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The Journal of Immunology, 2009, 182, 433 -445
Copyright © 2009 by The American Association of Immunologists, Inc.
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MicroRNA-221–222 Regulate the Cell Cycle in Mast Cells1

Ramon J. Mayoral2,*, Matthew E. Pipkin2,{dagger}, Mikhail Pachkov{ddagger}, Erik van Nimwegen{ddagger}, Anjana Rao{dagger} and Silvia Monticelli3,*

* Institute for Research in Biomedicine, Bellinzona, Switzerland; {dagger} Immune Disease Institute and Harvard Medical School, Boston, MA 02115; and {ddagger} Biozentrum, University of Basel, Basel, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods5
 Results
 Discussion
 Disclosures
 References
 
MicroRNAs (miRNAs) constitute a large family of small noncoding RNAs that have emerged as key posttranscriptional regulators in a wide variety of organisms. Because any one miRNA can potentially regulate expression of a distinct set of genes, differential miRNA expression can shape the repertoire of proteins that are actually expressed during development and differentiation or disease. Here, we have used mast cells as a model to investigate the role of miRNAs in differentiated innate immune cells and found that miR-221–222 are significantly up-regulated upon mast cell activation. Using both bioinformatics and experimental approaches, we identified some signaling pathways, transcription factors, and potential cis-regulatory regions that control miR-221–222 transcription. Overexpression of miR-221–222 in a model mast cell line perturbed cell morphology and cell cycle regulation without altering viability. While in stimulated cells miR-221–222 partially counteracted expression of the cell-cycle inhibitor p27kip1, we found that in the mouse alternative splicing results in two p27kip1 mRNA isoforms that differ in their 3' untranslated region, only one of which is subject to miR-221–222 regulation. Additionally, transgenic expression of miR-221–222 from bacterial artificial chromosome clones in embryonic stem cells dramatically reduced cell proliferation and severely impaired their accumulation. Our study provides further insights on miR-221–222 transcriptional regulation as well as evidences that miR-221–222 regulate cell cycle checkpoints in mast cells in response to acute activation stimuli.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods5
 Results
 Discussion
 Disclosures
 References
 
MicroRNAs (miRNAs)4 are a class of highly conserved, small noncoding RNAs that function mostly as endogenous translational repressors of protein-coding mRNAs. The ~22 nucleotides (nt) mature miRNAs are derived from pre-miRNAs of 60–70 nt that are in turn cleaved from longer primary transcripts (pri-miRNAs) (reviewed in Ref. 1). In mammals, miRNAs are predicted to control the activity of ~30% of all protein-coding genes, and they have been shown to participate in the regulation of almost every cellular process investigated so far (reviewed in Ref. 2). In particular, miRNAs appear ideally suited to rapidly adjust protein concentrations in cells, as expected to be required during response to changes in the cellular environment as well as during cell differentiation, a process controlled by an intricate network of growth and transcription factors that simultaneously regulate the commitment, proliferation, apoptosis, and maturation of progenitor cells. Accordingly, certain miRNAs are expressed in a stage-specific fashion (3, 4, 5). Moreover, although many miRNAs are ubiquitously or widely expressed, a relatively small set of miRNAs accounts for most of the differences in miRNA profiles between cell lineages and tissues (4, 6).

Consistent with the discovery that miRNAs can modulate proliferation, altered miRNA expression has been found to affect cancer development (1). Although selected miRNAs are up-regulated in cancer cells, global miRNA abundance seems to be generally reduced in tumors (7). Down-regulation of miRNAs probably contributes to neoplastic transformation by allowing increased expression of proteins with oncogenic potential. Of note, it has been recently shown that repression of tumor-suppressing miRNAs is a fundamental component of the Myc tumorigenic program (8), highlighting the interplay between miRNAs and transcription factors in regulating cell proliferation and oncogenic transformation. Despite growing knowledge of miRNA biology, little is known about the transcriptional regulation of miRNAs themselves. Given that most miRNAs, like protein-coding genes, are transcribed by RNA polymerase II (9) and that many miRNAs are located within the introns of protein-coding genes (10), it would not be surprising if the regulation of miRNA gene transcription was similar to the regulation of protein-coding genes as already described for a few miRNAs (11, 12, 13, 14).

While the role for miRNAs in development and differentiation is well established, there are only few examples of a role for miRNAs in fully differentiated cells (12, 15, 16). Possible miRNA roles include the maintenance of cell identity, as well as the modulation of cell proliferation and effector functions. Mast cells are cells of the immune system that reside in most tissues and derive from hematopoietic precursors in the bone marrow (17). Here, we have used mast cells as a model to investigate the role of miRNAs in a differentiated cell type. We performed miRNA arrays on differentiated mast cells, under resting or stimulated conditions, and we identified miR-221–222 as the only family of miRNAs significantly up-regulated upon cell activation. Using both bioinformatics and experimental approaches, we characterized the transcriptional requirements for the miR-221–222 gene, and through analysis of the pattern of DNaseI hypersensitivity (HS), we identified potential cis-regulatory regions that might control mast cell development and activation. Furthermore, by overexpressing miR-221–222, individually or in combination, we found that these two miRNAs cooperate in regulating cell cycle and cell proliferation in mast cells. Overexpression of miR-221–222 had a modest effect on the expression of the known target p27Kip1; we show that such partial effect on p27Kip1 was due to a splice variant of p27Kip1 that does not contain miR-221–222 binding sites in its 3' untranslated region (UTR). Finally, cell cycle regulation is likely to be a more general effect of miR-221–222, as transgenic expression of miR-221–222 from bacterial artificial chromosome (BAC) clones in embryonic stem (ES) cells dramatically reduced cell proliferation and severely impaired their accumulation. Our study provides evidence that miR-221–222 can be regulators of the cell cycle in a cell type- and activation-dependent manner.


   Materials and Methods5
Top
 Abstract
 Introduction
 Materials and Methods5
Results
 Discussion
 Disclosures
 References
 
Cell cultures and mast cell differentiation

The MC-9 mast cell line was obtained from the American Type Culture Collection and cultured in IMDM media containing 50% WEHI-3 conditioned supernatant (containing IL-3). Bone marrow-derived mast cells (BMMCs) were differentiated from bone marrow isolated from tibia and femur of 6- to 8-wk-old mice as described (18); no noticeable differences in miRNA expression were observed depending on the mouse strains used (either C57BL/6, Balb-c, or CD-1). Recombinant murine stem cell factor (SCF) was purchased from PeproTech and used at 20 ng/ml. The IKK inhibitor BMS-345541 was purchased from Calbiochem and used at 10 µM final concentration. Where noted, cells were stimulated with 20 nM PMA, 2 µM ionomycin, and pretreated with 1 µM cyclosporine A (CsA). Th1-D5, Th2-D10, and primary CD4+ T cells were isolated and cultured exactly as described (19, 20, 21).

