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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Immunity. Author manuscript; available in PMC Jul 23, 2011.
Published in final edited form as:
PMCID: PMC2911227

MicroRNA-34a perturbs B lymphocyte development by repressing the Forkhead Box Transcription Factor Foxp1


MicroRNAs (miRNAs) can influence lineage choice or affect critical developmental checkpoints during hematopoiesis. To search for a role of the recently described p53-induced microRNA, miR-34a, in hematopoiesis, we performed gain-of-function analysis in murine bone marrow. Constitutive expression of miR-34a led to a block in B cell development at the pro-B cell to pre-B cell transition, leading to a reduction in mature B cells. This block appeared to be mediated primarily by inhibited expression of the Forkhead transcription factor, Foxp1. We demonstrated that Foxp1 was a direct target of miR-34a in a 3′-untranslated region (UTR)-dependent fashion. Knockdown of Foxp1 by siRNA recapitulated the B cell developmental phenotype induced by miR-34a, whereas co-transduction of Foxp1 lacking its 3′UTR with miR-34a rescued B cell maturation. Lastly, knockdown of miR-34a resulted in increased amounts of Foxp1 and mature B cells. These findings identify a role for miR-34a in connecting the p53 network with suppression of Foxp1, a known B cell oncogene.


Antigen-independent B cell development from hematopoietic stem and progenitor cells is a complex process, closely coordinated with the generation of functional antigen receptors (Hardy and Hayakawa, 2001). B cells are the end product of an ordered series of developmental steps punctuated by checkpoints following the rearrangement of the immunoglobulin heavy (IgH) and light chain (IgL) loci. Defined stages of committed B cell precursors include pro-B cells, during which B cells rearrange IgH, pre-B cells, during which IgL is rearranged, and finally immature and mature B cells expressing variable amounts of surface immunoglobulin (IgM). A complex sequence of molecular events orchestrates successful V(D)J rearrangement in B cells. It includes the activation of several transcription factors, notably PU.1, E2a, Ebf, and Pax5 (Busslinger, 2004). A recent addition to the list of transcription factors required for early B cell development is Foxp1 (Hu et al., 2006). By using a recombination activating gene-2 (Rag2) complementation system, loss of this transcription factor was found to cause a block in early B cell development. Furthermore, a functional role in coordinating the development of B cells was suggested by Foxp1’s ability to bind to enhancer elements within the Rag gene loci (Hu et al., 2006). Foxp1 is a forkhead transcription factor with functions in tissue and cell-type specific gene expression and its targeted deletion is lethal in embryogenesis, mainly due to cardiac defects (Shu et al., 2001; Wang et al., 2004).

During B cell development, multiple checkpoints follow the rearrangement of the immunoglobulin loci (Meffre et al., 2000) that are partially dependent on p53 (encoded by Trp53). Trp53−/− mice show decreased apoptosis of pro-B cells, as well as accumulation of pre-B cells and immature B cells (Alizadeh et al., 2000; Lu and Osmond, 2000). Additionally, in SCID mice, deficiency of Trp53 leads to a massive accumulation of pro-B cells (Guidos et al., 1996). Presumably, such cells would normally undergo apoptosis as consequence of p53’s action in coordinating the response of cells to DNA damage. The apoptosis of pro-B cells depends on the ratio of pro-apoptotic Bax to the pro-survival Bcl-2 protein, and this ratio is disrupted in Trp53−/− mice.

Recently, the miR-34 family of miRNAs was discovered to be transcriptionally induced by p53 (Chang et al., 2007; He et al., 2007; Raver-Shapira et al., 2007). These miRNAs act by suppressing genes important in cell cycle progression, anti-apoptotic functions, and regulation of cell growth. As might be expected, expression of these miRNAs is altered in a broad range of cancers, with the most common scenario being one in which both p53 and miR-34 are downregulated (reviewed in (Hermeking, 2009)). In cancers of the hematopoietic system, profiling studies have suggested a role for one member of the miR-34 family, miR-34a, in chronic lymphocytic leukemia and acute myeloid leukemia (Isken et al., 2008; Mraz et al., 2009; Zenz et al., 2009). In the case of chronic lymphocytic leukemia, miR-34a alterations have been described both in association with and independent of p53 deletions.

