Knockdown of Fbxo7 Reveals Its Regulatory Role in Proliferation and Differentiation of Haematopoietic Precursor Cells

Research Article 2175 Knockdown of Fbxo7 reveals its regulatory role in proliferation and differentiation of haematopoietic precursor cells

El 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])

Summary Fbxo7 is an unusual F-box protein because most of its interacting proteins are not substrates for ubiquitin-mediated degradation. Fbxo7 directly binds p27 and Cdk6, enhances the level of cyclin D–Cdk6 complexes, and its overexpression causes Cdk6-dependent transformation 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, decreased cell size and shortened G1 phase. Analysis of cell cycle regulators showed that cells had decreased levels of p27, and increased levels of S phase cyclins and Cdk2 activity. Also, Fbxo7 protein levels correlated inversely with those of CD43, suggesting direct regulation of its expression and, therefore, of B cell maturation. Alterations to Cdk6 protein levels did not affect the cell cycle, indicating that Cdk6 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 was assessed by creating a transgenic mouse with a LacZ insertion into the Fbxo7 locus. Homozygous Fbxo7LacZ mice showed significantly increased pro-B cell and pro-erythroblast populations, consistent with Fbxo7 having an anti-proliferative function and/or a role in promoting maturation of precursor cells.

Key words: Fbxo7, Cdk6, Cell cycle, Differentiation, Pro-B cell

Introduction Another FBP that interacts with G1–S regulatory proteins is F-

Journal of Cell Science During the cell cycle, ubiquitin-mediated proteolysis provides a box protein 7 (Fbxo7). In contrast to the destabilising effects of swift and precise means to regulate the abundance of cell cycle other FBPs, Fbxo7 acts as a specific assembly factor for cyclin D– regulatory proteins, including cyclins and cyclin-dependent kinase Cdk6 complexes. Fbxo7 interacts directly with both Cdk6 and p27, (Cdk) inhibitors. This mechanism for regulating the turnover of and cooperatively increases cyclin D3 interactions with Cdk6 in proteins is mediated through ubiquitin ligases, which transfer vitro (Laman et al., 2005). In vivo, Fbxo7 overexpression in ubiquitin to target proteins, enabling their timely destruction immortalised murine fibroblasts leads to their Cdk6-dependent (Hershko, 1997; Hershko and Ciechanover, 1998; Nakayama and transformation. These cells had increased levels of cyclin D–Cdk6 Nakayama, 2006; Reed, 2006; Reed, 2003). E3 ubiquitin-ligases complexes and E2F activity, and formed tumours in athymic nude promote the specific attachment of poly-ubiquitin chains, which mice (Laman et al., 2005). Many mitogen and cytokine signalling then triggers proteolysis by the 26S proteasome. SCF-type (Skp1– pathways converge on cyclin D–Cdk activity, which – when Cullin–F-box) E3 ubiquitin ligases are multi-subunit complexes overstimulated – promotes oncogenesis. Because of its Cdk6- that consisting of Skp1, Cullin, Rbx1 and an F-box protein (FBP) dependent transforming activity, we propose that Fbxo7 functions (Ang and Wade, 2005; Deshaies, 1999; Jackson and Eldridge, as an oncogene. 2002; Nakayama and Nakayama, 2005; Vodermaier, 2004). It is In U2OS and NIH3T3 cells, Fbxo7 has shown selectivity for the FBP, which has a crucial role in specifically recruiting the Cdk6 over Cdk2 and Cdk4. Although the biochemical properties target substrate, usually directed by post-translational modification of Cdk4 and Cdk6 are similar, more-recent studies have indicated of the substrate (Cenciarelli et al., 1999; Hermand, 2006; Ho et al., that differences can be discerned in their selective binding to co- 2008; Jin et al., 2004; Winston et al., 1999). In cell cycle regulation, factors (Sugimoto et al., 1999; Laman et al., 2005), preference for several FBPs that regulate G1–S phase regulators have been phosphorylation sites in pRb in vitro (Takaki et al., 2005), intensively studied. These include the prototypical S-phase kinase- sensitivities to INK4 family members (Tourigny et al., 2002; Jones associated protein 2 (Skp2, also known as Fbxl1) and the F-box et al., 2007) and in vivo partnering with D-type cyclins (Ely et al., and WD repeat domain containing 7 (Fbxw7) that, respectively, 2005). Also, in studies of knockout mice, tissue-specific defects regulate the levels of the Cdk inhibitors cyclin E and cyclin- are seen: Cdk4-knockout mice have impaired development of dependent kinase inhibitor 1B (CDKN1B; also known as and pancreatic -islet cells (Rane et al., 1999), whereas Cdk6-null mice hereafter referred to as p27). In addition, three FBPs – Fbxo4, show deficiencies in haematopoiesis (Malumbres et al., 2004; Hu Fbxw8, Fbxo31 – promote the ubiquitin-mediated degradation of et al., 2009). In addition, whereas Cdk4–Cdk6 or Cdk2–Cdk4 cyclin D1 (Lin et al., 2006; Santra et al., 2009; Okabe et al., 2006). double-knockout mice are embryonic lethal, Cdk2–Cdk4 double- 2176 Journal of Cell Science 124 (13)

knockout mice are viable (Barriere et al., 2007; Malumbres et al., expression of Fbxo7 would alter the proliferation rate or substitute 2004; Berthet et al., 2006). Moreover, there is mounting evidence for IL-3 signalling. Ba/F3 cells were infected either with a retrovirus for a specific role for Cdk6 in differentiation, especially in neuronal expressing human FBXO7–IRES–GFP or the empty vector, and and haematopoietic cells (Ericson et al., 2003; Slomiany et al., fluorescence-activated cell sorting (FACS) sorted for GFP+ cells. 2006; Fujimoto et al., 2007; Grossel and Hinds, 2006; Matushansky Immunoblotting of lysates from polyclonal cell lines demonstrated et al., 2003). These studies support the idea that the G1 Cdks have the presence of exogenous Fbxo7 expression, and the increased overlapping, yet distinct, activities. mobility on SDS-PAGE of human Fbxo7 compared with the Fbxo7 also directly interacts with and stabilizes p27, although endogenous murine Fbxo7 (Fig. 1A). We first tested whether Fbxo7 does not require p27 to increase levels of cyclin D–Cdk6 increased Fbxo7 levels would enhance the proliferation rate which (Laman et al., 2005). p27 was first identified as an inhibitor of was assessed by seeding cells at equal densities and counting live cyclin-dependent kinases but it also acts as an assembly factor for cells at periodic intervals. However, both cell lines proliferated at cyclin D–Cdk complexes. More recently, p27 has been shown to the same rate (Fig. 1B). In addition, the proportion of live and dead have roles that extend beyond direct cell cycle regulation to functions cells was measured, and no significant differences were observed in differentiation, motility and migration, and cytoskeletal signalling between control and Fbxo7-expressing Ba/F3 cells (Fig. 1C). Next, (reviewed in Borriello et al., 2007). As such, p27 has both tumour the ability of Fbxo7 to substitute for IL-3 signalling was tested by suppressor and oncogenic activities – dependent on its subcellular withdrawing it from the growth medium and measuring cell localization and cell type (Besson et al., 2007; Chu et al., 2008). viability. As seen in Fig. 1D, vector control and Fbxo7-expressing Because Fbxo7 exhibited a preference for Cdk6, we reasoned cells lost viability with essentially the same kinetics. We noticed that it might have different functions in haematopoietic cells where that 48 hours post withdrawal ~5% more Fbxo7-expressing cells Cdk6 activity predominates (Laman, 2006). In this study, we tested were alive compared with those expressing the vector control; the activity of Fbxo7 in B cell lines. In contrast to our findings in however, this small effect, although statistically significant fibroblasts and osteosarcoma cells, data presented here indicate (P0.027), was transient, and suggests that Fbxo7 has only a weak that Fbxo7 is not a transforming gene in pro-B cells. Instead, the ability to provide survival signals to Ba/F3 cells. Thus Fbxo7 reduction of Fbxo7 protein levels led to an increased rate of expression did not relieve IL-3 dependence in Ba/F3 cells nor was proliferation and changed the expression of B cell markers to it rate-limiting for their proliferation. resemble a less-mature state. In addition, a mouse model presenting reduced Fbxo7 protein levels showed increased populations of Decreasing Fbxo7 levels enhanced Ba/F3 cell proliferation precursor pro-B cells and pro-erythroblasts. These data are We next determined whether Fbxo7 is required for proliferation or consistent with a model, in which Fbxo7 has an anti-proliferative viability. To achieve this, Ba/F3 cells were infected with retroviruses role in precursor cells and also a cell-cycle-independent regulatory that encoded miR-30-based short-hairpin RNA targeting the role in the maturation of haematopoietic cells. expression of Fbxo7 mRNA or an empty vector. Cell lines were cloned by limiting dilution under antibiotic selection and screened Results by immunoblotting lysates to test for Fbxo7 expression. Data for Increased Fbxo7 levels do not transform Ba/F3 cells two independent clonal lines are shown in Fig. 2A. Expression of