RNA extraction, RT-PCR, and analysis of miRNA expression

Northern blots for miRNAs were performed exactly as described (4). Briefly, total cellular RNA was prepared using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. Total RNA (25 µg) was separated on 12–15% denaturing urea-polyacrylamide gels in 0.5x Tris-borate-EDTA (TBE) buffer. RNA was transferred on a Nytran SuPerCharge membrane (Schleicher & Schuell Microscience) via wet transfer in 0.5x TBE buffer at 4°C, followed by cross-linking to the membrane in a UV Stratalinker (Stratagene). Membrane hybridization was performed overnight at 39°C with DNA-oligo probes (complementary to the mature miRNA sequence as reported in the miRBase database; see Ref22 and microrna.sanger.ac.uk/sequences/) radiolabeled with T4 polynucleotide kinase (New England Biolabs). Membranes were washed at 37°C, three times for 10 min with 2x SSC/0.1% SDS and once for 5 min with 0.1x SSC/0.1% SDS. Band intensities were quantified using a PhosphorImager and ImageQuant 5.0 software (Molecular Dynamics). Quantitative RT-PCR (qRT-PCR) was performed with a high-specificity miRNA qRT-PCR detection kit (Stratagene) following the manufacturer’s instructions. For TaqMan relative quantification of miRNA levels, first-strand synthesis was performed with a miRNA-specific primer on 10 ng of total RNA using a miRNA first-strand synthesis kit from Applied Biosystems. TaqMan PCR was performed for miR-221 and snoRNA202 as endogenous control using TaqMan miRNA assays from Applied Biosystems and a 9600 Applied Biosystems real-time PCR machine following exactly the manufacturer’s instruction. The first-strand synthesis reaction for semiquantitative RT-PCR was performed with a SuperScript kit from Invitrogen, following the manufacturer’s instruction.

Plasmid generation

The dual-promoter lentiviral vector Tween was obtained from Dr. De Maria and Dr. Bonci (23), and the sequences corresponding to the murine miR-221 and miR-222, plus 150 bp on each side,were amplified from a BAC clone and cloned together or independently downstream of the CMV promoter using common cloning techniques.

Transfections and transductions

MC-9 cells were transduced with lentiviral particles produced in 293FT cells cotransfected with lentiviral Tween vectors and packaging vectors; linear polyethylenimine (Sigma-Aldrich) was used as transfecting reagent. 293FT cells were grown in DMEM media supplemented with 10% FBS, nonessential amino acids, and glutamine. For transfection, 40 µg of DNA was diluted in 2 ml of Opti-MEM with a ratio 4:3:1 of transfer vector (Tween)/packaging coding vector (psPAX)/envelope coding vector (pMD2.G). Following a 5-min incubation at room temperature, 90 µl of a sterile solution of polyethylenimine (pH 7.6) at a concentration of 1 mg/ml was added to the cocktail, and the mixture was added to the cells after 10 min of incubation at room temperature. Viral particle-containing supernatant was harvested 36 and 48 h posttransfection. After filtering through a 0.45-µm low-binding protein filter, the viral particles were pelleted on a sucrose gradient. Concentrated lentiviruses were added to the MC-9 cell media supplemented with 1 µg/ml of polybrene.

BrdU incorporation and cell proliferation assays

For BrdU incorporation assay, cells were allowed to incorporate BrdU for 30–45 min at 37°C. Cells were cultured at various densities and in various conditions (with or without IL-3 withdrawal for 48 h) to rule out the effect of different culture conditions on BrdU incorporation. BrdU incorporation was detected using a labeling and detection kit from BD Biosciences. For propidium iodide staining and DNA content analysis, 106 cells were fixed in 70% ethanol for 45 min on ice, followed by incubation for 30 min at 37°C with 100 µg/ml RNaseA and 40 µg/ml propidium iodide. Cells were analyzed at the FACS immediately after staining. For thymidine incorporation assays, 50,000 cells were cultured for 4 h in a 96-well plate with 0.4 µCi [3H]thymidine per well. Cells were then harvested and the amount of incorporated radioactivity determined with a beta counter.

DNaseI hypersensitivity assay

DNaseI hypersensitivity assay was performed exactly as described (19, 24). Briefly, nuclei were isolated and aliquoted from BMMCs differentiated for 4 wk with IL-3. Increasing amounts of DNase I (Worthington Biochemical) were added to the nuclei aliquots and incubated at room temperature for 3 min. Subsequent purification of genomic DNA, Southern blotting, and hybridization were performed exactly as described (19). All probes used were designed to hybridize to one end of the restriction fragment to be analyzed and were generated by PCR from BAC clones or genomic DNA.

Western blots and Abs

Total protein extracts were prepared by lysis in Laemmli sample buffer and immediate boiling for 10 min. Samples were run on 10–15% SDS-polyacrylamide gels and proteins were transferred on nitrocellulose. Immunodetection was performed with p27 C-19 and β-tubulin H-235 (Santa Cruz Biotechnologies). Abs used for Fc{epsilon}RI cross-linking were mouse IgE (clone SPE-7, Sigma-Aldrich) and rat anti-mouse anti-IgE (BD Pharmingen).

Computational analysis

MotEvo (25) is a Bayesian algorithm that takes as input a collection of weight matrices, representing the sequence specificities of a collection of transcription factors, and predicts binding sites for these weight matrices in multiple alignments of intergenic DNA of related species. MotEvo uses an explicit model for the evolution of transcription factor binding sites that takes into account the phylogenetic relationships between the species, the possibility that functional sites may occur in only a subset of the species, and recognizes that some regions may be conserved for reasons other than the occurrence of binding sites for the collection of weight matrices with which it searches. MotEvo was run with a collection of >200 weight matrices representing mammalian transcription factors on multiple alignments of the DNaseI hypersensitive regions and orthologous regions in human, cow, dog, horse, rhesus macaque, and opossum. The density of predicted sites for each weight matrix was then compared with the density of predicted sites in proximal promoters (from 300 bp upstream of transcription start to 100 bp downstream of transcription start) of all mouse reference sequence (RefSeq) transcripts.