A role for miR-34a in normal hematopoiesis has not yet been defined. Such a role has been described for many miRNAs, with multiple recent studies describing the impact of miRNAs on lineage determinations during hematopoietic differentiation (reviewed in (Baltimore et al., 2008)). Indeed, these were some of the first functions described for miRNAs in mammalian systems, with miR-181 promoting B-lineage differentiation (Chen et al., 2004). The functional effects of miRNAs may depend on the regulation of a few critical factors that control gene expression, thereby affecting lineage choice (Xiao and Rajewsky, 2009). The range of targets ascribed to miR-34a suggests that it might have a role in the regulation of hematopoietic development. In fact, a role in B cell development was hypothesized following the demonstration that Bcl-2 is a direct target of miR-34a regulation (Bommer et al., 2007). It was particularly striking that an imbalance between Bax and Bcl-2 could lead to a defect in pro-B cell apoptosis in Trp53−/− mice. Here, we attempt to understand how miR-34a may be involved in hematopoiesis. We found that constitutive expression of miR-34a causes a partial block in B cell development, while its knockdown resulted in increased B cells. The key mediator of the miR-34a effect appears to be the Foxp1 transcription factor, which we showed to be a direct target of miR-34a. Knockdown of Foxp1 with an siRNA mimicked the effect of overexpression of miR-34a. Thus miR-34a appears to play an important role in the orchestration of B cell development in the mouse.


Constitutive expression of miR-34a in the bone marrow leads to a decrease of B lymphocytes

To explore the role of miR-34a in hematopoietic differentiation, we designed retroviral vectors that express the microRNA. The vector co-expresses miR-34a and green fluorescent protein (GFP) and exploits the miR-155 format vector that we have previously utilized successfully to give strong expression of microRNA and siRNA (Fig 1A and (O’Connell et al., 2009)). The vector produced pre-miR-34a and mature miR-34a as demonstrated by RNA blotting (Fig 1B) or RT-qPCR (Supp Fig 1A), in addition to GFP (Supp Fig 1B). After transduction of murine bone marrow cells with miR-34a and control vectors followed by reconstitution of lethally irradiated syngeneic C57/B6 mice, we analyzed the recipient’s bone marrow 2 months later. GFP positive cells were readily observed in both the control and miR-34a-transduced marrow recipients (hereafter referred to as “miR-34a mice”; Fig 1C). Expression of miR-34a was found to be elevated in primary bone marrow cells from the miR-34a mice (Fig 1D). The bone marrow was stained with a combination of antibodies to delineate the various hematopoietic lineages, including mature B cells (defined as being CD19+IgM+), myeloid cells (CD11b+), T cells (CD3ε+), and erythroid precursors (Ter119+). A significant reduction in mature B cells, as a proportion of transduced cells (approximately 30% lower), was observed (Fig 1E). We did not observe statistically significant changes in myeloid cells (Fig 1F), erythroid precursors (Fig 1G) or T cells (Fig 1H). These differences were observed consistently across three experiments. Thus, constitutive expression of miR-34a causes a specific reduction in the number of mature B cells.

Figure 1
Constitutive expression of miR-34a in the bone marrow compartment leads to a decrease in mature B-lymphocytes

miR-34a expression results in a block in B cell development at the pro-B to pre-B cell stage in the bone marrow

To determine whether the observed reduction in mature B cells was the consequence of a specific developmental abnormality in B cell ontogeny, flow cytometric analyses were performed to delineate the various stages of B cell maturation. We found a significant (approximately two-fold) increase in pro-B cells (CD19+c-kit+IgM) in miR-34a mice (Fig 2A). Conversely, there was a significant reduction in pre-B cells (B220+CD43IgM) in miR-34a mice (Fig 2B). This pattern indicates a developmental retardation at the pro-B to pre-B transition when B cells are passing through the pre-BCR checkpoint. A similar trend was observed when alternate stains were used to delineate pro-B cells (including CD19+CD43+AA4.1+ and CD43+B220+IgM) and pre-B cells (CD19+CD25+IgM; data not shown). The numbers of pro- and pre-B cells in miR-34a mice correlated inversely with the expression pattern of miR-34a in the normal B-lineage cells, where relatively high miR-34a was observed at the pro-B cell stage with lower amounts in pre-B cells (Fig 2C). A second miR-34a expressing vector, which uses the native stem-loop structure and flanking regions and produces mature miR-34a at lower amounts, induced a similar phenotype (Supplemental Figure 2A, 2C and 2D). The decrease in bone marrow B cells resulted in decreased circulating B lymphocytes (Fig 2D), but splenic populations of B cells were largely restored in miR-34a overexpressing mice (Fig 2E). This likely represents homeostatic expansion of B cells in the periphery by mechanisms that are independent of miR-34a expression. Thus interrupting the physiological downregulation of miR-34a by constitutive expression results in a partial block in B-cell development.

