infected with retroviruses expressing a pRETROSuper short hairpin RNA that targets Cdk6expression or with the empty vector. Actin was used as a loading control. (F)Immunoblot of endogenous Cdk6 associated with cyclin D2 in immunoprecipitationsfrom cell lysates of Ba/F3 cells with reduced Cdk6.
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tested the effect of Fbxo7 overexpression on cell surface markerexpression. To achieve this, an expression construct encodingFBXO7–IRES–GFP was transiently transfected into Ba/F3 cellsthat were then stained for CD43 and CD25; the percentages ofCD43hi and CD25hi GFP+ cells were measured by FACS. Consistentwith a direct effect of Fbxo7 on CD43 expression, its Fbxo7overexpression caused a 65.9% decrease in CD43hi expressingcells (n3). CD25 expression, however, was also reduced 39.2%when Fbxo7 was overexpressed, suggesting that Fbxo7 expressionhad an indirect effect on its levels (Fig. 6C). These data suggestthat Fbxo7 directly modulates CD43 expression; however, becauseFbxo7 overexpression does not increase the proliferation rate, theeffect of Fbxo7 on CD43 appears to be independent of its effectson the duration of the cell cycle.
Reduced levels of Cdk6 increased those of CD43To further investigate the link between cell cycle delay and theexpression of these markers of differentiation, the effect oflengthening the cell cycle was tested by transiently transfectingp27-IRES-GFP into Ba/F3 cells and assessing the effect on CD43and CD25 surface expression. Although a longer G1 phase might
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Fig. 5. Reduced levels of p27 in Ba/F3 cells increases therate of proliferation and decreases cell size.(A)Immunoblots for cell cycle regulatory proteins of celllysates from independent clonal lines stably expressing a miR-30-based hairpin RNA that targets p27 expression or theempty vector control. (B)Proliferation rates of cell lines withendogenous or reduced levels of p27. (C)Cell sizes of celllines shown in B. (D)Representative FACS plots (from n3)of an asynchronous cell population of cells, fixed and theirDNA content quantified by PI staining to determine thepercentages of cells in indicated cell cycle phase. **P<0.01,***P<0.001.
(CD43hi) that were on average 2.8-fold and 2.4-fold increasedcompared with the vector control (Fig. 6A). CD43 is asialoglycoprotein with a role in cell-cell adhesion and proliferation.During B cell differentiation it is expressed by early precursorcells (pre-pro-B and pro-B cells), and then rapidly downregulatedupon progression to pre-B and B cell stages with VDJrearrangement (Hardy et al., 1991). CD25 encodes the IL-2receptor -chain, which steadily increases during these stages ofB cell differentiation (Rolink et al., 1994). Thus pro-B cells haveCD43hi and low CD25 (CD25lo) expression, whereas small pre-Bcells have CD43lo CD25hi expression (Rolink et al., 1994; Zola etal., 1991). The expression of CD25 was also different in thesecells. The number of CD25hi cells decreased by 61.7% in Fbxo7-knockdown cells compared with vector control cells (Fig. 6B).These data demonstrate that the reduction of Fbxo7, whichincreased the proliferation rate, also altered the expression of cellsurface markers. Furthermore, the change in their expression levelssuggest that cells had reverted to a less-mature state, implying thatFbxo7 normally regulates differentiation at the pro-B cell stage.
As the reduction of Fbxo7 lead to a cell surface markerphenotype that suggested a reversal of differentiation, we also
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be expected to alter these markers in order to reflect increaseddifferentiation (i.e. decrease of CD43 and increase of CD25expression), no changes to the expression of either marker wereobserved in cells transfected with the construct encoding p27 (Fig.6C). Conversely, the expression of CD43 was assayed in the cellline with reduced p27 levels – which had a shorter G1 phase – alsohad no significant increase in CD43 staining (Fig. 6D).