   Results
Top
 Abstract
 Introduction
 Materials and Methods5
 Results
Discussion
 Disclosures
 References
 
miR-221–222 are transcriptionally induced in mast cells

To investigate the roles of acutely-inducible miRNAs in differentiated cells, we used BMMCs obtained by culturing bone marrow precursors for 4–6 wk in vitro with IL-3 (18). The cells were either left resting or stimulated for various amount of time with PMA and ionomycin, after which miRNA content was analyzed using triplicate arrays (not shown) (4). In three independent bone marrow cultures, miR-221–222 were the only miRNA family that increased upon cell stimulation. miR-221 and miR-222 are considered part of the same family, as they share the same "seed" sequence (Fig. 1A) (26). PipMaker (27) analysis of the genomic location of the mature miR-221 and miR-222 sequences revealed that these two miRNAs are both located on human and mouse chromosome X, ~600 bp apart, and are highly conserved across different species (Fig. 1B).


Figure 1
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  		FIGURE 1. Increased levels of miR-221–222 in activated mast cells. A, Alignment of mature miR-221 and miR-222 sequences. The "seed" sequence is shaded in gray. B, PipMaker (27 ) analysis of conservation of 3.18 kb of genomic sequence surrounding the miR-221 and miR-222 sequences in five different species. Each dot represents sequence identity. Only regions that were at least 50% identical are plotted. C, Northern blot of 4- to 8-wk-differentiated murine BMMCs left resting or stimulated for 1 or 24 h with PMA and ionomycin. D5 and D10 are respectively Th1 and Th2 clones, while naive T CD4+ lymphocytes were isolated from spleen and lymph nodes of 6-wk-old mice. D, TaqMan PCR for miR-221 expression in primary Th1, Th2, and BMMC cells. OT-II CD4+ lymphocytes were cultured for 6 days in Th1- and Th2-skewing conditions and were restimulated for 6 h with PMA and ionomycin. Th1 cells were 98% IFN-{gamma}+ with no IL4+ cells as assessed by intracellular cytokine staining (not shown), while Th2 cells were 70% IL4+ and 2% IFN-{gamma}+. Four-week IL-3-differentiated BMMCs were either left resting or were stimulated for 6 h with PMA and ionomycin. Total RNA was extracted and first-strand synthesis was performed with a miRNA-specific primer on 10 ng of total RNA. TaqMan PCR was performed for miR-221 and snoRNA202 as endogenous control. E, Northern blot of differentiated BMMCs cultured for 1 h in the presence of IgE only (3 µg/ml), followed by cross-linking with an anti-IgE Ab (2–6 µg/ml) for 24 h. BMMCs were also stimulated for the same amount of time with PMA and ionomycin. In both C and E the same blot was stripped and reprobed for the indicated miRNAs, as well as for an Arg-tRNA as loading control.

 
Up-regulation of miR-221–222 in stimulated BMMC was confirmed by Northern blot analysis (Fig. 1C). miR-221–222 were expressed at basal levels in resting BMMCs, detectably up-regulated within 1 h, and strongly induced by 24 h of stimulation with PMA/ionomycin (Fig. 1C, left panels). Moderate miR-221–222 expression was observed in the Th1 clone D5, but not in the Th2 clone D10 or in primary resting murine naive CD4+ T cells (Fig. 1C, right panels). Primary CD4+ cells were also cultured for 6 days in Th1- and Th2-skewing conditions and were restimulated for 6 h with PMA and ionomycin. Total RNA was extracted and miR-221 expression levels were assessed by TaqMan PCR. BMMCs were also treated and analyzed in the same way for comparison. As shown in Fig. 1D, miR-221 was expressed at higher levels in resting BMMC than in T lymphocytes, and it was further up-regulated as expected upon PMA and ionomycin stimulation. Conversely, Th2 cells had essentially undetectable basal levels of expression, which did not change dramatically upon stimulation. Finally, Th1 cells showed an intermediate level of expression, with low basal levels that were moderately induced upon PMA and ionomycin stimulation. miR-221–222 up-regulation was also observed in BMMCs upon cross-linking of the surface high-affinity IgE receptor Fc{epsilon}RI, which represents a more physiological way to stimulate mast cells (Fig. 1E). However, as shown before for other genes in mast cells, including cytokine genes (24), Fc{epsilon}RI cross-linking was a weaker stimulus compared with PMA and ionomycin, and the latter stimulus was therefore used in most subsequent experiments.

The relatively slow kinetics of miR-221–222 up-regulation (as compared, for example, to the kinetics of expression of the cytokine genes Il-13 and Il-4 in the same cell type, peaking by 1 h of stimulation) (24) suggested a requirement for protein synthesis, for either miRNA transcription or processing. We therefore performed experiments in which the protein synthesis inhibitor cycloheximide (CHX) was briefly added to BMMCs, either before or after stimulation with PMA/ionomycin for 24 h (supplemental Fig. 1A).6 As expected, PMA/ionomycin stimulation led to a clear increase in the amount of both pre-miR-222 (~70 nt) and mature miR-222 (~22 nt) over that observed in unstimulated cells (supplemental Fig. 1A, compare lanes 1, 2, and 4). In contrast, treatment with CHX for 2 h, either before or after the 24-h stimulation with PMA/ionomycin, reduced the level of mature miR-222 and correspondingly increased the level of pre-miR-222 over that in control-stimulated cells (supplemental Fig. 1A, compare lanes 3 and 5 with lane 4), indicating that continuous protein synthesis is required to achieve maximum miR-221–222 processing. Because prolonged CHX treatment can be toxic to cells, the duration of exposure of the cells to CHX was limited to 2 h, and cell toxicity due to CHX treatment over the course of the experiment was assessed by annexin V and 7-aminoactinomycin D staining. These experiments showed no increase in cell death in CHX-treated cells, compared to the DMSO-treated sample (supplemental Fig. 1B).

To determine whether the accumulation of mature miRNAs upon stimulation was due to induction of pri-miRNA transcription or to other mechanisms such as increased processing, we analyzed expression of the precursors pre- and pri-miR-221–222. Peak accumulation of pre-miR-222 (~70 nt) was observed at 4 h, before the peak of accumulation of mature miRNA (~22 nt) at 24 h (Fig. 2A, compare lanes 2 and 3); moreover, miR-221 and miR-222 originate from the same primary transcript, as we detected pri-miR-221–222 in activated BMMCs by 4–8 h, both by RT-PCR (Fig. 2B) as well as by regular Northern blots run on an agarose gel (supplemental Fig. 2). We conclude that the increase in miR-221–222 levels is due to transcriptional induction, suggesting a role for miR-221–222 during cell effector functions and/or proliferation of differentiated BMMCs; however, the basal expression observed in resting BMMCs is consistent with additional roles for these miRNAs in the maintenance of cell identity and during the differentiation process.