Figure 2
miR-34a expression causes an increase in pro-B cells and a decrease in pre-B cells

Foxp1 is a bona fide target of miR-34a

To understand what targets of miR-34a might mediate the B cell developmental block, we examined predicted targets using the TargetScan database (Lewis et al., 2005). From this list of targets, we identified Foxp1, which is predicted to have two 7-mer sites in the 3′UTR. Because Foxp1 has previously been implicated in B cell development, we examined whether this gene is a target of miR-34a. The 3′UTR of Foxp1 is schematically represented in Fig. 3A, showing the more 5′ of the 2 conserved miR-34a sites. To determine if Foxp1 might represent a direct target of miR-34a, luciferase assays were performed as previously described (O’Connell et al., 2008). Cells co-transfected with pcDNA3-miR-34a and luciferase linked to the FOXP1-3′UTR showed repression of luciferase compared to control (Fig 3B). When the conserved site in the FOXP1 3′UTR was mutated (as depicted in Fig 3A), luciferase expression was derepressed (Figure 3B; FOXP1mt). As controls, we examined repression of a UTR containing two repeats of the antisense sequence to miR-34a (2 repeats of antisense 22-mer), which showed the greatest repression (Fig 3B, a.s. 2mer), as well as repression of a BCL2-3′UTR containing construct, a previously reported miR-34a target (Fig 3B; BCL2 and BCL2mt;(Bommer et al., 2007)). As negative controls, we utilized the 3′UTR of two genes, Cebpb (encoding CEBPβ) and Sfpi1 (encoding PU.1), which were repressed by miR-155, as well as a control luciferase construct with no UTR (Fig 3B, (O’Connell et al., 2008)). We have also examined the second UTR site which does not show repression in a similar assay (data not shown).

Figure 3
Foxp1 is a bona fide target of miR-34a

To examine the effects of miR-34a expression on endogenous Foxp1, we utilized two cell lines at a similar stage of differentiation- NALM6, which is a human pre-B cell line, and 70Z/3, which is a murine pre-B cell line. First, we assayed NALM6 single cell clones that had been transduced with a lentivector expressing miR-34a (Supp Fig 1C). This vector expressed GFP as well as miR-34a by RNA blot analysis and RT-qPCR (Supp Fig 1D–F). Analysis of the single cell clones revealed repression of FOXP1 and overexpression of miR-34a RNA (Figure 3C and 3D, respectively). In murine 70Z/3 cells, we observed a similar repression of Foxp1 at the protein level within 4 days of transduction (Fig 3E). Analysis of Foxp1 RNA in B cell subsets showed an inverse correlation with miR-34a amounts at the pro- and pre-B cell stages (Fig 3F; compare with Fig 2C). Having observed repression of Foxp1 in cell lines, we also examined Foxp1 mRNA expression in whole bone marrow from miR-34a mice. Foxp1 mRNA is significantly downregulated in the bone marrow of mice transduced with miR-34a (Fig 3G), with no significant change observed in L32 control RNA (Fig 3H). It is notable that this difference was observed in whole bone marrow, where only about half of the cells had been transduced with miR-34a. Hence, repression of Foxp1 by miR-34a is demonstrated in human and mouse cell lines, as well as in primary murine bone marrow, and is mediated through a conserved site in the 3′UTR of Foxp1.

Direct repression of Foxp1 recapitulates constitutive miR-34a expression

Previous reports have indicated that the complete lack of Foxp1 results in a profound B cell developmental block that generates a near-total lack of mature B cells (Hu et al., 2006). To examine whether the phenotype of miR-34a expression might be explained by the repression of Foxp1, we developed constructs to express a siRNA against Foxp1 (Foxp1si) in the same format as the construct expressing miR-34a (Fig 4A). Testing of this construct in 70Z/3 cells showed that Foxp1 mRNA was repressed about 50%, similar to the repression observed in miR-34a-expressing cells (Fig 4B). In addition, repression by Foxp1si was similar to that by miR-34a at the protein level, where Foxp1 expression was reduced by about 50% (Fig 4B, right hand panel, compare to Fig 3E, right hand panel). Retroviral transduction and bone marrow transfer showed a high degree of reconstitution of the marrow by Foxp1si-expressing cells (Fig 4C). Foxp1 RNA was reduced by about 50% in the marrow of mice reconstituted with this vector (Fig 4D). With this degree of repression, a dramatic increase in pro-B cells (about 3.5-fold) was observed (Fig 4E) along with a corresponding decrease in pre-B cells (Fig 4F). In contrast, retrovirally mediated knockdown of Bcl-2 in the bone marrow by a similar vector failed to recapitulate the phenotype observed in the miR-34a overexpressing mice, despite the demonstration that Bcl-2 is a direct target of miR-34a (Supp Fig 3A and B). Instead, there were major reductions in all stages of B cell differentiation and a mild reduction in myeloid cells in the bone marrow (Supp Fig 3C, D, E, and F). Hence, the block in B cell development is largely recapitulated in a specific manner by the repression of Foxp1 using a siRNA.