In addition to testing the effect of altering the levels of p27, theexpression levels of CD43 were measured in cells with reducedlevels of Cdk6, because this kinase has a specific role indifferentiation (Grossel and Hinds, 2006). Cells with reducedamounts of Cdk6 were also immunostained for CD43 and werefound to have on average a 2.3-fold increase in CD43hi cells overthree independent experiments compared with vector control (Fig.6E). This was surprising because reducing Cdk6 levels in Ba/F3cells did not alter the proliferation rate of cells (Fig. 4G), supportingthe idea that increased CD43hi expression did not require a more-rapid cell cycle or a shortened G1 phase. Together, these datademonstrate that reducing the expression of Fbxo7 or Cdk6 hadthe same effect of increasing CD43 expression, despite only Fbxo7-
knockdown cells affecting the rate of proliferation. In addition,cells with alterations to p27 levels had unchanged CD43 expression.In sum, these data suggest that Fbxo7 acts as a positive regulatorof pro-B cell differentiation because its levels are inverselycorrelated with CD43 expression, and that the effect of Fbxo7expression on CD43 is not due to changes in the cell cycle.
Reduction of Fbxo7 levels enhanced erythropoietin-induced differentiation of Ba/F3 cells along the erythroidlineageBecause Fbxo7 appeared to positively regulate pro-B celldifferentiation, we next investigated whether differentiation ofanother haematopoietic cell lineage is also affected by Fbxo7levels. We exploited the potential of Ba/F3 cells to be differentiatedalong the erythroid lineage because of expression of erythroid-specific transcriptional factors, including GATA-1, NF-E2 andEKLF (Krosl et al., 1995). Cells will partially differentiate whenengineered to express the erythropoietin receptor (Epo-R) andcultured in the presence of erythropoietin (Epo). They cannot,however, terminally differentiate and do not synthesize
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Fig. 6. Changes to cell surface marker expression in cellswith reduced or overexpressed levels of Fbxo7.Representative FACS plots of cells with stably reducedFbxo7 expression stained for CD43-PE (A) or CD25-PE(B); GFP is on the x axis (FL-1), PE is on the y axis (FL-2).(C)Representative FACS plots from Ba/F3 cells transientlytransfected with empty vector, Fbxo7–IRES–GFP or p27–IRES–GFP, and stained for CD43-PE or CD25-PE-Cy7 asindicated. Analysis was first gated on GFP-positive cells(representative plot on left). Then, this gated population wasanalysed for FSC on the x axis and CD43-PE or CD25-PE-Cy7 on the y axis. Representative FACS plots for cells withstable reduction of p27 (D) where GFP is on the x axis (FL-1) and CD43-PE is on the y axis (FL-2), and of cells withstable reduction of Cdk6 expression (E), where FSC is onthe x axis and CD43-PE is on the y axis.
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haemoglobin. Instead, expression of -globin mRNA is used tomeasure their state of differentiation (Krosl et al., 1995; Carroll etal., 1995; Krosl et al., 1996). To test directly the effect of Fbxo7on cytokine-induced differentiation, we engineered expression ofEpo-R–IRES–GFP in these cells (see Materials and Methods), andcreated cell lines with reduced Fbxo7 by subsequently infectingBa/F3 Epo-R-expressing (Ba/F3-Epo-R) cells with retrovirusesencoding the short-hairpin that targets Fbxo7 or with a non-silencingcontrol. Two independent clonal lines were created, and thereduction in Fbxo7 protein expression was verified byimmunoblotting (Fig. 7A). Cells were cultured in the presence ofEpo, total RNA was isolated and semi-quantitative RT-PCR
performed to assess -globin mRNA levels. Samples from cellswith reduced Fbxo7 showed a 10-fold increase in the amount of -globin message as compared with vector (Fig. 7B), suggesting anegative regulatory role for Fbxo7 in this engineered model oferythroid differentiation.