Figure 2
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  		FIGURE 2. miR-221–222 are transcriptionally up-regulated in activated mast cells. A, Time course of stimulation with PMA and ionomycin of differentiated BMMCs. Ethidium bromide staining of the gel is shown as loading control. B, RT-PCR on RNA extracted from differentiated BMMCs stimulated for the indicated times with PMA and ionomycin. Actin mRNA was used as a loading control. The RT-PCR shown is representative of three independent experiments. A schematic representation (not to scale) of the primers used to detect the primary transcript is shown on top.

 
miR-221–222 induction requires both calcineurin and NF-{kappa}B pathways

Since we established that the observed up-regulation of miR-221–222 in activated BMMCs was due to transcriptional activation, we studied the role of selected candidate transcription factors in regulating miR-221–222 expression. PMA and ionomycin were both capable individually to induce miR-221–222 expression (Fig. 3A), indicating that multiple signaling pathways need to be activated to achieve maximum miRNA expression.


Figure 3
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  		FIGURE 3. Transcriptional requirements for miR-221–222. A, Northern blot of 4- to 8-wk-differentiated BMMCs stimulated for 24 h with PMA and ionomycin alone or in combination. Cells were also either left resting or were treated with CsA. The same blot was stripped and reprobed for an Arg-tRNA as loading control. Quantification of the Northern blot via PhosporImager is indicated by the numbers underneath the gel. B, Primary BMMCs were differentiated for 4 wk from mice deleted for nfat1 and from control littermates, and they were either left resting or were stimulated for 24 h with ionomycin alone or in combination with PMA, with or without pretreatment for 20 min with CsA. Quantification of the Northern blot was performed with a PhosphorImager and it is shown in the graph. C, Northern blot of differentiated BMMCs pretreated for 1 h with an IKK inhibitor or DMSO vehicle followed by PMA and ionomycin stimulation for 24 h. The same blot was stripped and reprobed for the indicated miRNAs, as well as with an Arg-tRNA as loading control. The figure is representative of three independent experiments. D, Quantitative RT-PCR for miR-222 performed on BMMCs treated for 1 h with an IKK inhibitor or DMSO vehicle followed by PMA alone or PMA and ionomycin stimulation for 24 h. U6 was used to normalize expression. Expression in the differently treated samples is compared with the DMSO-treated sample, which is set to one.

 
Up-regulation of miR-221–222 (but not basal expression (Fig. 3A)) in response to ionomycin was blocked by pretreatment of wild-type BMMCs with CsA (Fig. 3B), showing involvement of the calcium-dependent serine/threonine phosphatase calcineurin (28). Calcineurin directly dephosphorylates and activates the NFAT family of transcription factors (29), and it can also modulate the function of a variety of other transcriptional regulators. However, BMMCs differentiated from the bone marrow of nfat1-deleted mice (30) behaved identically to BMMCs from control littermates in terms of up-regulating miR-222 expression (Fig. 3B, compare open and filled bars). These data suggest that NFAT family members other than NFAT1 expressed in mast cells (such as NFAT2) might compensate for the lack of NFAT1, as already shown for the cytokine genes Il-4 and Il-13 (18, 31). Alternatively, other CsA-sensitive transcriptional regulators could potentially have a role.

In addition to activation of the calcineurin/NFAT pathway, mast cells also activate a second major signaling/transcription pathway, the IKK/NF-{kappa}B pathway, upon acute stimulation (32). NF-{kappa}B is already known to influence miRNA transcription (14, 33). To investigate the possibility that NF-{kappa}B regulates miR-221–222 transcription in activated BMMCs, we analyzed RNA extracted from BMMCs pretreated with the IKK inhibitor BMS-345541 for 1 h before stimulation with PMA and ionomycin (Fig. 3C). miR-221 and miR-222 expression was greatly reduced by pretreatment with this inhibitor, whereas the expression of other miRNAs such as miR-26 was unaffected (Fig. 3C, top three panels). As a control we analyzed the expression of the two members of the miR-146 family, miR-146a and miR-146b (lower and upper bands in fourth panel of Fig. 3C). Induction of miR-146a, which is known to be NF-{kappa}B regulated (33), was completely abrogated by pretreatment with the IKK inhibitor, whereas induction of miR-146b was unaffected (Fig. 3C, fourth panel). The identity of the miR-146 bands and the inducibility of miR-146a, but not miR-146b, were confirmed by qRT-PCR using miRNA-specific primers (data not shown).

NF-{kappa}B is induced in many cell types by stimulation with PMA alone. To address this point in mast cells, we stimulated BMMCs either with PMA alone or with both PMA and ionomycin. Pretreatment with the IKK inhibitor completely blocked up-regulation of miR-221–222 in PMA-stimulated cells, but it had a partial effect on miR-221–222 up-regulation in response to both PMA and ionomycin, as detected by qRT-PCR (Fig. 3D). Taken together, these data suggest that as previously observed for BIC/miR-155 (14), both the calcineurin/NFAT pathway and the IKK/NF-{kappa}B pathway contribute to miR-221–222 induction in stimulated BMMCs.

DNaseI HS analysis of the miR-221–222 genomic locus identifies putative regulatory regions