Figure 4
Knockdown of Foxp1 recapitulates miR-34a-induced B-lineage abnormalities

Foxp1 cDNA lacking its 3′UTR is able to rescue the miR-34a mediated block in B cell development

Next, we tested whether complementation of Foxp1 by an exogenous cDNA not responsive to miR-34a could correct the B cell phenotype. MSCV based constructs containing the Foxp1 coding sequence were generated with and without miR-34a as depicted in Figure 5A, and the vector containing both miR-34a and Foxp1 was designated as the “rescue vector”. These constructs contained the entire coding sequence of Foxp1, followed by an internal ribosomal entry sequence (IRES) to allow for GFP expression. The resultant constructs, when used to infect 70Z/3 cells, overexpressed Foxp1 at the protein level, at about 1.5–2 times endogenous levels (Fig 5B), and also overexpressed miR-34a (Fig 5C). Retroviral transduction and bone marrow transfer was completed as before, and we detected constitutive expression of Foxp1 RNA by RT-qPCR (Fig 5D). In addition, miR-34a was constitutively expressed in mice receiving the rescue vector, Foxp1+34a, in a quantitatively similar manner to that observed when miR-34a was expressed by itself (Fig 5E). In the bone marrow, pro-B cell numbers were significantly decreased in animals receiving the rescue vector as compared to those receiving miR-34a alone, and similar to pro-B cell numbers observed in the mice receiving the control vector (Fig 5F). Similarly, pre-B cell numbers were comparable to those in control mice, with a significant increase in animals receiving the rescue vector compared with those animals receiving miR-34a alone (Fig 5G). These findings indicate that B cell maturation was largely rescued by provision of Foxp1 lacking its 3′UTR in murine bone marrow. In contrast, such an effect was not observed when BCL2 was utilized in a similar “rescue experiment” (constructs depicted in Supp Fig 5A). Despite BCL2 being a direct target of miR-34a and the detection of transduced BCL2 in the bone marrow, the number of pro-B cells remained elevated, and the number of pre-B cells did not increase (Supp Fig 5 B,C,D, and E). Hence, the dependence of the miR-34a mediated B cell phenotype on Foxp1 is demonstrated in both loss-of-function and rescue contexts, indicating that Foxp1 is a highly specific target of miR-34a during B cell differentiation.

Figure 5
Foxp1 expression rescues the miR-34a induced B cell developmental abnormality

Knockdown of miR-34a results in increased numbers of mature B cells in the bone marrow

Our next step in understanding the role of miR-34a in B cells was to analyze the effects of miR-34a loss of function. We did this by attaching a so-called “sponge” sequence to a reporter (Gentner et al., 2009). In this approach, four consecutive synthetic miR-34a target sequences separated by four 4-6-nucleotide spacer sequences were cloned downstream of GFP in the previously described MGP vector (MGP-anti-34as; Fig 6A and Supplemental Table). Retrovirus generated from this construct was used to transduce 70Z/3 cells, which were immunoblotted for Foxp1. The cells expressing anti-34as showed an approximately 1.4-fold derepression of Foxp1 at the protein level (Fig 6B and data not shown). Luciferase assays performed using synthetic 34a target sites (ie., a perfectly complementary sequence; 34a-2mer) as well as the Foxp1 3′UTR in the reporter constructs demonstrated derepression as a consequence of expression of anti-34as (Fig 6C and 6D, respectively). This derepression was seen both as an effect on endogenous miR-34a activity (left two bars in Figs. 6C and 6D) and an effect on exogenously overexpressed miR-34a (right two bars in Figs. 6C–6D). The derepression was specific in that no derepression was seen if there was no UTR in the reporter plasmid or an irrelevant UTR (Fig 6E and data not shown). Next, we utilized this construct in a bone marrow transfer experiment, and found reproducible GFP expression in the recipient mice 2 months after transfer (Fig 6F). Examination of the bone marrow revealed an increase in the mature B cell population in mice with miR-34a knockdown (Fig 6G versus 6H). These differences were statistically significant (Fig 6I). Minor differences were seen in the pro-B and pre-B cell populations, but these were not significant. In the bone marrow of mice transduced with anti-34as, Foxp1 RNA levels were modestly, but significantly derepressed (Fig 6J). This set of findings is best explained by the fact that pro-B and pre-B cells are transient states during B cell development-cells flux through these states, accumulating as mature B cells. The numbers of cells in these transient compartments in miR-34a knockdown mice are therefore not a sensitive measure of how rapidly cells are moving through the compartment whereas the number of mature B cells is a more sensitive measure. Because Foxp1 RNA levels seem so important in determining the course of B cell development, we analyzed pooled data from two experiments in which we overexpressed Foxp1 (see Fig 5), finding that indeed, it is the mature B cell pool (CD19+IgM+) that is increased upon modest overexpression of Foxp1 (Fig 6K). Thus, miR-34a knockdown causes a phenotype that is the opposite of that seen with constitutive expression, suggesting a role for miR-34a in the physiological regulation of B cell development.