Journal of Cell Science To address whether Fbxo7 is capable of transforming short hairpin RNA achieved a significant reduction in Fbxo7 levels, haematopoietic cells, we used the pro-B cell line Ba/F3, which arguing against the idea that high levels of Fbxo7 are required for depends on IL-3 for survival, and tested whether exogenous proliferation or viability. In fact, during cloning, we noticed that

Fig. 1. Increased Fbxo7 expression in Ba/F3 cells does not affect growth or proliferation. (A)Immunoblot of cell lysates from Ba/F3 cells infected with retroviruses that express either human FBXO7 or the empty vector. Actin was used as a control for equivalent loading. (B)Time course of the proliferation rate of cells seeded at equal densities and counted on the day indicated. (C)Percentages of viable and non-viable cells in an asynchronous culture assessed by PI staining of live cells and by FACS (mean ± s.d., n3). (D)Kinetics of cell survival upon withdrawal of IL-3 from the culture medium. Cells were seeded at equal densities in medium without IL-3 and the number of viable cells was determined as indicated; *P<0.05. Regulatory role of Fbxo7 in precursor cells 2177

cultures with reduced Fbxo7 protein levels proliferated more rapidly with vector control cells. In addition, although we have previously and were smaller than control cells. By measuring the rate of shown in fibroblasts and osteosarcoma cells that Fbxo7 is required proliferation (P<0.01 on day 3 and P<0.001 on day 17) and the cell for Cdk6 association with D-type cyclins (Laman et al., 2005), in volume (P<0.001), we confirmed the statistical significance of Ba/F3 cells, the amount of Cdk6 associated with endogenous cyclin these observations (Fig. 2B,C). D2 and D3 was unaffected by the reduction of Fbxo7 levels as To investigate the molecular basis for the Ba/F3 cell cycle tested by in vivo co-immunoprecipitation assays (Fig. 2E). We phenotypes, protein levels of other cell cycle regulators were also conclude, therefore, that Cdk6 association with D-type cyclins determined. Immunoblotting the lysates from control and Fbxo7- does not depend on Fbxo7 in this cell type. Together, these data knockdown cells showed that the total levels of subunits (cyclin indicate that reducing Fbxo7 protein levels augmented Cdk2 D2, cyclin D3 and Cdk4, Cdk6) were not significantly changed; activity, and promoted a rapid G1 to S phase transition and a however, protein expression of the S phase cyclins E and A was smaller cell size. markedly increased, whereas p27 protein levels were reduced (Fig. As cells acquire much of their mass during G1 phase, cell cycle 2A). These alterations suggested that Cdk2 activity was profiling was performed. Asynchronous populations of cells were predominantly affected in these cells. To test this, Cdk2 kinase fixed, stained with propidium iodide (PI) and assayed by FACS to assays were performed. In independent experiments endogenous determine DNA content (n10). Representative plots shown in Cdk2 activity was quantified as being 25% (data not shown) and Fig. 2F demonstrate that, compared with vector control, Fbxo7- 52% (Fig. 2D) increased in cells with reduced Fbxo7 as compared knockdown cells had fewer cells with 2N DNA content in G1 Journal of Cell Science

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 cycle proteins 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 have reduced 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 endogenous levels of Fbxo7. pRb was used as a substrate and the amount of incorporated [-33P]ATP was quantified on a phosphoimager (Cyclone). (E)Immunoblotting of endogenous 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 n10) 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 PI staining of live cells and by FACS (mean ± s.d., n3). (H)Immunoblotting for pocket proteins in cell lysates of Ba/F3 cells with reduced and endogenous levels of Fbxo7. **P<0.01, ***P<0.001. 2178 Journal of Cell Science 124 (13)

phase and more cells with >2N DNA content in S and G2–M phases. These data indicate that the more-rapid proliferation and smaller size of the Fbxo7-knockdown cells are due to a shorter G1 phase. The possibility that cells with less Fbxo7 protein resist cell death during culturing, which would account for the increased cell number, was investigated by assaying live cell exclusion of PI; however, the percentages of viable cells were similar for both cell lines (Fig. 2G). The inactivation of pocket proteins (pRb, p107 and p130) is rate-limiting for transition through G1 and passage through the restriction point. Consistent with the observed shortened G1 phase, immunoblotting the pocket proteins in lysates from cells with reduced Fbxo7 levels also showed their levels decreased compared 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 to Ba/F3 cells, cell size and proliferation rates were tested in other types of cell that have reduced Fbxo7 levels. The human pre-B cell leukemic cell line Nalm-6 was infected with a retrovirus encoding a miR-30-based short hairpin RNA that targets FBXO7 expression. Immunoblotting lysates from FBXO7-knockdown cells showed that levels of Cdk2, Cdk4, Cdk6 and cyclin D3 were unaffected, whereas levels of cyclin A were elevated and those of p27 were reduced when compared with vector control (Fig. 3A). In agreement with the results for the Ba/F3 cells, the reduction of Fbxo7 protein levels also caused a statistically significant increase in the rate of proliferation and a decrease in the volume of these cells (P<0.001) (Fig. 3B,C). Similarly, in the human cervical carcinoma cell line C-33A, reduction of Fbxo7 protein levels by using a different miR- 30 short hairpin RNA construct resulted in cells with a faster proliferation rate than in vector control cells (P value <0.05) (Fig. 3D). Cells with less Fbxo7 were also smaller than vector control cells, although the reduction in cell size was more variable and not statistically significant (P0.052) (Fig. 3E). Immunoblotting showed a reduction in Fbxo7 and a small decrease in p27, but no

Journal of Cell Science reduction in the levels of cyclin E in this cell line (Fig. 3F). Thus, in human and mouse B cell lines, and in an adherent human Fig. 3. Reduced Fbxo7 levels in Nalm-6 and C-33A cells increases their epithelial cell line, reduction of Fbxo7 caused cells to proliferate proliferation rate. Immunoblot of various cell cycle proteins in lysates from faster and become smaller in size, indicating that the proliferation Nalm-6 cells (A) or C-33A cells (F) infected with retroviruses expressing of several cell types is regulated by Fbxo7. different miR-30-based hairpin RNAs that target FBXO7 expression. Time course of the proliferation rate of an empty vector control cell line and either Nalm-6 (B) or C-33A (D) cell lines with reduced Fbxo7 protein expression. Changes to Cdk6 expression did not alter the size or Cells were seeded at equal densities and counted on the indicated day. Cell proliferation of Ba/F3 cells size measurements of Nalm-6 (C) or C-33A (E) cells seeded at equal densities. We next wanted to investigate the role of Cdk6 in the cell cycle of *P<0.05, ***P<0.001. Ba/F3 cells. We first tested whether overexpression of Cdk6 affects proliferation rates and cell size. A polyclonal population of cells that overexpress Cdk6 was generated by retroviral infection of independent clonal cell lines were assayed for Cdk6 protein viruses that encode CDK6-IRES-GFP, and by using FACS sorting expression and, in both cases, its levels were substantially reduced for GFP+ cells. Immunoblotting of lysates from these cells showed while levels of Fbxo7 and p27 were unchanged (Fig. 4E). In that levels of Cdk6 were approximately fivefold increased, whereas addition, in vivo co-immunoprecipitation assays showed the lack the levels of the cell cycle regulators Fbxo7, p27 and cyclin E were of Cdk6 associated with cyclin D2 (Fig. 4F). However, when the the same as for the vector control cells (Fig. 4A). In addition, we rate of proliferation and the cell volume were measured, these used in vivo co-immunoprecipitation assays to test the amount of characteristics were similar between control cells and those with Cdk6 associated with cyclin D2. Overexpression of Cdk6 alone reduced Cdk6 (Fig. 4G,H). Taken together, these data indicate that did not change its association with cyclin D2, arguing that Cdk6 Cdk6 levels do not influence cell growth or proliferation or impact levels are in excess and not limiting its association with cyclin D2 on the amount of Fbxo7 or p27. Thus Cdk6 is neither rate-limiting (Fig. 4B). We found that proliferation rate and cell size for cells nor essential for the proliferation of Ba/F3 cells. that overexpress Cdk6 are virtually identical to those of vector control (Fig. 4C,D). Reduced Fbxo7 levels cannot bypass cell cycle inhibition Cdk6 requirement for cell proliferation was tested by using caused by increased levels of p27 in Ba/F3 cells retroviruses that encode short-hairpin RNAs targeting Cdk6 or the Fbxo7 interacted directly with the N-terminus of p27 and stabilized empty vector to infect Ba/F3 cells to create stable cell lines. Two its levels in U2OS cells (Laman et al., 2005) and also in the cell Regulatory role of Fbxo7 in precursor cells 2179