To identify regions potentially involved in transcriptional regulation of pri-miR-221–222, we analyzed the pattern of DNaseI HS in the miR-221–222 locus, both in primary mast cells, which do express these miRNAs even at basal levels, and in primary Th2 lymphocytes, which do not express significant levels of miR-221–222 in either resting or stimulated conditions (see Fig. 1, C and D). As shown in Fig. 4 and supplemental Figs. 3 and 4, most of the sites in the 30-kb region upstream of the miR-222 sequence were exclusively mast cell-specific, suggesting that they may be important cell type-specific regulatory regions for miR-221–222 transcription. Similarly, inducible sites present only in mast cells might represent cell-specific enhancers (DH VII and VIII in Fig. 4D). Moreover, most of these regions mapped closely to conserved noncoding sequences in the genome as shown by the University of California at Santa Cruz (UCSC) Genome Browser (genome.ucsc.edu), highlighting again their importance as regulatory regions (34). Transient transfection experiments with reporter plasmids did not identify a region with unequivocal promoter activity (data not shown), possibly because to be active these regions need to reside in their native chromatin context. We used the MotEvo algorithm (25) to predict transcription factor binding sites within 1000 nucleotides around each DNaseI HS site. This analysis revealed that sites for the promyelocytic leukemia zinc finger (ZBTB16) are the most enriched in these regions relative to the promoters of all mouse genes. In our analysis of the DNaseI HS regions, we also found that besides the ZBTB16 sites, there was also a strong overrepresentation of sites for the CDX and MEF2 transcription factors. Furthermore, binding sites were predicted for NFAT at sites IV, V, VI, VII, VIII, and X, but not at site III, while a NF-{kappa}B site was predicted close to site IV, which would correlate with the observed NF-{kappa}B dependence of miR-221–222 transcriptional up-regulation. Nevertheless, in contrast to the ZBTB16, CDX, and MEF2 sites, the sites for NFAT and NF-{kappa}B were not significantly enriched at the DNaseI HS regions relative to the promoters of all mouse genes. The functional role of the identified DNaseI HS and transcription factors binding sites are currently being tested.


Figure 4
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  		FIGURE 4. DNase I HS pattern in the miR-221–222 locus. A, DNase HS analysis in unstimulated primary murine Th2 cells differentiated for 1 wk and BMMCs. The probe recognized a 17.9-kb BamHI fragment encompassing the miR-221 and miR-222 sequences (shown in the schematic representation in D). The filled triangles indicate increasing amounts of DNaseI enzyme. B, The same blot from A was stripped and reprobed with a probe recognizing the BamHI fragment farther upstream in the miR-221–222 locus (24.3 kb in the schematic representation in D). C, DNase HS analysis in primary murine BMMCs differentiated in vitro for 4–8 wk and either left resting or stimulated for 24 h with PMA and ionomycin. The probe used recognized the 17.7-kb KpnI fragment shown in D. D, Schematic representation of all of the DNaseI HS sites identified in the 33 kb of genomic sequence upstream of miR-221 and miR-222. Identified sites are superimposed on an analysis of sequence conservation performed in UCSC Genome Browser. Putative NF-{kappa}B and NFAT binding sites identified by bioinformatics analysis are also indicated.

 
miR-221–222 regulate proliferation in mast cells

To investigate the role of miR-221–222 in mast cells, we cloned miR-221 and miR-222 (either individually or together) and their surrounding sequences in the Tween lentiviral-based vector (23), generating the Tween miR-221 (T-221), Tween miR-222 (T-222), and the Tween miR-221–222 (T-221–222) vectors. These vectors as well as the empty Tween vector were used to transduce the mast cell line MC-9. After transduction, ~50% of the cells were GFP+, and they were further enriched to >90% GFP+ cells by cell sorting. These cells expressed miR-221 and miR-222 at significantly higher than endogenous levels (Fig. 5A), as also confirmed by Northern blot analysis (not shown). Cell cycle analysis by propidium iodide staining showed that individual expression (or more strikingly, combined expression) of miR-221 and miR-222 led to an increased number of cells in the prominent G1/G0 peak, with correspondently fewer cells in G2/M (Fig. 5B; quantified in Fig. 5C). There were no obvious differences in viability as assessed by annexin V and 7AAD staining, even when cell death was induced with CHX treatment in various experimental conditions (not shown).


Figure 5
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  		FIGURE 5. A higher proportion of mast cells stably expressing miR-221–222 is in G1/G0. A, MC-9 cells transduced with miR-221–222 lentiviral vectors were sorted for GFP expression; analysis of miR-221 and miR-222 expression was performed by qRT-PCR on GFP+ MC-9 cells. B, Propidium iodide staining and DNA content analysis of transduced MC-9 cells. C, Quantification of the percentages of transduced MC-9 cells in the different stages of the cell cycle. Each bar is the mean of four independent experiments.

 
miR-221–222 expression also led to an increase in the fraction of cells in a less granular cell population defined by low side scatter (SSClow) (Fig. 6A; quantified in Fig. 6B). Analysis of the sorted GFP+SSClow and GFP+SSChigh subsets showed that both populations were Fc{epsilon}RI+, c-KIT+, granzyme B+, and they expressed the mast cell protease enzyme MCP-5, as assessed by FACS analysis or RT-PCR (not shown). Furthermore, propidium iodide staining of the sorted GFP+SSClow and GFP+SSChigh subsets showed that the less granular cells correspond to cells that are mainly in G1/G0 (Fig. 6C). When recultured after sorting, both subsets reestablished the parental extent of heterogeneity after a few days in separate cultures, suggesting that the observed differences in cell size and granularity reflect different stages of the cell cycle. The sorted SSClow population was still capable of cycling and growing similarly to the unsorted population, ruling out the possibility that these cells left the cell cycle and are mainly resting G0 cells.


Figure 6
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  		FIGURE 6. SSClow cells are mainly in G1/G0. A, MC-9 cells untransduced (untrans) or transduced with either vector alone (Tween) or miRNA-expressing vectors were analyzed by FACS for presence of two subsets with different side scatter (SSChigh and SSClow). B, Quantification of the percentages of GFP+SSChigh vs GFP+SSClow subsets in the transduced cells. Each bar is the mean of five different measurements on live GFP+ cells. C, Propidium iodide staining and DNA content analysis of the sorted SSClow cells.

 
To further investigate the effect of miR-221–222 overexpression on the cell cycle, we analyzed the DNA content profile of cells cultured under different conditions (Fig. 7). Untreated cells overexpressing miR-221–222, individually or together, showed an increase in the number of cells in G1/G0 as described above (Fig. 7, top row). This miRNA-dependent increase in G1/G0 cell numbers was maintained when the cells were stimulated with PMA and ionomycin for 24 h; this stimulation also induced some cell death (as shown by the increase in the percentage in cells in sub-G0), which was not dramatically different in cells expressing or not expressing miRNAs (Fig. 7, second row). This was confirmed by induction of cell death in various conditions and annexin V/7AAD staining (not shown). Even when cells were cultured for 72 h in the absence of the survival and proliferation-inducing factor IL-3, we observed an increase in cell death as expected, but the number of surviving cells predominantly arrested in G1/G0 was again greater in cells overexpressing miR-222, alone or in combination with miR-221 (Fig. 7, third row). Re-addition of IL-3 for 18 h after the 72-h withdrawal released the block in the cell cycle, and accordingly a higher percentage of cells was found in G2/M phases of the cell cycle (Fig. 7, fourth row). Conversely, addition of SCF (c-KIT ligand), a known proliferative factor for mast cells (35), pushed most of the cells into G2/M (Fig. 7, fifth row). Remarkably, in all of these culture conditions, we could still clearly observe that more cells expressing miR-221–222 remained in G1/G0 as compared with the control cells.