Figure 6
Knockdown of miR-34a using a “sponge” strategy is effective and results in increased mature B cells in the bone marrow


We describe here a role for miR-34a in B cell development that is largely explained by its repression of Foxp1. The block induced by miR-34a specifically involves Foxp1 because it is rescued by Foxp1 expression and not expression of another miR-34a target, BCL2. In early B cell development, loss of Foxp1 is known to cause a block at the pro-B cell to pre-B cell transition, and a near complete absence of B-lymphocytes in the peripheral lymphoid tissues (Hu et al., 2006). Additionally, Foxp1 downregulation is essential to monocytic differentiation, indicating a role in directing hematopoietic progenitors towards a B cell fate and away from the myeloid fate (Shi et al., 2008; Shi et al., 2004). By partial repression of Foxp1 in the bone marrow, constitutive miR-34a expression slows passage through the pro-B cell to pre-B cell transition, resulting in a lower number of circulating B cells. Conversely, loss of miR-34a function results in increased numbers of mature B cells (accompanied by modestly elevated amounts of Foxp1), implying a less stringent or faster transit through this checkpoint. The role of miR-34a and Foxp1 seem to be in controlling the output of the B cell developmental pathway, with mature B cells being the most sensitive measure of increased flux through this pathway. In differentiated B cells, FOXP1 appears to act as a B cell oncogene, whose dysregulated expression can be a consequence of translocations to the IGH locus, in cases of marginal zone lymphoma, and through amplification in many cases of diffuse large B cell lymphoma (Lenz et al., 2008; Streubel et al., 2005; Wlodarska et al., 2005). Thus, Foxp1 is both needed for B cell differentiation and a danger in mature B cell life. We speculate that the effects of miR-34a on Foxp1 may be the mode through which p53 suppresses this potentially oncogenic protein in post-germinal center B cells. Such an effect would parallel the previously described role of miR-34a as a direct connection between the tumor suppressor p53 and the oncogenic protein Bcl-2.

The effect of miR-34a on the B cell developmental pathway is consistent with previously reported abnormalities seen with a deficiency of p53, namely an increased number of pre-B cells as well as B cells, with the latter finding also a consequence of loss of miR-34a function (Lu and Osmond, 2000). With miR-34a constitutively expressed, we find the opposite- an increase in pro-B cells and a decrease in pre-B and B cells. Our findings imply a connection between p53 and Foxp1 via the action of miR-34a. In early B cell development, Foxp1 activates several B cell factors, such as E2a and Pax-5 and binds to the enhancers of the Rag genes (Hu et al., 2006). This activity of Foxp1 may be dependent on the downregulation of miR-34a, releasing repression of Foxp1, and allowing B cell development to proceed. We interrupt this physiological downregulation by constitutive expression, resulting in an accumulation of pro-B cells. With miR-34a loss-of-function, mature B cells are increased, likely arising from the loss of a miR-34a dependent checkpoint. The idea that Foxp1 expression is important for the output of B cells from the bone marrow is borne out by the following: (i) reciprocal changes in Foxp1 levels with miR-34a gain- and loss-of-function, (ii) mature B cells are increased in the marrow of mice that overexpress Foxp1 and (iii) a partial arrest at the pro-B to pre-B transition is found in Foxp1si-expressing mice.