The rate of proliferation of Epo-R–IRES–GFP control cells andcells with reduced Fbxo7 was also determined by a growth curveperformed on cells induced to differentiate by growth in Epo. Ashas been previously reported for Ba/F3–Epo–R cells, a transientslowing of growth was observed when cells were withdrawn fromIL-3 and induced to differentiate by the addition of Epo. However,cells with reduced Fbxo7 levels were less delayed, recovered morerapidly and proliferated faster than vector control cells (Fig. 7C,D).This was especially apparent when growth rates of the cell lines inthe first 24 hours were compared. Fbxo7-knockdown cells seededinto Epo showed little delay, proliferating almost as quickly ascells seeded back into IL-3 after starvation (Fig. 7D). By 42 and48 hours after cytokine withdrawal, the differences in cell numberwere statistically significant (P0.0131 and P0.0158) (Fig. 7C).These data demonstrate that reduction in Fbxo7 levels results inmore-robust differentiation in response to Epo along an erythroidlineage, despite cells proliferating faster than vector control cells.Therefore, these data argue against the requirement of a slower cellcycle to allow erythroid differentiation in Ba/F3-Epo-R cells.
Mice with reduced levels of Fbxo7 show increasednumbers of pro-B cells and pro-erythroblastsOur data indicate that Fbxo7 has a cell-type specific anti-proliferative role (Ba/F3, Nalm-6 and C-33A cells) and also positiveand negative regulatory roles in the differentiation of pro-B anderythroid cell lineages, respectively. As these in vitro experimentswere conducted mainly by using a Ba/F3 cell line model, we alsowanted to assess the effect of Fbxo7 on differentiation in a morephysiological setting. To achieve this, a transgenic mouse wasengineered using targeted ES cells from the International KnockoutMouse Consortium (EUCOMM ID: 23037). These ES cells havean insertion of the LacZ gene with a splice acceptor site betweenexon 3 and exon 4 of isoform 1 of Fbxo7, and this Fbxo7LacZ allele,which disrupts expression of all Fbxo7 isoforms by splicing in theLacZ-coding sequences. Four chimeric mice were generated byinjection of ES cells into blastocysts, two of which showed germlinetransmission of the Fbxo7LacZ transgene. Heterozygous animalswere bred to homozygosity, and lysates from various tissues wereimmunoblotted for the expression of Fbxo7, showing a gene–dosage-dependent reduction in Fbxo7 expression in theheterozygous and homozygous animals (Fig. 8A). Fbxo7 proteinexpression was, however, detectable upon very long exposure ofimmunoblots in the cerebellum demonstrating the production ofsome wild-type Fbxo7 mRNA (data not shown). Our full phenotypecharacterisation of these mice will be reported elsewhere(manuscript in preparation).
To address the effect of reduced Fbxo7 expression on B celldevelopment in vivo, splenocytes were harvested from age-matchedwild-type (n2) and homozygous (n2) littermates, and stained forB220, CD43 and CD25 to measure the pro-B and pre-B cellpopulations. Animals homozygous for Fbxo7LacZ showed astatistically significant increase in the percentage of CD43hiCD25lo
pro-B cells as compared with wild-type controls; however, thelater pre-B cell stages showed no significant differences (Fig.8B,C). The increase in the percentage of pro-B cells seen in vivocould be due to increased proliferation of pro-B cells or a block in
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Fig. 7. Fbxo7 regulates differentiation. (A)Immunoblots for Fbxo7expression in cell lysates from independent clonal cell lines of Ba/F3 cellsexpressing the Epo-R and infected with retroviruses expressing a miR-30-based hairpin RNA which targets Fbxo7 expression. A non-specific band wasused as an internal loading control. (B)Semi-quantitative RT-PCR for -globinand cyclophilin from cells which have been induced to differentiate by theaddition of Epo for 3 days. (C)Increase in cell numbers over 3 days duringwhich time cells were starved of all cytokines and then induced to differentiatein Epo or to proliferate in IL-3. (D)Data from the first 24 hours of the graphshown in C. *P<0.05.
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maturation from the pro-B to pre-B cell stages. These data supportthe results described for Ba/F3 cells and suggest that Fbxo7 usuallyhas an anti-proliferation or a positive role in promoting thedifferentiation of pro-B cells.