Figure 7
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  		FIGURE 7. The effect of miR-221–222 expression on the cell cycle persists under various culture conditions. MC-9 cells transduced with a control vector (Tween) or miR-221–222 (alone or in combination) expressing constructs were left untreated (first row) or treated with either 1) PMA and ionomycin for 24h (second row), 2) IL-3 withdrawal for 72 h (third row), 3) IL-3 withdrawal for 72 h followed by re-addition of IL-3 for another 18 h (fourth row), or 4) 20 ng/ml of SCF for 18 h (fifth row). After treatment, cells were stained with propidium iodide and analyzed for DNA content.

 
To further study the effect of miR-221–222 overexpression on the cell cycle in MC-9 cells, we performed thymidine-incorporation (Fig. 8A) and BrdU-incorporation assays (Fig. 8, B and C). Untransduced MC-9 cells, and cells transduced with either the empty Tween vector or the miRNA-expressing vectors were cultured for 4 h in the presence of [3H]thymidine (Fig. 8A) or for 30 min in the presence of BrdU (Fig. 8B). Proliferation was then analyzed by counting the incorporated radioactivity or by staining with an anti-BrdU Ab respectively. As shown in Fig. 8A, proliferation as assessed by thymidine incorporation was slightly, but repeatedly, reduced in cells that overexpressed miR-221–222 in combination, while the effect of the individual miRNAs was marginal in this system. Accordingly, a BrdU-incorporation assay (Fig. 8B) showed again the presence of higher number of cells in G1/G0 when miR-222 was overexpressed alone or in combination with miR-221, but also reduced percentages of cells in S phase when miR-221–222 were overexpressed. Since these cells were not synchronized, and the best way to partially synchronize them turned out to be prolonged IL-3 withdrawal (see also Fig. 7, third row), we performed the same BrdU-incorporation assay on MC-9 cells that were cultured without IL-3 for 48 h; IL-3 was then re-added for 14 h and BrdU was added for the final 30 min of culture. As shown in Fig. 8C, in these conditions the percentage of cells in G1/G0 was increased, but the percentage of cells that entered the S phase was much more clearly reduced when miR-222 was expressed alone or in combination with miR-221.


Figure 8
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  		FIGURE 8. Mast cells overexpressing miR-221–222 are impaired in proliferation. A, Thymidine incorporation assay of MC-9 cells untransduced (untr.) or transduced with either the empty Tween vector or the miR-221-, miR-222-, and miR-221–222-expressing vectors. Cells were cultured for 4 h in a 96-well plates with 0.4 µCi [3H]thymidine per well. Cells were then harvested and the amount of incorporated radioactivity was determined with a beta counter. The graph shows three independent experiments, each one performed in quadruplicate. B, BrdU incorporation assay of MC-9 cells transduced with either the empty Tween vector or the miR-221-, miR-222-, and miR-221–222-expressing vectors. Cells were cultured for 45 min in the presence of 10 µM BrdU before fixation and staining with an anti-BrdU-allophycocyanin Ab and 7AAD. C, BrdU incorporation assay of MC-9 cells transduced with either the empty Tween vector or the miR-221-, miR-222-, and miR-221–222-expressing vectors. Cells were cultured for 48 h in the absence of IL-3; IL-3 was then re-added for 14 h and 10 µM BrdU was added for the last 30 min of culture. Cells were then fixed and stained with an anti-BrdU-allophycocyanin Ab and 7AAD. Because of the high level of cell death (up to 50%) due to IL-3 withdrawal, dead cells were excluded from the analysis in these particular conditions.

 
To further examine the role of miR-221–222, we generated ES cells bearing BAC transgenes that encompassed the miR-221–222 locus. To do this, we engineered, by homologous recombination in bacteria, BAC clones containing almost 200 kb of genomic murine miR-221–222 locus (miR+), and as a control, the same vector containing all of the surrounding sequences except for 1.5 kb spanning pre-miR-221 and pre-miR-222 (miR–; schematic in supplemental Fig. 5A), and ES cells were transfected with the BAC transgenes and selected.

Notably, miR+ BAC-transgenic ES cells expressing miR-221–222 proliferated far less well than did control miR– BAC transgenic ES cells lacking the miRNAs. The impaired proliferation of miR+ transgenic ES cells was confirmed by cell counting and BrdU incorporation (supplemental Fig. 5, B and C).

Thus, the effect of miR-221–222 overexpression is to change the propensity of the cells to enter the cell cycle and to reduce proliferation, possibly by acting on proteins involved in different cell cycle checkpoints.

Splice variants of murine p27Kip1 mRNA differ in their 3' UTR

The cell cycle regulator p27Kip1 is an established target for miR-221–222 in humans (12, 15, 36). Accordingly, we could confirm that miR-221–222 directly regulated p27Kip1 expression in HeLa cells as expected, both in reporter assay and at the level of endogenous protein (data not shown). Next, we overexpressed miR-221–222 in MC-9 cells and analyzed p27Kip1 protein expression under resting and stimulated conditions (Fig. 9A). We found that p27Kip1 protein expression was induced substantially over the basal amount found in resting cells. Overexpression of miR-221–222 had essentially no effect on the basal levels of p27Kip1 expression; however, we observed a clear, albeit incomplete, inhibition of p27Kip1 protein expression in stimulated cells. Given the ability of miR-221–222 to dramatically reduce p27Kip1 expression in human cells from previous studies and our hands, we were surprised at the incomplete effect on p27Kip1 expression in mouse cells. To investigate this discrepancy further, we compared the human and mouse p27Kip1 splice variants as published in the UCSC Genome Browser and Ensembl (www.ensembl.org) databases. Unexpectedly, we found that all of the known human splice variants retained at least one putative miR-221–222 binding site. In striking contrast, we found that two major splice variants were predicted in the mouse, which shared identical 5' UTR and coding sequence, but possessed alternatively spliced 3' UTR (schematic in Fig. 9B). Of these, one splice variant ("+ miR" in Fig. 9) contained two putative miR-221–222 binding sites, while the second ("no miR" in Fig. 9) had no miR-221–222 potential binding sites that we could identify. These observations were confirmed by analysis of the p27Kip1 splice variants sequences for miR-221–222 target predictions using the RNA22 algorithm (cbcsrv.watson.ibm.com/rna22.html) (37).