The effects of miRNAs in B cell development have been assessed globally as well as in terms of specific miRNAs. The deletion of Dicer at the earliest stages of B cell development leads to a block in development at the pro-B to pre-B transition (Koralov et al., 2008). In this case, bioinformatic analysis showed that upregulated genes in Dicer-deficient B-lineage cells were likely those targeted by the miR-17~92 cluster, and that ablation of the pro-apoptotic protein Bim (a target of miR-17~92) or transgenic overexpression of Bcl-2 could partially rescue the developmental block. This target specificity is distinct from the block induced by miR-34a, because Bcl-2 increased survival of miR-34a expressing pro-B cells but did not significantly promote their differentiation. B cell developmental blocks have also been seen following the disruption of specific miRNAs. Early expression of miR-150 led to a block in B cell development, while its deletion led to expansions of certain B cell subsets (Xiao et al., 2007; Zhou et al., 2007). In this case, the relevant target appeared to be c-Myb, a transcription factor involved in many stages of lymphocyte differentiation. Overexpression of the miR-17~92 cluster led to a B-lymphoproliferative disease, likely as a consequence of suppression of the tumor suppressor gene PTEN and Bim (Xiao et al., 2008). Other miRNAs with effects of B cell development include miR-181a and miR-155, which promote early B cell development and the germinal center response, respectively (Chen et al., 2004; Rodriguez et al., 2007; Thai et al., 2007; Vigorito et al., 2007). Specific targeting by miR-155 in the context of B cell development has been extensively investigated and it appears that multiple targets are responsible for miR-155-induced phenotypes (Chen et al., 2008; Pedersen et al., 2008; Vigorito et al., 2007).

The specificity of miRNA targeting remains incompletely understood. On the one hand, miRNAs, including miR-34a, are predicted to target hundreds of genes by computational algorithms. On the other hand, in particular developmental contexts, one or a few genes are most important in the causation of a miRNA-mediated phenotype. Such observations have been made in both loss-of-function and gain-of-function contexts, indicating the general applicability of this principle for particular miRNAs. Our study extends these observations by demonstrating a specificity of miR-34a targeting for Foxp1 in early B-lymphoid development. This is based on both recapitulation and rescue of the phenotype by Foxp1 loss- and gain-of-function, respectively. The basis of such specificity is likely the fact that certain cell fate decisions hinge on small changes in the expression of critical lineage specification genes, and miRNAs can cause such changes. From this, it can be inferred that the study of miRNAs in hematopoietic development may illuminate critical points in cell fate decisions, and bring novel insights into this complex and dynamic developmental system.

Our findings may be of relevance in human disease as others have observed that miR-34a is functionally lost (either directly or as a consequence of p53 deletion) in chronic lymphocytic leukemia but is overexpressed in acute myeloid leukemia (Isken et al., 2008; Mraz et al., 2009; Zenz et al., 2009). In our study overexpression of miR-34a led to disruptions in B cell development, but not myeloid development. We have confirmed independently that miR-34a is overexpressed in AML by RT-qPCR (data not shown) and that its overexpression does not correlate with that of miR-155. Since we did not observe a myeloproliferative disorder in miR-34a-overexpressing mice, any mechanism whereby miR-34a contributes to leukemia is likely to be distinct from that observed in miR-155 expressing mice (O’Connell et al., 2008). Our findings suggest one possibility- the expression of miR-34a may affect the phenotype of the leukemic cells by blocking cells transformed by other oncogenic lesions from differentiating into B- lineage cells. Indeed, some recent studies indicate that there may be a progenitor with lymphoid characteristics that gives rise to acute myeloid leukemia (Deshpande et al., 2006). Our results suggest that miRNA expression may be one mechanism whereby the leukemia assumes a distinctive lymphoid or myeloid phenotype. Such phenotypic specification certainly has clinical relevance, as acute myeloid leukemia in general remains the more difficult of the acute leukemias to treat. However, further work needs to be done to understand the role that miR-34a plays in leukemia and how its action or inhibition can be parlayed into novel therapeutic strategies.

Materials and Methods

Cell Culture

All cells were cultured in a sterile incubator maintained at 37°C with 5% CO2. 293T cells were cultured in complete DMEM with 10% FBS, 100 units/ml penicillin and 100 units/ml streptomycin. WEHI-231 and 70Z/3 cells were cultured in complete RPMI with 10% FBS, 100 units/ml penicillin, 100 units/ml streptomycin and 50 μM 2-Mercaptoethanol.