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 by infecting Ba/F3 cells with retroviruses expressing human CDK6–IRES–GFP and sorted for GFP expression. (B)Immunoblot of endogenous Cdk6 associated with cyclin 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 levels on 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 cycle regulatory proteins in lysates from independent clones of Ba/F3 cells infected with retroviruses expressing a pRETROSuper short hairpin RNA that targets Cdk6 expression or with the empty vector. Actin was used as a loading control. (F)Immunoblot of endogenous Cdk6 associated with cyclin D2 in immunoprecipitations from cell lysates of Ba/F3 cells with reduced Cdk6.

types tested here (Fig. 2A, Fig. 3A,F). To test directly whether decreased percentage of cells in G1 phase and a commensurate Fbxo7 is required to stabilize levels of p27, we investigated whether increased percentage of cells in S phase are shown in Fig. 5D.

Journal of Cell Science Fbxo7-knockdown cells become resistant to the cell cycle inhibitory Furthermore, when the expression of other cell cycle regulators effects of exogenous p27 expression. For this, a plasmid containing was assayed, no changes were seen in the expression levels of the coding region of p27 fused to dsRED was transfected into cyclin D2, Cdk4, Cdk6, Fbxo7 or cyclin E (Fig. 5A). These data Ba/F3 cells with reduced levels of Fbxo7. The dsRED-positive demonstrate that, in Ba/F3 cells, reduction of p27 protein alone cells were collected by FACS sorting, and DNA content and cell affects the distribution of cell cycle phases, proliferation rate and volume were measured. Exogenous expression of p27 cell size, but not the total levels of cell cycle regulators. Thus p27 approximately doubled the percentage of cells containing 2N DNA also has an anti-proliferative role in Ba/F3 cells. (G1 phase) (38% compared with 19.5% in untransfected cells), cells expressing p27-dsRED also showed a 13% increase in cell Ba/F3 cells with reduced levels of Fbxo7 showed size (data not shown). These data argue against the idea that Fbxo7 increased levels of CD43 is required for p27 to function as a cell cycle inhibitor, because It has been reported that lengthening the G1 phase can increase reduction in Fbxo7 levels did not overcome a p27-induced block the probability of differentiation (Johnson et al., 1993; Carroll et to G1–S phase progression. al., 1995), whereas shortening the G1 phase decreases the probability of differentiation and favours proliferation (Kato and Decreased p27 levels in Ba/F3 cells increased their Sherr, 1993). Reducing the cellular levels of Fbxo7 shortened the proliferation rate and reduced cell size duration of G1 phase, so we wanted to investigate any effects Because a reduction of Fbxo7 levels led to a decrease in p27 in Fbxo7 might have on differentiation. Stages of B cell Ba/F3 cells (Fig. 2A), we next investigated the phenotypic effects differentiation can be distinguished by the expression of cell of decreasing p27 levels specifically in Ba/F3 cells. Cell lines were surface antigens, so a subset of these common surface markers created that stably express a miR-30-based short-hairpin RNA was assayed, including B220, B7.1, B7.2, CD43, CD25, MHC targeting p27 expression. Two independent clonal cell lines with II(Ia) (Zola et al., 1991). Ba/F3 empty vector control cells and reduced p27 protein levels were created (Fig. 5A). When rate of cells with reduced levels of Fbxo7 were stained with fluorescent proliferation and cell size were assayed, cells with less p27 protein antibody conjugates and analysed by FACS. Expression of most showed statistically significant increased rates of proliferation of the above markers was not changed; however, staining for (P<0.001 from day 4 onwards) (Fig. 5B) and a smaller size CD43 showed differences between cells with endogenous or (P0.005) (Fig. 5C). The DNA content was also measured by reduced levels of Fbxo7 (Fig. 6A). In separate experiments, Ba/F3 FACS analysis (n3); representative plots demonstrating the cells with stable Fbxo7 knockdown expressed high levels of CD43 2180 Journal of Cell Science 124 (13)

Fig. 5. Reduced levels of p27 in Ba/F3 cells increases the rate of proliferation and decreases cell size. (A)Immunoblots for cell cycle regulatory proteins of cell lysates from independent clonal lines stably expressing a miR- 30-based hairpin RNA that targets p27 expression or the empty vector control. (B)Proliferation rates of cell lines with endogenous or reduced levels of p27. (C)Cell sizes of cell

Journal of Cell Science lines shown in B. (D)Representative FACS plots (from n3) of an asynchronous cell population of cells, fixed and their DNA content quantified by PI staining to determine the percentages 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 increased tested the effect of Fbxo7 overexpression on cell surface marker compared with the vector control (Fig. 6A). CD43 is a expression. To achieve this, an expression construct encoding sialoglycoprotein with a role in cell-cell adhesion and proliferation. FBXO7–IRES–GFP was transiently transfected into Ba/F3 cells During B cell differentiation it is expressed by early precursor that were then stained for CD43 and CD25; the percentages of cells (pre-pro-B and pro-B cells), and then rapidly downregulated CD43hi and CD25hi GFP+ cells were measured by FACS. Consistent upon progression to pre-B and B cell stages with VDJ with a direct effect of Fbxo7 on CD43 expression, its Fbxo7 rearrangement (Hardy et al., 1991). CD25 encodes the IL-2 overexpression caused a 65.9% decrease in CD43hi expressing receptor -chain, which steadily increases during these stages of cells (n3). CD25 expression, however, was also reduced 39.2% B cell differentiation (Rolink et al., 1994). Thus pro-B cells have when Fbxo7 was overexpressed, suggesting that Fbxo7 expression CD43hi and low CD25 (CD25lo) expression, whereas small pre-B had an indirect effect on its levels (Fig. 6C). These data suggest cells have CD43lo CD25hi expression (Rolink et al., 1994; Zola et that Fbxo7 directly modulates CD43 expression; however, because al., 1991). The expression of CD25 was also different in these Fbxo7 overexpression does not increase the proliferation rate, the cells. The number of CD25hi cells decreased by 61.7% in Fbxo7- effect of Fbxo7 on CD43 appears to be independent of its effects knockdown cells compared with vector control cells (Fig. 6B). on the duration of the cell cycle. These data demonstrate that the reduction of Fbxo7, which increased the proliferation rate, also altered the expression of cell Reduced levels of Cdk6 increased those of CD43 surface markers. Furthermore, the change in their expression levels To further investigate the link between cell cycle delay and the suggest that cells had reverted to a less-mature state, implying that expression of these markers of differentiation, the effect of Fbxo7 normally regulates differentiation at the pro-B cell stage. lengthening the cell cycle was tested by transiently transfecting As the reduction of Fbxo7 lead to a cell surface marker p27-IRES-GFP into Ba/F3 cells and assessing the effect on CD43 phenotype that suggested a reversal of differentiation, we also and CD25 surface expression. Although a longer G1 phase might Regulatory role of Fbxo7 in precursor cells 2181