Figure 9
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  		FIGURE 9. Murine mast cells express two splice variants of p27Kip1 mRNA that differ in the 3' UTR. A, MC-9 cells were transduced with either vector alone (Tween) or miRNA-expressing vectors, and were treated with PMA and ionomycin for 24 h or left resting. After lysis in Laemmli sample buffer, Western blot analysis was performed for either p27Kip1 or β-tubulin as loading control. B, Schematic representation of two murine p27Kip1 splice variants: the open boxes represent the 5' and 3' UTRs; the filled boxes represent the coding sequence and the two gray bars the miR-221–222 binding sites. The positions of the primers used in the RT-PCR in panels C and D are also indicated. C, RT-PCR analysis of the expression of the two p27Kip1 splice variants indicated in B. Total RNA was extracted from MC-9 cells untransduced (unt) or transduced with either the empty vector (Tween) or the miR-expressing vectors. After reverse transcription, PCR was performed with the primers indicated in the schematic in B. Actin mRNA was used as control. D, Untransduced MC-9 cells were either stimulated with PMA and ionomycin for 36 h or left resting; total RNA was extracted and RT-PCR was performed as in C.

 
We therefore analyzed expression of these two isoforms in mast cells by RT-PCR (Fig. 9C). Consistent with the negligible effect of miR-221–222 on basal p27Kip1 protein expression, the major splice variant expressed in unstimulated MC-9 cells appeared to be the one that lacked the miR-221–222 binding sites. However, in cells transduced with the miR-221–222 constructs the expression of p27Kip1 was increased, perhaps because cells in an altered cell cycle state accumulated in these cultures (see Figs. 6 and 7). Accordingly, we analyzed expression of the different p27Kip1 splice variants in cells that were sorted for the SSChigh and SSClow populations and found that the sorted SSClow cells expressed higher levels of the "+ miR" isoform compared with the SSChigh population, while the levels of "no miR" isoform were comparable in the two populations (not shown). This suggests that the increased levels of p27Kip1 mRNA observed when miR-221–222 were overexpressed are indeed due to the altered cell cycle. Both splice variants of the p27Kip1 mRNA were also expressed in primary BMMC and murine ES cells (data not shown). Most interestingly, the splice variant that possesses the miR-221–222 binding sites was primarily induced upon PMA and ionomycin stimulation of MC-9 cells (Fig. 9D). These data suggest that the miR-221–222-regulated p27Kip1 splice form that is induced by stimulation is responsible for the up-regulated p27Kip1 protein expression that was observed upon PMA and ionomycin treatment, and they explain why overexpression of miR-221–222 had a negligible effect on basal p27Kip1 expression, but was able to partially inhibit p27Kip1 protein expression after stimulation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods5
 Results
 Discussion
Disclosures
 References
 
Mast cells can function as effector cells during innate and adaptive immune responses through the direct or indirect action of a wide variety of mast cell-derived products (reviewed in Ref. 38). In vertebrates, mast cells are widely distributed throughout the vascularized tissues, in particular near surfaces that are exposed to the environment, including the skin, airways, and gastrointestinal tract, making them well positioned to be one of the first cell types of the immune system to interact with environmental Ags and allergens. Mast cells are long-lived cells that, similar to monocytes and macrophages, can reenter the cell cycle and proliferate following appropriate stimulation. Increased recruitment and/or retention, as well as the local maturation of mast cell progenitors, can also contribute to the expansion of mast cell populations in the tissue (38). Given their importance in participating in the immune responses, it is essential to understand the molecular basis of the development, proliferation, and functions of mast cells. Our results point toward a role for miR-221–222 in regulating cell cycle checkpoints in mast cells. Furthermore, we showed that miR-221–222 are transcriptionally inducible in mast cells, and we have defined some transcriptional requirements and mapped potential regulatory elements. A possible role for miR-221–222 in mast cell differentiation and maintenance of cell identity remains to be investigated, which will be the subject of further analysis.

Specifically, we identified putative mast cell-specific transcriptional regulatory regions through DNase I hypersensitivity analysis of the miR-221–222 locus; it will be interesting to perform a functional analysis of these regions to unveil the details of miR-221–222 transcriptional regulation in these cells. As for the transcription factors involved in miR-221–222 regulation, using bioinformatics and experimental approaches, we identified some important regulators of miR-221–222 transcription (e.g., the calcineurin and NF-{kappa}B pathways). Other transcription factors important for mast cell development and/or functions are PU.1, GATA-1, and GATA-2 (39, 40, 41, 42). Pu.1–/– cells are hematopoietic progenitors that upon reconstitution of PU.1 expression can be differentiated to macrophages, neutrophils, mast cells, or B lymphoid cells (reviewed in Ref. 43). To analyze the role of PU.1 in miR-221–222 regulation, we used Pu.1–/– cells (39) that were reconstituted with either GFP or PU.1 through retroviral transduction, but to date we could not identify a role for this transcription factor in miR-221–222 regulation of transcription. Along the same line, so far we could not uncover any effect on miR-221–222 that was dependent on GATA-1 and GATA-2. Conversely, through bioinformatics analysis we found that ZBTB16 binding sites are most enriched in the DNaseI HS regions upstream from the miR-221–222 sequences, compared with promoters of all mouse genes. ZBTB16 (PLZF) is a zinc finger transcription factor expressed at relatively high levels in CD34+ hematopoietic precursors in the bone marrow, as well as in immature erythroid, lymphoid, and myeloid cells (reviewed in Ref. 44). When cells are induced to differentiate, ZBTB16 levels generally decline, suggesting that down-regulation of ZBTB16 may be required for terminal cell differentiation. Interestingly, in a model of an IL-3-dependent myeloid cell line, ZBTB16 expression inhibited transit through the cell cycle, blocking cells in G0/G1, inhibited differentiation, and yielded cells with a more immature immunophenotypic profile (45). It has also been demonstrated that ZBTB16 exerts a transcriptional repression activity by binding to specific promoter sequences followed by the recruitment of histone deacetylases (46). While a role for ZBTB16 in mast cell differentiation has not been directly investigated so far, it has been shown that it physically interacts with GATA-1, and that it is important in megakaryocytic development (47). Most notably, ZBTB16 has been recently demonstrated to be a transcriptional repressor of miR-221–222 and miR-146a expression in melanoma and megakaryopoiesis, respectively (12, 13). These observation make ZBTB16 an attractive candidate for further studies aimed at elucidating the molecular circuitry underlying miR-221–222 expression and, more generally, mast cell differentiation and proliferation. We conclude that while the factors required for basal transcription remain to be identified, both the NFAT and NF-{kappa}B pathways (and possibly other transcription factors) are required for miR-221–222 induction of transcription.