DNA constructs

The MGP vector system has been described previously, and further detailed cloning strategies are available upon request (O’Connell et al., 2009). Table S1 contains sequence information for all oligonucleotides used to build new constructs. MiR-34a and siRNAs were built using the Invitrogen BlockiT pol II miR RNAi strategy as described previously, using the miR-155 arms for processing (O’Connell et al., 2009). SiRNA sequences were predicted using the Invitrogen BlockiT miR RNAi designer. In addition, a 300-base pair sequence of human genomic DNA encoding the native miR-34a mature sequence, endogenous stem-loop structure and flanking sequences was PCR-amplified using the 34a.NotI.f and 34a.XhoI.r primers and cloned into the MGP vector using the NotI and XhoI sites, as well as into pcDNA3 using 34a.EcoRI.f and 34a.XhoI.r primers and cloning into the EcoRI and XhoI sites. The MGP-based vector is referred to as MGP-miR-34a-lo. The last set of expression vectors, utilized for expression of miR-34a in human cell lines, was developed from the FUGW vector system (Lois et al., 2002). For this purpose, miR-34a genomic sequence was cut out of pcDNA3-34a and blunt-ligated into the FUGW EcoRI site. For reporter assays, two regions of the FoxP1 3′ UTR were amplified by PCR, each containing a putative miR-34a binding site as determined by TargetScan (Lewis et al., 2005). The 5′ segment was 637 base pairs and the 3′ segment was 760 base pairs. 2000 base pairs of the BCL2 3′ UTR was also PCR-amplified. These PCR products were cloned into pmiReport (Ambion, Austin, TX). The miR-34a seed regions in both the FoxP1 3′UTR 5′ segment and the BCL2 3′ UTR segment were mutated using PCR to yield Foxp1mt and BCL2mt constructs. A positive control insert consisting of two miR-34a antisense sites (antisense 2-mer) was constructed by annealing oligonucleotides with SpeI and HindIII sticky ends and cloning into pmiReport. For the rescue vectors, cDNAs for Foxp1 and BCL2 were PCR-amplified from pCMV-Sport-Foxp1 (Open Biosystems) and pDNR-Dual-BCL2 (Harvard PlasmID Core Facility), and cloned between the BglII and XhoI sites in MSCV-IRES-GFP, yielding MIG/Foxp1 and MIG/BCL2, respectively. The cDNAs contain the entire coding sequence for Foxp1 (listed in supplemental table 1) and BCL2, respectively. miR-34a was cloned by PCR amplification from pcDNA3-34a and cloned between the XhoI and EcoRI sites in the MIG vectors, yielding MIG/Foxp1+34a and MIG/BCL2+34a vectors respectively. The MGP/anti-34as construct was generated by annealing the 34a-spacer-f and 34a-spacer-r oligonucleotides (designed with XhoI/NotI sticky ends) and ligating into the MGP vector with XhoI and NotI. The spacers between each miR-34a binding site were the same as those previously published (Gentner et al., 2009).

Mice and Bone marrow reconstitution experiments

C57BL/6 mice were bred and housed in the Caltech Office of Laboratory Animal Resources (OLAR) facility. The Caltech Institutional Animal Care and Use Committee (IACUC) approved all experiments related to mice. Virus production and reconstitution experiments were performed as previously described (O’Connell et al., 2008). Experimental groups of 4–5 mice received each of the vectors described. Recipient mice were monitored for health and a peripheral blood sample removed at 1 month for flow cytometric analysis. Mice were sacrificed 2 months after reconstitution and were examined by morphology, FACS and RT-qPCR as described below. All experiments were repeated at least twice and in most cases, 3 times, as described in figure legends.

Flow cytometry

Cells were isolated from the relevant tissue, homogenized, and red blood cells were lysed using RBC lysis buffer (Biolegend). Cells were stained with the following Fluorophore-conjugated antibodies (all from Ebioscience): CD3ε, CD11b, CD19, B220, CD43, CD117 (c-kit), CD25, IgM, Gr-1, Ter-119, AA4.1, CD4, and CD8α, in various combinations to delineate the hematopoietic lineages and various stages of B cell differentiation. Cells were sorted using a FACSCalibur (Becton Dickinson) and all data were analyzed using FloJo software (Treestar). Specific gating strategies to delineate various B cell populations are available upon request. Data were presented as the percentage of GFP positive cells in the relevant hematopoietic compartment. These data were also compared against the GFP-negative cells within the same mouse.