Fig. 6. Changes to cell surface marker expression in cells with reduced or overexpressed levels of Fbxo7. Representative FACS plots of cells with stably reduced Fbxo7 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 transiently transfected with empty vector, Fbxo7–IRES–GFP or p27– IRES–GFP, and stained for CD43-PE or CD25-PE-Cy7 as indicated. Analysis was first gated on GFP-positive cells (representative plot on left). Then, this gated population was analysed for FSC on the x axis and CD43-PE or CD25-PE- Journal of Cell Science Cy7 on the y axis. Representative FACS plots for cells with stable 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 with stable reduction of Cdk6 expression (E), where FSC is on the x axis and CD43-PE is on the y axis.

be expected to alter these markers in order to reflect increased knockdown cells affecting the rate of proliferation. In addition, differentiation (i.e. decrease of CD43 and increase of CD25 cells with alterations to p27 levels had unchanged CD43 expression. expression), no changes to the expression of either marker were In sum, these data suggest that Fbxo7 acts as a positive regulator observed in cells transfected with the construct encoding p27 (Fig. of pro-B cell differentiation because its levels are inversely 6C). Conversely, the expression of CD43 was assayed in the cell correlated with CD43 expression, and that the effect of Fbxo7 line with reduced p27 levels – which had a shorter G1 phase – also expression on CD43 is not due to changes in the cell cycle. had no significant increase in CD43 staining (Fig. 6D). In addition to testing the effect of altering the levels of p27, the Reduction of Fbxo7 levels enhanced erythropoietin- expression levels of CD43 were measured in cells with reduced induced differentiation of Ba/F3 cells along the erythroid levels of Cdk6, because this kinase has a specific role in lineage differentiation (Grossel and Hinds, 2006). Cells with reduced Because Fbxo7 appeared to positively regulate pro-B cell amounts of Cdk6 were also immunostained for CD43 and were differentiation, we next investigated whether differentiation of found to have on average a 2.3-fold increase in CD43hi cells over another haematopoietic cell lineage is also affected by Fbxo7 three independent experiments compared with vector control (Fig. levels. We exploited the potential of Ba/F3 cells to be differentiated 6E). This was surprising because reducing Cdk6 levels in Ba/F3 along the erythroid lineage because of expression of erythroid- cells did not alter the proliferation rate of cells (Fig. 4G), supporting specific transcriptional factors, including GATA-1, NF-E2 and the idea that increased CD43hi expression did not require a more- EKLF (Krosl et al., 1995). Cells will partially differentiate when rapid cell cycle or a shortened G1 phase. Together, these data engineered to express the erythropoietin receptor (Epo-R) and demonstrate that reducing the expression of Fbxo7 or Cdk6 had cultured in the presence of erythropoietin (Epo). They cannot, the same effect of increasing CD43 expression, despite only Fbxo7- however, terminally differentiate and do not synthesize 2182 Journal of Cell Science 124 (13)

performed to assess -globin mRNA levels. Samples from cells with reduced Fbxo7 showed a 10-fold increase in the amount of - globin message as compared with vector (Fig. 7B), suggesting a negative regulatory role for Fbxo7 in this engineered model of erythroid differentiation. The rate of proliferation of Epo-R–IRES–GFP control cells and cells with reduced Fbxo7 was also determined by a growth curve performed on cells induced to differentiate by growth in Epo. As has been previously reported for Ba/F3–Epo–R cells, a transient slowing of growth was observed when cells were withdrawn from IL-3 and induced to differentiate by the addition of Epo. However, cells with reduced Fbxo7 levels were less delayed, recovered more rapidly and proliferated faster than vector control cells (Fig. 7C,D). This was especially apparent when growth rates of the cell lines in the first 24 hours were compared. Fbxo7-knockdown cells seeded into Epo showed little delay, proliferating almost as quickly as cells seeded back into IL-3 after starvation (Fig. 7D). By 42 and 48 hours after cytokine withdrawal, the differences in cell number were statistically significant (P0.0131 and P0.0158) (Fig. 7C). These data demonstrate that reduction in Fbxo7 levels results in more-robust differentiation in response to Epo along an erythroid lineage, despite cells proliferating faster than vector control cells. Therefore, these data argue against the requirement of a slower cell cycle to allow erythroid differentiation in Ba/F3-Epo-R cells.

Mice with reduced levels of Fbxo7 show increased numbers of pro-B cells and pro-erythroblasts Our data indicate that Fbxo7 has a cell-type specific anti- proliferative role (Ba/F3, Nalm-6 and C-33A cells) and also positive and negative regulatory roles in the differentiation of pro-B and erythroid cell lineages, respectively. As these in vitro experiments were conducted mainly by using a Ba/F3 cell line model, we also wanted to assess the effect of Fbxo7 on differentiation in a more physiological setting. To achieve this, a transgenic mouse was

Journal of Cell Science engineered using targeted ES cells from the International Knockout Mouse Consortium (EUCOMM ID: 23037). These ES cells have an insertion of the LacZ gene with a splice acceptor site between exon 3 and exon 4 of isoform 1 of Fbxo7, and this Fbxo7LacZ allele, Fig. 7. Fbxo7 regulates differentiation. (A)Immunoblots for Fbxo7 which disrupts expression of all Fbxo7 isoforms by splicing in the expression in cell lysates from independent clonal cell lines of Ba/F3 cells LacZ-coding sequences. Four chimeric mice were generated by expressing the Epo-R and infected with retroviruses expressing a miR-30- injection of ES cells into blastocysts, two of which showed germline based hairpin RNA which targets Fbxo7 expression. A non-specific band was transmission of the Fbxo7LacZ transgene. Heterozygous animals used as an internal loading control. (B)Semi-quantitative RT-PCR for -globin and cyclophilin from cells which have been induced to differentiate by the were bred to homozygosity, and lysates from various tissues were addition of Epo for 3 days. (C)Increase in cell numbers over 3 days during immunoblotted for the expression of Fbxo7, showing a gene– which time cells were starved of all cytokines and then induced to differentiate dosage-dependent reduction in Fbxo7 expression in the in Epo or to proliferate in IL-3. (D)Data from the first 24 hours of the graph heterozygous and homozygous animals (Fig. 8A). Fbxo7 protein shown in C. *P<0.05. expression was, however, detectable upon very long exposure of immunoblots in the cerebellum demonstrating the production of some wild-type Fbxo7 mRNA (data not shown). Our full phenotype haemoglobin. Instead, expression of -globin mRNA is used to characterisation of these mice will be reported elsewhere measure their state of differentiation (Krosl et al., 1995; Carroll et (manuscript in preparation). al., 1995; Krosl et al., 1996). To test directly the effect of Fbxo7 To address the effect of reduced Fbxo7 expression on B cell on cytokine-induced differentiation, we engineered expression of development in vivo, splenocytes were harvested from age-matched Epo-R–IRES–GFP in these cells (see Materials and Methods), and wild-type (n2) and homozygous (n2) littermates, and stained for created cell lines with reduced Fbxo7 by subsequently infecting B220, CD43 and CD25 to measure the pro-B and pre-B cell Ba/F3 Epo-R-expressing (Ba/F3-Epo-R) cells with retroviruses populations. Animals homozygous for Fbxo7LacZ showed a encoding the short-hairpin that targets Fbxo7 or with a non-silencing statistically significant increase in the percentage of CD43hiCD25lo control. Two independent clonal lines were created, and the pro-B cells as compared with wild-type controls; however, the reduction in Fbxo7 protein expression was verified by later pre-B cell stages showed no significant differences (Fig. immunoblotting (Fig. 7A). Cells were cultured in the presence of 8B,C). The increase in the percentage of pro-B cells seen in vivo Epo, total RNA was isolated and semi-quantitative RT-PCR could be due to increased proliferation of pro-B cells or a block in Regulatory role of Fbxo7 in precursor cells 2183