From the functional point of view, overexpression of miR-221–222 in mast cells determined an alteration in the cell cycle and decreased proliferation. Surprisingly, we observed an effect on the cell cycle that was greatest when miR-221 and miR-222 were simultaneously expressed, while the effect of the individual miRNAs was reduced. More specifically, in some experiments the effect of miR-221 overexpression alone was marginal, if any. We think that this may be due to differences in the levels of expression (the T-221 vector expresses miR-221 at low levels) (Fig. 5A and data not shown), but it may also indicate that even though these miRNAs share the same seed sequence, they might have some redundant but also some nonoverlapping functions, with some mRNA targets being differentially regulated by miR-221 and miR-222. This hypothesis remains to be tested.

The cell cycle regulator p27Kip1 and the prosurvival surface receptor c-KIT are both established targets for miR-221–222 (12, 15, 36, 48). During human erythropoiesis and erythroleukemic cell growth, miR-221–222 are down-regulated, permitting c-KIT protein production and leading to an expansion of early erythroblasts (48). Conversely, high levels of miR-221–222 in human glioblastomas and melanoma correlate with low levels of p27Kip1 protein (12, 15, 36) and a higher proliferation rate. Most strikingly, miR-222 was shown to be down-modulated in endometrioid and clear cell ovarian carcinoma, as compared with normal tissues, but not in the serous type of the same carcinoma (49). These results confirm that miRNA activity can be very dependent on the cellular environment, and that miRNA-mediated control can display specificity in terms of functional restriction to a particular cellular contest or differentiation pathway (50). Remarkably, we found that in the mouse two different splice variants for p27Kip1 mRNA are expressed, only one of which has the potential to be regulated by miR-221–222. To the best of our knowledge, all of the work published so far aimed at demonstrating a role for miR-221–222 in regulating p27Kip1 levels was performed in the human system; murine p27Kip1 regulation might therefore be different. More specifically, we found that the splice variant that possesses the miR-221–222 binding sites was primarily induced in the same conditions of stimulation that also induced miR-221–222 expression, indicating a possible role for miR-221–222 in regulating p27Kip1 upon induction, and therefore allowing p27Kip1 levels to return to basal levels after cell stimulation. Interestingly, it has been recently shown that upon stimulation, proliferating CD4+ T lymphocytes express mRNAs with shortened 3' UTRs and fewer miRNA target sites, suggesting that UTR-based mRNA regulation plays distinct roles in the regulatory networks of nonproliferating or slowly proliferating cells as compared with actively proliferating cells (51).

Our hypothesis is that within a given set of transcription and regulatory factors, alteration of miR-221–222 levels can either favor or block cell proliferation, thus influencing the cell cycle. Even though miR-221–222 have a clear role in regulating the cell cycle, the mechanism of their effect can be very dependent on the cell type and on the relative abundance of miRNAs and their targets. Moreover, recent publications have shown that a single miRNA can dampen levels of hundreds of proteins by impeding their translation, and they have found that the algorithms for predicting miRNA targets varied in their predictive abilities (52, 53), suggesting that the combined effect on many different targets may be what determines the final phenotypical outcome of miRNA expression. Of note, it has also been shown that miRNA functions can oscillate between repression and activation in coordination with the cell cycle (54): in proliferating cells miRNAs can repress translation, whereas in G1/G0 arrest they mediate activation.

While miR-221–222 down-regulation may represent an important "hit" during the tumorigenic transformation of some cells, restoration of miR-221–222 levels of expression in fully transformed cells may be not sufficient to reduce the proliferation rate. Accordingly, we found that the mastocytoma cell line P815 expressed low levels of miR-221–222, which were not up-regulated upon cell stimulation. Transduction of P815 cells and overexpression of miR-221–222 were nevertheless unable to influence the cell cycle in these cells (data not shown). Although genetic abnormalities have been reported for some forms of mastocytosis, little is known concerning pathogenetic factors that contribute to the development of disease variants and disease progression (55). The description of new molecular mechanisms that might contribute to the pathogenesis of mastocytosis has the potential to form the basis of novel therapeutic approaches.

Overall our findings contribute to reveal an unanticipated versatility of miRNAs in response to the cellular environment, with important implications for our understanding of the role of miRNAs in complex processes such as cell development and carcinogenesis.


   Acknowledgments
 
We offer special thanks to M. Zavolan for help with the bioinformatics analysis, as well as for discussions and sharing ideas. A special thanks also to J. Nardone for the PipMaker analysis. We also thank D. Tennen, P. Lazlo, and H. Singh for the Pu.1–/– cell lines, R. De Maria and D. Bonci for the Tween vector, as well as D. Jarrossay and T. Pertel for technical help and discussions, and finally K. M. Ansel, A. Lanzavecchia, and F. Sallusto for discussions and critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods5
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a start-up grant from the Institute for Research in Biomedicine Foundation to S.M. and National Institutes of Health Grants AI44432 and AI070788 to A.R. M.E.P. is supported by a fellowship from the National Cancer Institute F32 CA126247-01. R.J.M. was temporarily supported by a fellowship from the Ceresio Foundation and is now a recipient of a San Raffaele "Vita e Salute" University predoctoral fellowship. Back

2 R.J.M. and M.E.P. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Silvia Monticelli, Institute for Research in Biomedicine, Via Vincenzo Vela 6, CH-6500, Bellinzona, Switzerland. E-mail address: silvia.monticelli{at}irb.unisi.ch Back

4 Abbreviations used in this paper: miRNA, microRNA; BMMC, bone marrow-derived mast cell; CHX, cycloheximide; CsA, cyclosporin A; ES, embryonic stem; HS, hypersensitivity; nt, nucleotide; pri-, primary; qRT-PCR, quantitative RT-PCR; 7AAD, 7-aminoactinomycin D; SCF, stem cell factor; SSC, side scatter; UTR, untranslated region; BAC, bacterial artificial chromosome. Back

5 The online version of the article contains Extended Methods. Back

6 The online version of this article contains supplemental material. Back

Received for publication June 23, 2008. Accepted for publication October 23, 2008.


   References
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 Abstract
 Introduction
 Materials and Methods5
 Results
 Discussion
 Disclosures
 References
 

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