Cell sorting for RNA analysis at various stages of B cell differentiation

Bone marrows were extracted from 2 mice, lysed in RBC lysis buffer, and spun down. Cells were then resuspended in lymphocyte FACS buffer (1X Hanks Buffered Salt Solution, 10mM HEPES, 2.5 mg/mL BSA), filtered through a 40μm mesh, blocked with FcBlock (Becton Dickinson), and depleted on a magnetic column for CD11b, Gr-1, Ter119, CD3ε, and NK1.1 using biotinylated antibodies and streptavidin-MACS beads, as per manufacturer’s instructions (Miltenyi). Cells were then stained with c-kit, B220, CD43, IgM, Streptavidin, and CD138, and sorted on a BD Biosciences FACSVantage flow cytometer (Caltech Core facility). 4 populations of cells were collected: Lin- (B220-, IgM-, Streptavidin-, and CD138-); pro-B cells (c-kit+B220+CD43+IgM-streptividin-), pre-B cells (B220+CD43-c-kit-IgM-streptavidin-) and B cells (B220+c-kit-CD43-IgM+strepatavidin-). RNA was isolated by TRIzol (Invitrogen) purification and subjected to RT-qPCR as described below. These sorts were repeated three times, with similar results.

Sequence alignments

The miR-34a seed region and Foxp1 3′ UTR sequences from human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus) and dog (Canis familiaris) were obtained and aligned using TargetScan (Lewis et al., 2005).

Experiments with cell lines

VSV-G-pseudotyped FUGW lentiviruses were made using TransIT-293 based transient transfections of 293T cells. The viruses were then used to infect NALM6 human pre-B cells by spin-infection at 1,200×g for 90 minutes at 30C, supplemented with 10μg/mL polybrene (Chemicon). Single cell clones were derived by subjecting the cells to limiting dilution assays. Clones were maintained in RPMI supplemented with 10% FBS and antibiotics. For examination of Foxp1 levels in murine cells, MSCV-based retroviruses were prepared as described previously, pseudotyped with pCL-Eco, and used to infect 70Z/3 murine pre-B cells. Cells were expanded for 3–4 days, and then utilized for RNA purification or protein extraction for Western Blotting.

Luciferase Reporter assays

2 × 105 293T cells were cultured for 18 hours and subsequently transfected with relevant plasmids with TransIT 293. Transfected plasmids included pcDNA3, pcDNA3-34a, β-gal expression vector, and pmiReport vectors. For the derepression assays with the sponge construct, we added in MGP (empty vector) or MGP/anti-34as. After 48 hours, cells were lysed using Reporter Lysis Buffer (Promega, Madison, WI) and subjected to luciferase reporter assays as previously described (O’Connell et al., 2008).

RNA preparation and quantitation

RNA was isolated using TRIzol as per manufacturer’s instructions. SYBR Green based quantitative real-time PCR (qPCR) was performed using the 7300 Real-time PCR system (Applied Biosystems, Foster City, CA) to assay miR-34a, 5s, FoxP1 mRNA, BCL2 mRNA and L32 mRNA levels as described previously (O’Connell et al., 2008). Mature miR-34a and 5s RNA were assayed using a miRVana miRNA detection kit as per manufacturer’s instructions (Ambion). Human FOXP1, BCL2 and L32 mRNA were detected using specific primers and utilizing the SybrGreen system (Applied Biosystems) (Table S1).


Total cell extracts were fractionated by electrophoresis on 10% SDS polyacrylamide gel and electroblotted to Trans-Blot nitrocellulose membrane (Bio-Rad, Hercules, CA) using a semi-dry transfer apparatus (Bio-Rad). Protein detection was performed using the following antibodies: FoxP1 (ab16645) (Abcam, Cambridge, MA), β-Actin (A1978) (Sigma, St. Louis, MO); GAPDH (sc-47724), α-Tubulin (sc-5286), goat anti-mouse HRP-conjugated secondary antibody (sc-2005), goat anti-rabbit HRP-conjugated secondary antibody (sc-2004) (Santa Cruz Biotechnology, Santa Cruz, CA). Expression intensities were determined using Scion Image software.

Statistical tests

All statistical analyses were performed using Microsoft Excel. Expression and flow cytometric data were first analyzed by an F-test to determine whether the distributions were homo- or heteroscedastic. Then, the correct type of unpaired, two-tailed T-test was applied to determine if the distributions were statistically different. T-tests that returned p-values of less than 0.05 were considered to be statistically significant.

Supplementary Material



We would like to thank members of the Baltimore laboratory, Dr. Kenneth Dorshkind, and Dr. Ellen Rothenberg for helpful discussions. This work was supported by career development award 1K08CA133521 from the National Cancer Institute and a clinical fellowship training grant from the California Institute of Regenerative Medicine/Eli and Edythe Broad Center for Regenerative Medicine and Stem cell Research at UCLA (D.S.R.), the Irvington Institute Fellowship Program of the Cancer Research Institute (R.M.O.), the graduate research fellowship program of the National Science Foundation (A.A.C.), and National Institutes of Health (1R01AI079243-01; D.B.).


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