Fig. 8. Mice with LacZ insertion into the Fbxo7 locus show increased numbers of pro-B cells and pro-erythroblasts. (A)Immunoblots of lysates prepared from the organs of mice inheriting one or two Fbxo7LacZ alleles. (B)FACS analysis of B220+, CD43, CD25 populations in splenocytes of WT and homozygous Fbxo7LacZ 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 cell populations (pro-B: B220+, CD43hi, CD25lo; small pre-B: B220+, CD43hi, CD25hi; large pre-B: B220+, CD43lo, CD25hi) (n2). (D)Number of CFU-E colonies present in bone marrow of WT and homozygous Fbxo7LacZ animals upon seeding into colony formation assays containing Epo (n 2 mice per genotype, each Journal of Cell Science  seeded in triplicate). (E)FACS analysis of erythroid populations in bone marow of WT and homozygous Fbxo7LacZ animals by Ter119 staining on the x-axis and CD71 on the y-axis (n2). (F)Percentages of erythroblast populations as indicated. *P<0.05, **P<0.01.

maturation from the pro-B to pre-B cell stages. These data support differentiation after which cells exit the bone marrow and the results described for Ba/F3 cells and suggest that Fbxo7 usually differentiate to reticulocytes and erythrocytes (Fig. 8E,F). These has an anti-proliferation or a positive role in promoting the data indicate that pro-erythroblasts cells are blocked in their differentiation of pro-B cells. differentiation, eventually leading to a decrease in the numbers of To address the effect of Fbxo7 on erythroid differentiation, the later stage cells or, alternatively, that Fbxo7 acts in a different number of erythroid colony-forming units (CFU-Es) was assessed capacity at later stages of erythropoiesis, potentially required to by seeding bone marrow cells into methylcellulose medium promote proliferation or survival at a later stage in maturation. containing Epo. Cells from mice homozygous for Fbxo7LacZ formed almost twice as many colonies, indicating a significant increase in Discussion the number of CFU-Es (P0.0033) in these mice as compared with In a previous study using immortalised fibroblasts, overexpression wild-type animals (Fig. 8D). These data suggest that Fbxo7 of Fbxo7 protein increased cyclin D–Cdk6 complexes and caused normally has an anti-proliferative role in pro-erythroblasts or a role Cdk6-dependent transformation without affecting the cell cycle or in promoting pro-erythroblast differentiation. Bone marrow from proliferation (Laman et al., 2005). In addition, in osteosarcoma wild-type and homozygous animals was also stained for CD71 and cells, a reduction of Fbxo7 expression led to decreased amounts of Ter119 protein expression, because together they demarcate the cyclin D–Cdk6 complexes, also without altering the cell cycle later stages of erythroblast differentiation (Fig. 8E,F). Consistent (Laman et al., 2005), which argues for a direct role of Fbxo7 in with data showing an increase in CFU-Es in homozygous Fbxo7LacZ promoting the assembly of cyclin D–Cdk6 complexes. We report animals, we also detected a statistically significant increase in the here that, in an immortalised pro-B cell line, overexpression of number of earlier stage pro-erythroblasts (CD71hiTer119lo) Fbxo7 does not transform cells, enhance cell proliferation or (P0.017) as compared with wild-type littermates. Surprisingly, viability or affect the levels of cyclin D–Cdk6 complexes. Instead, they also had fewer late normoblasts (orthochromatophilic the reduction of its levels accelerated the rate of proliferation, erythroblasts) (CD71–Ter119hi) (P0.025), the stage of decreased cell size, and shortened G1 phase, all indicative of an 2184 Journal of Cell Science 124 (13)

anti-proliferative function for Fbxo7 in pro-B cells. This effect was pro-B to pre-B cell stage, due to the inability to downregulate not specific to Ba/F3 cells, because reduction of Fbxo7 also CD43 in the absence of Fbxo7. In the erythroid lineages, a increased the proliferation rate of Nalm-6 and C-33A cells. Cell significant increase in the number of CFU-Es and pro-erythroblasts cycle analysis showed that a decrease in Fbxo7 caused a rapid was observed in the bone marrow of homozygous Fbxo7LacZ mice. transit through G1 phase. Cells also had higher Cdk2 activity, This, again, could reflect either increased proliferation of these stemming from increased cyclin E and cyclin A, reduced p27 and lineage-committed precursor cells or a block in the differentiation an overall reduction in the levels of pocket proteins. One at this stage in erythropoiesis. In considering these two models we interpretation of these findings is that, in Ba/F3 cells, p27 has a notice that, in the Ba/F3 Epo-R erythroid differentiation model, the main role in restraining S phase entry, which is supported by the reduction in Fbxo7 protein expression enhanced both Epo-induced finding that independently decreasing its levels results in smaller differentiation and proliferation. This lends support to the model in cells and a faster rate of proliferation – even without increased which increased proliferation, rather than a block in differentiation, cyclin E levels. In addition, changes to Cdk6 levels had no explains the in vivo findings. However, a reduction in cell number detectable effect on cell cycle parameters, suggesting that, although at the late normoblast stage was also observed and might mean Cdk6 is abundant in pro-B cells, it is not rate-limiting for cell cycle that, when Fbxo7 levels are reduced, erythroid differentiation is progression under the experimental conditions used here. Together, inhibited. Alternatively, Fbxo7 might have different pro- these results support the idea that Fbxo7 has different cell cycle proliferation or pro-survival functions later on in erythropoiesis. activities, and transforming or anti-proliferative properties in a How cell cycle regulatory proteins like Fbxo7 and Cdk6 variety of cell types, and we speculate that the ultimate effect of influence differentiation and cell cycle, and at which maturation changes to its expression levels depends on the G1 phase circuitry stage this occurs is an interesting question for future study. There of that cell type or, possibly, to the stage of differentiation of the are experimental data that indicate direct binding of Cdk6 to cell (stem or progenitor cell versus mature cell). We notice, for transcription factors that regulate differentiation, suggesting a cell- example, that in differentiating embryonal carcinoma P19 cells, cycle-independent function (Fujimoto et al., 2007), whereas other proliferation correlated with the activity of the cyclin-E–cyclin-A– studies show that downregulation of Cdk6 protein is crucial for Cdk2 but not cyclin-D–Cdk4–Cdk6–p27 complexes (Bryja et al., terminal differentiation, presumably by allowing a permanent 2008). withdrawal from the cell cycle (Matushansky et al., 2003). In Because Fbxo7 affects G1 phase and cell proliferation, we addition, it is possible that these proteins have different regulatory reasoned it might also affect cell differentiation – which is roles, cell cycle and/or transcriptional functions at different times temporally linked to G1. A long-standing correlation exists between in cell maturation. In summary, Fbxo7 impacts on both cell cycle a prolonged G1 phase that enhances the differentiation potential and differentiation in a cell-type specific manner, which indicates and a shortened G1 phase that diminishes it (Kato and Sherr, 1993; that Fbxo7 has an important and sensitive role in integrating cell Carroll et al., 1995; Johnson et al., 1993). However, there is signalling and functionality with cell cycle progression during experimental evidence in a variety of cell types, including haematopoiesis. lymphocytes (Rush et al., 2002; Lea et al., 2003), which show that these processes can be separated and need not proceed Materials and Methods