To address the effect of Fbxo7 on erythroid differentiation, thenumber of erythroid colony-forming units (CFU-Es) was assessedby seeding bone marrow cells into methylcellulose mediumcontaining Epo. Cells from mice homozygous for Fbxo7LacZ formedalmost twice as many colonies, indicating a significant increase inthe number of CFU-Es (P0.0033) in these mice as compared withwild-type animals (Fig. 8D). These data suggest that Fbxo7normally has an anti-proliferative role in pro-erythroblasts or a rolein promoting pro-erythroblast differentiation. Bone marrow fromwild-type and homozygous animals was also stained for CD71 andTer119 protein expression, because together they demarcate thelater stages of erythroblast differentiation (Fig. 8E,F). Consistentwith data showing an increase in CFU-Es in homozygous Fbxo7LacZ
animals, we also detected a statistically significant increase in thenumber of earlier stage pro-erythroblasts (CD71hiTer119lo)(P0.017) as compared with wild-type littermates. Surprisingly,they also had fewer late normoblasts (orthochromatophilicerythroblasts) (CD71–Ter119hi) (P0.025), the stage of
differentiation after which cells exit the bone marrow anddifferentiate to reticulocytes and erythrocytes (Fig. 8E,F). Thesedata indicate that pro-erythroblasts cells are blocked in theirdifferentiation, eventually leading to a decrease in the numbers oflater stage cells or, alternatively, that Fbxo7 acts in a differentcapacity at later stages of erythropoiesis, potentially required topromote proliferation or survival at a later stage in maturation.
DiscussionIn a previous study using immortalised fibroblasts, overexpressionof Fbxo7 protein increased cyclin D–Cdk6 complexes and causedCdk6-dependent transformation without affecting the cell cycle orproliferation (Laman et al., 2005). In addition, in osteosarcomacells, a reduction of Fbxo7 expression led to decreased amounts ofcyclin D–Cdk6 complexes, also without altering the cell cycle(Laman et al., 2005), which argues for a direct role of Fbxo7 inpromoting the assembly of cyclin D–Cdk6 complexes. We reporthere that, in an immortalised pro-B cell line, overexpression ofFbxo7 does not transform cells, enhance cell proliferation orviability or affect the levels of cyclin D–Cdk6 complexes. Instead,the reduction of its levels accelerated the rate of proliferation,decreased cell size, and shortened G1 phase, all indicative of an
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Fig. 8. Mice with LacZ insertion into the Fbxo7 locus show increased numbers of pro-B cells and pro-erythroblasts. (A)Immunoblots of lysates preparedfrom the organs of mice inheriting one or two Fbxo7LacZ alleles. (B)FACS analysis of B220+, CD43, CD25 populations in splenocytes of WT and homozygousFbxo7LacZ animals. Analysis was first gated on B220+ cells and then analysed for CD43 on the x axis and CD25 on the y axis. (C)Percentages of precursor B cellpopulations (pro-B: B220+, CD43hi, CD25lo; small pre-B: B220+, CD43hi, CD25hi; large pre-B: B220+, CD43lo, CD25hi) (n2). (D)Number of CFU-E coloniespresent in bone marrow of WT and homozygous Fbxo7LacZ animals upon seeding into colony formation assays containing Epo (n2 mice per genotype, eachseeded in triplicate). (E)FACS analysis of erythroid populations in bone marow of WT and homozygous Fbxo7LacZ animals by Ter119 staining on the x-axis andCD71 on the y-axis (n2). (F)Percentages of erythroblast populations as indicated. *P<0.05, **P<0.01.
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anti-proliferative function for Fbxo7 in pro-B cells. This effect wasnot specific to Ba/F3 cells, because reduction of Fbxo7 alsoincreased the proliferation rate of Nalm-6 and C-33A cells. Cellcycle analysis showed that a decrease in Fbxo7 caused a rapidtransit through G1 phase. Cells also had higher Cdk2 activity,stemming from increased cyclin E and cyclin A, reduced p27 andan overall reduction in the levels of pocket proteins. Oneinterpretation of these findings is that, in Ba/F3 cells, p27 has amain role in restraining S phase entry, which is supported by thefinding that independently decreasing its levels results in smallercells and a faster rate of proliferation – even without increasedcyclin E levels. In addition, changes to Cdk6 levels had nodetectable effect on cell cycle parameters, suggesting that, althoughCdk6 is abundant in pro-B cells, it is not rate-limiting for cell cycleprogression under the experimental conditions used here. Together,these results support the idea that Fbxo7 has different cell cycleactivities, and transforming or anti-proliferative properties in avariety of cell types, and we speculate that the ultimate effect ofchanges to its expression levels depends on the G1 phase circuitryof that cell type or, possibly, to the stage of differentiation of thecell (stem or progenitor cell versus mature cell). We notice, forexample, that in differentiating embryonal carcinoma P19 cells,proliferation correlated with the activity of the cyclin-E–cyclin-A–Cdk2 but not cyclin-D–Cdk4–Cdk6–p27 complexes (Bryja et al.,2008).