Journal of Cell Science simultaneously (Brown et al., 2003). Experiments here are Cell culture consistent with the idea that Fbxo7 promotes the differentiation of Murine Ba/F3 pro-B cells were maintained in complete RPMI-1640 medium pro-B cells: overexpression of Fbxo7 decreased CD43, which is supplemented with 10% foetal calf serum (FCS; PAA Laboratories), 10% WEHI-3B conditioned medium, penicillin (50 U/ml), streptomycin (50 U/ml) (Gibco BRL), downregulated as B cell mature, and reduction of Fbxo7 levels and 10ng/ml recombinant murine IL-3 (Fitzgerald Laboratories). Human Nalm-6 increased its expression. This inverse correlation between Fbxo7 pre-B acute lymphoblastic leukaemia cells were maintained in RPMI-1640 medium and CD43 expression is suggestive of a direct regulatory supplemented with 10% FCS, penicillin (50 U/ml),and streptomycin (50 U/ml). relationship, but it is unlikely to be due to cell cycle alterations. Human C-33A cervical cancer cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, penicillin (50 U/ml), and Several lines of evidence support this. For example, although streptomycin (50 U/ml). Cells were incubated at 37°C in a humidified 5% CO2 Fbxo7 knockdown altered the cell cycle, the overexpression of atmosphere. Fbxo7 did not alter proliferation but did decrease CD43 expression. 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 that Moreover, in other experiments where cell cycle length was altered delivered a miR-30-based short hairpin RNA sequence targeting the genes of interest. by changes to p27, CD43 expression was not affected. However, Target sequences identified and validated by RNAiCodex (http://cancan.cshl.edu/cgi- Cdk6 knockdown, which had no effect on the cell cycle, increased bin/Codex/Codex.cgi) were purchased and hairpin-containing oligonucleotids were the frequency of CD43hi-expressing cells. Taken together these PCR amplified and subcloned into retroviral vectors. The sequences used were, for murine Fbxo7sh1: 5Ј-CGGAGATTGTGGTATTGATAAT-3Ј (HP_240439), human data argue that CD43 expression is regulated independently from Fbxo7sh1: 5Ј-CGCCCAGTCTGGTGTTTGGAAT (HP_3087), human Fbxo7sh2: cell cycle progression. A further implication of this finding is that 5Ј-CGCTGAGTCAATTCAAGATAAT-3Ј (HP_434828), murine p27: 5Ј- Fbxo7 also regulates CD43 signalling in other cell types that AGCCATTAGAGTCACTTTCCAT-3Ј (HP_288922). Either empty LMP or non- silencing vector PSMP (Open Biosystems) were used as negative controls. To express this marker, such as activated B cells, macrophages and T decrease Cdk6 protein levels, retroviruses containing pRETROSUPER-based short cells, a hypothesis that is currently under investigation. hairpin constructs were used as previously described (Laman et al., 2005) to interfere Consistent with the observations in Ba/F3 cells, quantification with Cdk6 transcription. Independent B cell clones were generated using limiting of pro-B cells (B220+, CD43hi, CD25lo) from spleens showed a dilution and grown in the presence of puromycin (2 g/ml); several clones were analysed. A different short hairpin construct was used to reduce FBXO7 gene statistically significant increase in the proportion of pro-B cells in expression in C-33A cells, and polyclonal cell lines were selected in the prescence mice that are deficient for Fbxo7 protein expression compared puromycin (2 g/ml). To overexpress proteins, cells were infected with retroviruses with wild-type mice. This increase was specific to pro-B cells and that encoded MSCV-IRES-GFP and expressed the gene of interest. Polyclonal cell could have resulted either from an increase in the proliferation of lines were detected by FACS for GFP-expressing cells. Ba/F3 cells expressing Epo-R were generated by retroviral infection with MSCV- these precursor cells due to the loss of the anti-proliferation function EpoR-GFP (kindly provided by Tony Green, Department of Hematology, University of Fbxo7 or, alternatively, to a block in the differentiation at the of Cambridge). Retroviruses encoding the Epo-R-IRES-GFP were used to infect Regulatory role of Fbxo7 in precursor cells 2185

Ba/F3 cells, which were sorted for GFP expression. Clones were subsequently measured using a nanodrop spectrophotomer (Nanodrop Technologies). 1 g of total selected for Epo-R expression by growing cells in medium that was supplemented RNA was used for each sample harvested at 3 days post induction of differentiation. with 0.05 U/ml erythropoietin (Epo) (Cell Sciences) but lacked IL-3. Ba/F3-Epo-R 2 l of random hexamers (Promega) were added to each sample, which was incubated cell lines were thereafter maintained in IL-3 during standard sub-culturing. at 65°C for 5 minutes and then kept on ice. 7 l of a master mix [4 l of 5ϫ RT For transient transfection, 5 g of DNA encoding p27or Fbxo7, or empty vector buffer, 0.5 l of RNasin (Promega), 2 l of 10 mM dNTP, 0.5 l of reverse plasmid DNA was nucleofected into 5ϫ106 Ba/F3 cells by using solution V and transcriptase (Roche)] was added to each sample, which was then incubated at 25°C program X-001, as per manufacturer’s instructions (Lonza). After 72 hours, live for 10 minutes, 42°C for 60 minutes and 70°C for 10 minutes. cDNA was used in GFP+ cells were immunostained (see below) and analysed by flow cytometry, using PCR reactions (95°C for 30 seconds, 50°C for 1 minute, 72°C for 1 minute, for PI (2.5 g/ml) as a live–dead discriminator. 25 cycles) using primers to amplify -globin (forward: 5Ј- To measure the rate of proliferation and the cell volume, cells were seeded in GACCCAGCGGTACTTTGATAGC-3Ј and reverse: 5Ј-TGAGGCTGTCCAAG - triplicate at a density of 0.3–0.5ϫ106 cells/ml (for B cells) or 2ϫ105 cells per well TGATTCA-3Ј) and cyclophilin (forward: 5Ј-CCTTGGGCCGCGTCTCCTT-3Ј and (C-33A cells), and cell numbers and volumes were assayed every 2 days using a reverse: 5Ј-CACCCTGGCACATGAATCCTG-3Ј) (Invitrogen). Quantification was Casy cell counter (Scharfe System). Three independent experiments were performed. carried out using a Molecular Imager Gel Documentation System using Quantity To measure cell viability, cells were exposed to 50 g/ml PI (Sigma Aldrich) at room One software (BioRad). temperature for 30 minutes prior to analysis by FACS (FACS Calibur, Becton Dickinson). To quantify DNA content, 1–2ϫ106 cells were washed in PBS and fixed Generation of Fbxo7LacZ mice in ice-cold 70% ethanol at 4°C for 30 minutes. Cells were washed in PBS, incubated Mouse embryonic stem (ES) cells heterozygous for the -galactosidase gene (LacZ) in 50 g/ml RNAse A and 100 g/ml propidium iodide (PI) for 30 minutes, and then targeting the Fbxo7 locus between exons 3 and 4 were generated by conditional analysed by FACS. For statistical analyses, data were analysed using a two-tailed ‘targeted trapping’ technology employed by the European Conditional Mouse Student’s t-test; values of P<0.05 or less were considered significant. Mutagenesis Program (www.eucomm.org). To generate chimeric mice, two independent Fbxo7LacZ/+ ES cell lines (EUCOMM ID: 23037) were obtained, Immunoprecipitations and immunoblotting karyotyped and injected into C57BL6/J blastocysts. One clone successfully produced For immunoprecipitations and kinase assays, cells were lysed in 50 mM HEPES pH four chimeric male mice, as determined by PCR analysis for the mutant transgene 7.4, 150 mM NaCl, 20 mM EDTA pH 8, 1 mM DTT, 10 mM -glycerophosphate, (primers available on request). Two of these males had germline transmission of the 0.5% Triton X-100, 10 mM NaF, 2 mM PMSF and protease inhibitor cocktail, then transgene and their offspring were bred to homozygosity (Fbxo7LacZ/LacZ). Experiments sonicated three times and centrifuged at 16100 g for 10 minutes at 4°C. Lysates were were conducted from heterozygous crosses to enable analysis of homozygous mice pre-cleared by rotation in the prescence of protein G-Sepharose beads at 4°C for 30 with wild-type littermate controls. Spleen and bone marrow were harvested from minutes prior to incubation with primary antibody and addition of protein G- mice aged 7–12 weeks. Sepharose beads for 4 hours at 4°C for immunoprecipitation. Beads were washed three times and co-immunoprecipitating proteins were detected by immunoblotting. We thank C. Bacon, R. Dickins, A. Green, N. Holmes, S. Lowe and For kinase assays, beads were washed in 50 mM HEPES/KOH pH 7.4, 1 mM A. Skoultchi for generously providing reagents, W. Mansfield, W. MnCl2, 10 mM MgCl2, 10 mM -glycerophosphate, 1 mM DTT, 1 mM PMSF prior to the addition of 0.6 g of purified pRb substrate (Abcam, ab56270), 1 Ci of [- Khaled and N. Miller for technical advice and assistance, and D. 33P]ATP and 200 mM ATP and incubating at 30°C for 1 hour. Proteins were resolved Lagos, N. Holmes, A. Philpott and members of the Laman lab for their using SDS-PAGE and gels fixed in 10% acetic acid/methanol, dried and quantified critical reading of this manuscript. Funding was provided by the using a Cyclone Phosphor Imager (PerkinElmer). Association for International Cancer Research, the BBSRC, Campod, Protein samples were prepared by lysing cell pellets in modified RIPA buffer (150 and Cancer Research UK. mM 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. References For immunoblotting, protein concentration was determined using protein assay Ang, X. L. and Wade, H. J. (2005). SCF-mediated protein degradation and cell cycle (Biorad). 50 g of total lysate was resolved by SDS-PAGE, and transferred onto control. Oncogene 24, 2860-2870. PVDF membranes, which were incubated in PBS with 0.05% Tween-20 and 5% Barriere, C., Santamaria, D., Cerqueira, A., Galan, J., Martin, A., Ortega, S., dried skimmed-milk powder. The following rabbit polyclonal primary antibodies Malumbres, M., Dubus, P. and Barbacid, M. (2007). Mice thrive without Cdk4 and were used: Cdk6 (c-12) (sc-177), Cdk4 (c-22) (sc-260), Cdk2 (M2) (sc-163), cyclin Cdk2. Mol. Oncol. 1, 72-83.

Journal of Cell Science A (H-32) (sc-751), cyclin E (M-20) (sc-481), cyclin D2 (c-17) (sc-181), cyclin D3 Berthet, C., Klarmann, K. D., Hilton, M. B., Suh, H. C., Keller, J. R., Kiyokawa, H. (c-16) (sc-182), p27 (c-19) (sc-528) (Santa Cruz Biotechnology Inc.) and actin and Kaldis, P. (2006). Combined loss of Cdk2 and Cdk4 results in embryonic lethality (Sigma Aldrich). The antibody against Fbxo7 has been described previously (Laman and Rb hypophosphorylation. Dev. Cell 10, 563-573. et al., 2005). Donkey anti-rabbit IgG conjugated to horseradish peroxidase was Besson, A., Hwang, H. C., Cicero, S., Donovan, S. L., Gurian-West, M., Johnson, D., obtained from Santa Cruz Biotechnology, Inc. Clurman, B. E., Dyer, M. A. and Roberts, J. M. (2007). Discovery of an oncogenic activity in p27Kip1 that causes stem cell expansion and a multiple tumor phenotype. Differentiation assays Genes Dev. 21, 1731-1746. The following fluorescent and biotin-conjugated antibodies were used to assay cell Borriello, A., Cucciolla, V., Oliva, A., Zappia, V. and Della, R. F. (2007). p27Kip1 surface markers: CD43-PE (clone S7; BD Pharmingen), CD25-PE or PE-Cy7 (both metabolism: a fascinating labyrinth. Cell Cycle 6, 1053-1061. clone PC61.5; eBioscience), B220-biotin (clone RA3-6B2; Pharmingen) or B220- Brown, G., Hughes, P. J. and Michell, R. H. (2003). Cell differentiation and proliferation- FITC (clone RA1-6B2; eBioscience), CD19-PE (clone 1D3; eBioscience), IgM- simultaneous but independent? Exp. Cell Res. 291, 282-288. biotin (clone II/41; eBioscience), CD71-biotin (clone RI7 217.1.4; Caltag), Ter119-PE Bryja, V., Pachernik, J., Vondracek, J., Soucek, K., Cajanek, L., Horvath, V., Holubcova, Z., Dvorak, P. and Hampl, A. (2008). Lineage specific composition of (clone Ter119; Caltag) and Streptavadin-APC (Caltag). All antibodies were used at ϫ cyclin D-CDK4/CDK6-p27 complexes reveals distinct functions of CDK4, CDK6 and 1:100 dilution in 100 l FACS buffer (1 PBS, 0.05% NaN3, 2.5% FBS) – except individual D-type cyclins in differentiating cells of embryonic origin. Cell Prolif. 41, ϫ 5 anti-Ter119, which was used at 1:50. For all staining procedures, 5 10 cells were 875-893. incubated with primary antibodies for 1 hour at 4°C, washed twice in FACS buffer Carroll, M., Zhu, Y. and D’Andrea, A. D. (1995). Erythropoietin-induced cellular and then either analysed immediately or incubated with secondary streptavidin-APC differentiation requires prolongation of the G1 phase of the cell cycle. Proc. Natl. Acad. antibody for 30 minutes at 4°C, followed by two washes. Cells were then collected Sci. USA 92, 2869-2873. and analysed by flow cytometry (Cyan ADP Analyser, DakoCytomation) using Cenciarelli, C., Chiaur, D. S., Guardavaccaro, D., Parks, W., Vidal, M. and Pagano, Summit 4.3 software (Beckman Coulter). To assay B-cell populations, spleen cell M. (1999). Identification of a family of human F-box proteins. Curr. Biol. 9, 1177-1179. suspensions were treated with 200 l NH4Cl red blood cell lysis solution (150 mM Chu, I. M., Hengst, L. and Slingerland, J. M. (2008). The Cdk inhibitor p27 in human NH4Cl, 10 mM KHCO3, 1 mM EDTA pH 7.3) for 1 minute, before being resuspended cancer: prognostic potential and relevance to anticancer therapy. Nat. Rev. Cancer 8, in 15 ml of complete medium. Cells were then Fc-blocked with anti-CD16/CD32 253-267. antibody (clone 93; eBioscience) for 20 minutes at 4°C prior to incubation with Deshaies, R. J. (1999). SCF and Cullin/Ring H2-based ubiquitin ligases. Annu. Rev. Cell primary antibody. For analysis of erythroid progenitors and for bone marrow staining, Dev. Biol. 15, 435-467. cells were not pre-treated with red blood lysis solution before Fc blocking and cell Ely, S., Di Liberto, M., Niesvizky, R., Baughn, L. B., Cho, H. J., Hatada, E. N., staining. For colony formation assays, 2ϫ105 bone marrow cells were plated with Knowles, D. M., Lane, J. and Chen-Kiang, S. (2005). Mutually exclusive cyclin- dependent kinase 4/cyclin D1 and cyclin-dependent kinase 6/cyclin D2 pairing inactivates 3U/ml Epo as per manufacturer’s instructions (M3234) (StemCell Technologies); retinoblastoma protein and promotes cell cycle dysregulation in multiple myeloma. CFU-E colonies were counted after 3 days. Cancer Res. 65, 11345-11353. EpoR-expressing Ba/F3 cells, with or without the short-hairpin RNA to target Ericson, K. K., Krull, D., Slomiany, P. and Grossel, M. J. (2003). Expression of cyclin- Fbxo7, were cultured in the presence of IL-3, Epo (0.05 U/ml or 0.5 U/ml), or IL-3 dependent kinase 6, but not cyclin-dependent kinase 4, alters morphology of cultured and Epo (0.5 U/ml) together. Cells were harvested at various time points after the mouse astrocytes. Mol. Cancer Res. 1, 654-664. addition of Epo, and RNA was extracted by lysing cells in Trizol and centrifugation Fujimoto, T., Anderson, K., Jacobsen, S. E., Nishikawa, S. I. and Nerlov, C. (2007). at 11,200 g for 10 minutes at 4°C. Chloroform extraction was carried out before Cdk6 blocks myeloid differentiation by interfering with Runx1 DNA binding and RNA isolation using the RNeasy mini kit (Qiagen) and RNA concentration was Runx1-C/EBPalpha interaction. EMBO J. 26, 2361-2370. 2186 Journal of Cell Science 124 (13)