Because Fbxo7 affects G1 phase and cell proliferation, wereasoned it might also affect cell differentiation – which istemporally linked to G1. A long-standing correlation exists betweena prolonged G1 phase that enhances the differentiation potentialand a shortened G1 phase that diminishes it (Kato and Sherr, 1993;Carroll et al., 1995; Johnson et al., 1993). However, there isexperimental evidence in a variety of cell types, includinglymphocytes (Rush et al., 2002; Lea et al., 2003), which show thatthese processes can be separated and need not proceedsimultaneously (Brown et al., 2003). Experiments here areconsistent with the idea that Fbxo7 promotes the differentiation ofpro-B cells: overexpression of Fbxo7 decreased CD43, which isdownregulated as B cell mature, and reduction of Fbxo7 levelsincreased its expression. This inverse correlation between Fbxo7and CD43 expression is suggestive of a direct regulatoryrelationship, but it is unlikely to be due to cell cycle alterations.Several lines of evidence support this. For example, althoughFbxo7 knockdown altered the cell cycle, the overexpression ofFbxo7 did not alter proliferation but did decrease CD43 expression.Moreover, in other experiments where cell cycle length was alteredby changes to p27, CD43 expression was not affected. However,Cdk6 knockdown, which had no effect on the cell cycle, increasedthe frequency of CD43hi-expressing cells. Taken together thesedata argue that CD43 expression is regulated independently fromcell cycle progression. A further implication of this finding is thatFbxo7 also regulates CD43 signalling in other cell types thatexpress this marker, such as activated B cells, macrophages and Tcells, a hypothesis that is currently under investigation.
Consistent with the observations in Ba/F3 cells, quantificationof pro-B cells (B220+, CD43hi, CD25lo) from spleens showed astatistically significant increase in the proportion of pro-B cells inmice that are deficient for Fbxo7 protein expression comparedwith wild-type mice. This increase was specific to pro-B cells andcould have resulted either from an increase in the proliferation ofthese precursor cells due to the loss of the anti-proliferation functionof Fbxo7 or, alternatively, to a block in the differentiation at the
pro-B to pre-B cell stage, due to the inability to downregulateCD43 in the absence of Fbxo7. In the erythroid lineages, asignificant increase in the number of CFU-Es and pro-erythroblastswas observed in the bone marrow of homozygous Fbxo7LacZ mice.This, again, could reflect either increased proliferation of theselineage-committed precursor cells or a block in the differentiationat this stage in erythropoiesis. In considering these two models wenotice that, in the Ba/F3 Epo-R erythroid differentiation model, thereduction in Fbxo7 protein expression enhanced both Epo-induceddifferentiation and proliferation. This lends support to the model inwhich increased proliferation, rather than a block in differentiation,explains the in vivo findings. However, a reduction in cell numberat the late normoblast stage was also observed and might meanthat, when Fbxo7 levels are reduced, erythroid differentiation isinhibited. Alternatively, Fbxo7 might have different pro-proliferation or pro-survival functions later on in erythropoiesis.
How cell cycle regulatory proteins like Fbxo7 and Cdk6influence differentiation and cell cycle, and at which maturationstage this occurs is an interesting question for future study. Thereare experimental data that indicate direct binding of Cdk6 totranscription factors that regulate differentiation, suggesting a cell-cycle-independent function (Fujimoto et al., 2007), whereas otherstudies show that downregulation of Cdk6 protein is crucial forterminal differentiation, presumably by allowing a permanentwithdrawal from the cell cycle (Matushansky et al., 2003). Inaddition, it is possible that these proteins have different regulatoryroles, cell cycle and/or transcriptional functions at different timesin cell maturation. In summary, Fbxo7 impacts on both cell cycleand differentiation in a cell-type specific manner, which indicatesthat Fbxo7 has an important and sensitive role in integrating cellsignalling and functionality with cell cycle progression duringhaematopoiesis.