Grossel, M. J. and Hinds, P. W. (2006). Beyond the cell cycle: a new role for Cdk6 in Matushansky, I., Radparvar, F. and Skoultchi, A. I. (2003). CDK6 blocks differentiation: differentiation. J. Cell. Biochem. 97, 485-493. coupling cell proliferation to the block to differentiation in leukemic cells. Oncogene Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J. D. and Hayakawa, K. (1991). 22, 4143-4149. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse Nakayama, K. I. and Nakayama, K. (2005). Regulation of the cell cycle by SCF-type bone marrow. J. Exp. Med. 173, 1213-1225. ubiquitin ligases. Semin. Cell Dev. Biol. 16, 323-333. Hermand, D. (2006). F-box proteins: more than baits for the SCF? Cell Div. 1, 30. Nakayama, K. I. and Nakayama, K. (2006). Ubiquitin ligases: cell-cycle control and Hershko, A. (1997). Roles of ubiquitin-mediated proteolysis in cell cycle control. Curr. cancer. Nat. Rev. Cancer 6, 369-381. Opin. Cell Biol. 9, 788-799. Okabe, H., Lee, S. H., Phuchareon, J., Albertson, D. G., McCormick, F. and Tetsu, Hershko, A. and Ciechanover, A. (1998). The ubiquitin system. Annu. Rev. Biochem. 67, O. (2006). A critical role for FBXW8 and MAPK in cyclin D1 degradation and cancer 425-479. cell proliferation. PLoS ONE 1, e128. Ho, M. S., Ou, C., Chan, Y. R., Chien, C. T. and Pi, H. (2008). The utility F-box for Rane, S. G., Dubus, P., Mettus, R. V., Galbreath, E. J., Boden, G., Reddy, E. P. and protein destruction. Cell. Mol. Life Sci. 65, 1977-2000. Barbacid, M. (1999). Loss of Cdk4 expression causes insulin-deficient diabetes and Hu, M. G., Deshpande, A., Enos, M., Mao, D., Hinds, E. A., Hu, G. F., Chang, R., Cdk4 activation results in beta-islet cell hyperplasia. Nat. Genet. 22, 44-52. Guo, Z., Dose, M., Mao, C. et al. (2009). A requirement for cyclin-dependent kinase Reed, S. I. (2003). Ratchets and clocks: the cell cycle, ubiquitylation and protein turnover. 6 in thymocyte development and tumorigenesis. Cancer Res. 69, 810-818. Nat. Rev. Mol. Cell Biol. 4, 855-864. Jackson, P. K. and Eldridge, A. G. (2002). The SCF ubiquitin ligase: an extended look. Reed, S. I. (2006). The ubiquitin-proteasome pathway in cell cycle control. Results Probl. Mol. Cell 9, 923-925. Cell Differ. 42, 147-181. Jin, J., Cardozo, T., Lovering, R. C., Elledge, S. J., Pagano, M. and Harper, J. W. Rolink, A., Grawunder, U., Winkler, T. H., Karasuyama, H. and Melchers, F. (1994). (2004). Systematic analysis and nomenclature of mammalian F-box proteins. Genes IL-2 receptor alpha chain (CD25, TAC) expression defines a crucial stage in pre-B cell Dev. 18, 2573-2580. development. Int. Immunol. 6, 1257-1264. Johnson, P., Chung, S. and Benchimol, S. (1993). Growth suppression of Friend virus- Rush, J. S., Hasbold, J. and Hodgkin, P. D. (2002). Cross-linking surface Ig delays transformed erythroleukemia cells by p53 protein is accompanied by hemoglobin CD40 ligand- and IL-4-induced B cell Ig class switching and reveals evidence for production and is sensitive to erythropoietin. Mol. Cell. Biol. 13, 1456-1463. independent regulation of B cell proliferation and differentiation. J. Immunol. 168, Jones, R., Ruas, M., Gregory, F., Moulin, S., Delia, D., Manoukian, S., Rowe, J., 2676-2682. Brookes, S. and Peters, G. (2007). A CDKN2A mutation in familial melanoma that Santra, M. K., Wajapeyee, N. and Green, M. R. (2009). F-box protein FBXO31 abrogates binding of p16INK4a to CDK4 but not CDK6. Cancer Res. 67, 9134-9141. mediates cyclin D1 degradation to induce G1 arrest after DNA damage. Nature 459, Kato, J. Y. and Sherr, C. J. (1993). Inhibition of granulocyte differentiation by G1 cyclins 722-725. D2 and D3 but not D1. Proc. Natl. Acad. Sci. USA 90, 11513-11517. Slomiany, P., Baker, T., Elliott, E. R. and Grossel, M. J. (2006). Changes in motility, Krosl, J., Damen, J. E., Krystal, G. and Humphries, R. K. (1995). Erythropoietin and gene expression and actin dynamics: Cdk6-induced cytoskeletal changes associated interleukin-3 induce distinct events in erythropoietin receptor-expressing BA/F3 cells. with differentiation in mouse astrocytes. J. Cell. Biochem. 99, 635-646. Blood 85, 50-56. Sugimoto, M., Nakamura, T., Ohtani, N., Hampson, L., Hampson, I. N., Shimamoto, Krosl, J., Damen, J. E., Krystal G. and Humphries, R. K. (1996). Interleukin-3 (IL-3) A., Furuichi, Y., Okumura, K., Niwa, S., Taya, Y. et al. (1999). Regulation of CDK4 inhibits erythropoietin-induced differentiation in Ba/F3 cells via the IL-3 receptor alpha activity by a novel CDK4-binding protein, p34(SEI-1). Genes Dev. 13, 3027-3033. subunit. J. Biol. Chem. 271, 27432-27437. Takaki, T., Fukasawa, K., Suzuki-Takahashi, I., Semba, K., Kitagawa, M., Taya, Y. Laman, H. (2006). Fbxo7 gets proactive with cyclin D/cdk6. Cell Cycle 5, 279-282. and Hirai, H. (2005). Preferences for phosphorylation sites in the retinoblastoma Laman, H., Funes, J. M., Ye, H., Henderson, S., Galinanes-Garcia, L., Hara, E., protein of D-type cyclin-dependent kinases, Cdk4 and Cdk6, in vitro. J. Biochem. 137, Knowles, P., McDonald, N. and Boshoff, C. (2005). Transforming activity of Fbxo7 381-386. is mediated specifically through regulation of cyclin D/cdk6. EMBO J. 24, 3104-3116. Tourigny, M. R., Ursini-Siegel, J., Lee, H., Toellner, K. M., Cunningham, A. F., Lea, N. C., Orr, S. J., Stoeber, K., Williams, G. H., Lam, E. W., Ibrahim, M. A., Mufti, Franklin, D. S., Ely, S., Chen, M., Qin, X. F., Xiong, Y. et al. (2002). CDK inhibitor G. J. and Thomas, N. S. (2003). Commitment point during G0->G1 that controls entry p18(INK4c) is required for the generation of functional plasma cells. Immunity 17, 179- into the cell cycle. Mol. Cell. Biol. 23, 2351-2361. 189. Lin, D. I., Barbash, O., Kumar, K. G., Weber, J. D., Harper, J. W., Klein-Szanto, A. Vodermaier, H. C. (2004). APC/C and SCF: controlling each other and the cell cycle. J., Rustgi, A., Fuchs, S. Y. and Diehl, J. A. (2006). Phosphorylation-dependent Curr. Biol. 14, R787-R796. ubiquitination of cyclin D1 by the SCF(FBX4-alphaB crystallin) complex. Mol. Cell Winston, J.T., Koepp, D. M., Zhu, C., Elledge, S. J. and Harper, J. W. (1999). A family 24, 355-366. of mammalian F-box proteins. Curr. Biol. 9, 1180-1182. Malumbres, M., Sotillo, R., Santamaria, D., Galan, J., Cerezo, A., Ortega, S., Dubus, Zola, H., Macardle, P. J., Flego, L. and Webster, J. (1991). The expression of sub- P. and Barbacid, M. (2004). Mammalian cells cycle without the D-type cyclin- population markers on B cells: a re-evaluation using high-sensitivity fluorescence flow

Journal of Cell Science dependent kinases Cdk4 and Cdk6. Cell 118, 493-504. cytometry. Dis. Markers 9, 103-118.