Materials and MethodsCell cultureMurine Ba/F3 pro-B cells were maintained in complete RPMI-1640 mediumsupplemented with 10% foetal calf serum (FCS; PAA Laboratories), 10% WEHI-3Bconditioned medium, penicillin (50 U/ml), streptomycin (50 U/ml) (Gibco BRL),and 10ng/ml recombinant murine IL-3 (Fitzgerald Laboratories). Human Nalm-6pre-B acute lymphoblastic leukaemia cells were maintained in RPMI-1640 mediumsupplemented with 10% FCS, penicillin (50 U/ml),and streptomycin (50 U/ml).Human C-33A cervical cancer cells were maintained in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10% FCS, penicillin (50 U/ml), andstreptomycin (50 U/ml). Cells were incubated at 37°C in a humidified 5% CO2
atmosphere.To reduce the protein levels of human or mouse Fbxo7, or mouse p27, cells were
infected with retrovirus that contained either an LMP or PSMP vector and thatdelivered a miR-30-based short hairpin RNA sequence targeting the genes of interest.Target sequences identified and validated by RNAiCodex (http://cancan.cshl.edu/cgi-bin/Codex/Codex.cgi) were purchased and hairpin-containing oligonucleotids werePCR amplified and subcloned into retroviral vectors. The sequences used were, formurine Fbxo7sh1: 5�-CGGAGATTGTGGTATTGATAAT-3� (HP_240439), humanFbxo7sh1: 5�-CGCCCAGTCTGGTGTTTGGAAT (HP_3087), human Fbxo7sh2:5�-CGCTGAGTCAATTCAAGATAAT-3� (HP_434828), murine p27: 5�-AGCCATTAGAGTCACTTTCCAT-3� (HP_288922). Either empty LMP or non-silencing vector PSMP (Open Biosystems) were used as negative controls. Todecrease Cdk6 protein levels, retroviruses containing pRETROSUPER-based shorthairpin constructs were used as previously described (Laman et al., 2005) to interferewith Cdk6 transcription. Independent B cell clones were generated using limitingdilution and grown in the presence of puromycin (2 g/ml); several clones wereanalysed. A different short hairpin construct was used to reduce FBXO7 geneexpression in C-33A cells, and polyclonal cell lines were selected in the prescencepuromycin (2 g/ml). To overexpress proteins, cells were infected with retrovirusesthat encoded MSCV-IRES-GFP and expressed the gene of interest. Polyclonal celllines were detected by FACS for GFP-expressing cells.
Ba/F3 cells expressing Epo-R were generated by retroviral infection with MSCV-EpoR-GFP (kindly provided by Tony Green, Department of Hematology, Universityof Cambridge). Retroviruses encoding the Epo-R-IRES-GFP were used to infect
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Ba/F3 cells, which were sorted for GFP expression. Clones were subsequentlyselected for Epo-R expression by growing cells in medium that was supplementedwith 0.05 U/ml erythropoietin (Epo) (Cell Sciences) but lacked IL-3. Ba/F3-Epo-Rcell lines were thereafter maintained in IL-3 during standard sub-culturing.
For transient transfection, 5 g of DNA encoding p27or Fbxo7, or empty vectorplasmid DNA was nucleofected into 5�106 Ba/F3 cells by using solution V andprogram X-001, as per manufacturer’s instructions (Lonza). After 72 hours, liveGFP+ cells were immunostained (see below) and analysed by flow cytometry, usingPI (2.5 g/ml) as a live–dead discriminator.
To measure the rate of proliferation and the cell volume, cells were seeded intriplicate at a density of 0.3–0.5�106 cells/ml (for B cells) or 2�105 cells per well(C-33A cells), and cell numbers and volumes were assayed every 2 days using aCasy cell counter (Scharfe System). Three independent experiments were performed.To measure cell viability, cells were exposed to 50 g/ml PI (Sigma Aldrich) at roomtemperature for 30 minutes prior to analysis by FACS (FACS Calibur, BectonDickinson). To quantify DNA content, 1–2�106 cells were washed in PBS and fixedin ice-cold 70% ethanol at 4°C for 30 minutes. Cells were washed in PBS, incubatedin 50 g/ml RNAse A and 100 g/ml propidium iodide (PI) for 30 minutes, and thenanalysed by FACS. For statistical analyses, data were analysed using a two-tailedStudent’s t-test; values of P<0.05 or less were considered significant.
Immunoprecipitations and immunoblottingFor immunoprecipitations and kinase assays, cells were lysed in 50 mM HEPES pH7.4, 150 mM NaCl, 20 mM EDTA pH 8, 1 mM DTT, 10 mM -glycerophosphate,0.5% Triton X-100, 10 mM NaF, 2 mM PMSF and protease inhibitor cocktail, thensonicated three times and centrifuged at 16100 g for 10 minutes at 4°C. Lysates werepre-cleared by rotation in the prescence of protein G-Sepharose beads at 4°C for 30minutes prior to incubation with primary antibody and addition of protein G-Sepharose beads for 4 hours at 4°C for immunoprecipitation. Beads were washedthree times and co-immunoprecipitating proteins were detected by immunoblotting.For kinase assays, beads were washed in 50 mM HEPES/KOH pH 7.4, 1 mMMnCl2, 10 mM MgCl2, 10 mM -glycerophosphate, 1 mM DTT, 1 mM PMSF priorto the addition of 0.6 g of purified pRb substrate (Abcam, ab56270), 1 Ci of [-33P]ATP and 200 mM ATP and incubating at 30°C for 1 hour. Proteins were resolvedusing SDS-PAGE and gels fixed in 10% acetic acid/methanol, dried and quantifiedusing a Cyclone Phosphor Imager (PerkinElmer).
Protein samples were prepared by lysing cell pellets in modified RIPA buffer (150mM NaCl, 1% NP40, 0.1% SDS, 50 mM Tris pH 7.5, protease inhibitor cocktail),incubating on ice for 15 minutes, and centrifuged at 16,100 g for 10 minutes at 4°C.For immunoblotting, protein concentration was determined using protein assay(Biorad). 50 g of total lysate was resolved by SDS-PAGE, and transferred ontoPVDF membranes, which were incubated in PBS with 0.05% Tween-20 and 5%dried skimmed-milk powder. The following rabbit polyclonal primary antibodieswere used: Cdk6 (c-12) (sc-177), Cdk4 (c-22) (sc-260), Cdk2 (M2) (sc-163), cyclinA (H-32) (sc-751), cyclin E (M-20) (sc-481), cyclin D2 (c-17) (sc-181), cyclin D3(c-16) (sc-182), p27 (c-19) (sc-528) (Santa Cruz Biotechnology Inc.) and actin(Sigma Aldrich). The antibody against Fbxo7 has been described previously (Lamanet al., 2005). Donkey anti-rabbit IgG conjugated to horseradish peroxidase wasobtained from Santa Cruz Biotechnology, Inc.
Differentiation assaysThe following fluorescent and biotin-conjugated antibodies were used to assay cellsurface markers: CD43-PE (clone S7; BD Pharmingen), CD25-PE or PE-Cy7 (bothclone PC61.5; eBioscience), B220-biotin (clone RA3-6B2; Pharmingen) or B220-FITC (clone RA1-6B2; eBioscience), CD19-PE (clone 1D3; eBioscience), IgM-biotin (clone II/41; eBioscience), CD71-biotin (clone RI7 217.1.4; Caltag), Ter119-PE(clone Ter119; Caltag) and Streptavadin-APC (Caltag). All antibodies were used at1:100 dilution in 100 l FACS buffer (1�PBS, 0.05% NaN3, 2.5% FBS) – exceptanti-Ter119, which was used at 1:50. For all staining procedures, 5�105 cells wereincubated with primary antibodies for 1 hour at 4°C, washed twice in FACS bufferand then either analysed immediately or incubated with secondary streptavidin-APCantibody for 30 minutes at 4°C, followed by two washes. Cells were then collectedand analysed by flow cytometry (Cyan ADP Analyser, DakoCytomation) usingSummit 4.3 software (Beckman Coulter). To assay B-cell populations, spleen cellsuspensions were treated with 200 l NH4Cl red blood cell lysis solution (150 mMNH4Cl, 10 mM KHCO3, 1 mM EDTA pH 7.3) for 1 minute, before being resuspendedin 15 ml of complete medium. Cells were then Fc-blocked with anti-CD16/CD32antibody (clone 93; eBioscience) for 20 minutes at 4°C prior to incubation withprimary antibody. For analysis of erythroid progenitors and for bone marrow staining,cells were not pre-treated with red blood lysis solution before Fc blocking and cellstaining. For colony formation assays, 2�105 bone marrow cells were plated with3U/ml Epo as per manufacturer’s instructions (M3234) (StemCell Technologies);CFU-E colonies were counted after 3 days.
EpoR-expressing Ba/F3 cells, with or without the short-hairpin RNA to targetFbxo7, were cultured in the presence of IL-3, Epo (0.05 U/ml or 0.5 U/ml), or IL-3and Epo (0.5 U/ml) together. Cells were harvested at various time points after theaddition of Epo, and RNA was extracted by lysing cells in Trizol and centrifugationat 11,200 g for 10 minutes at 4°C. Chloroform extraction was carried out beforeRNA isolation using the RNeasy mini kit (Qiagen) and RNA concentration was
measured using a nanodrop spectrophotomer (Nanodrop Technologies). 1 g of totalRNA was used for each sample harvested at 3 days post induction of differentiation.2 l of random hexamers (Promega) were added to each sample, which was incubatedat 65°C for 5 minutes and then kept on ice. 7 l of a master mix [4 l of 5� RTbuffer, 0.5 l of RNasin (Promega), 2 l of 10 mM dNTP, 0.5 l of reversetranscriptase (Roche)] was added to each sample, which was then incubated at 25°Cfor 10 minutes, 42°C for 60 minutes and 70°C for 10 minutes. cDNA was used inPCR reactions (95°C for 30 seconds, 50°C for 1 minute, 72°C for 1 minute, for 25 cycles) using primers to amplify -globin (forward: 5�-GACCCAGCGGTACTTTGATAGC-3� and reverse: 5�-TGAGGCTGTCCAAG -TGATTCA-3�) and cyclophilin (forward: 5�-CCTTGGGCCGCGTCTCCTT-3� andreverse: 5�-CACCCTGGCACATGAATCCTG-3�) (Invitrogen). Quantification wascarried out using a Molecular Imager Gel Documentation System using QuantityOne software (BioRad).
Generation of Fbxo7LacZ miceMouse embryonic stem (ES) cells heterozygous for the -galactosidase gene (LacZ)targeting the Fbxo7 locus between exons 3 and 4 were generated by conditional‘targeted trapping’ technology employed by the European Conditional MouseMutagenesis Program (www.eucomm.org). To generate chimeric mice, twoindependent Fbxo7LacZ/+ ES cell lines (EUCOMM ID: 23037) were obtained,karyotyped and injected into C57BL6/J blastocysts. One clone successfully producedfour chimeric male mice, as determined by PCR analysis for the mutant transgene(primers available on request). Two of these males had germline transmission of thetransgene and their offspring were bred to homozygosity (Fbxo7LacZ/LacZ). Experimentswere conducted from heterozygous crosses to enable analysis of homozygous micewith wild-type littermate controls. Spleen and bone marrow were harvested frommice aged 7–12 weeks.
We thank C. Bacon, R. Dickins, A. Green, N. Holmes, S. Lowe andA. Skoultchi for generously providing reagents, W. Mansfield, W.Khaled and N. Miller for technical advice and assistance, and D.Lagos, N. Holmes, A. Philpott and members of the Laman lab for theircritical reading of this manuscript. Funding was provided by theAssociation for International Cancer Research, the BBSRC, Campod,and Cancer Research UK.
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