The interplay between the master transcription
factor PU.1 and miR-424 regulates human
A. Rosa*, M. Ballarino*, A. Sorrentino†, O. Sthandier*, F. G. De Angelis*, M. Marchioni‡, B. Masella†, A. Guarini§,
A. Fatica*, C. Peschle†, and I. Bozzoni*‡¶
*Institute Pasteur Cenci-Bolognetti, Department of Genetics and Molecular Biology,‡Institute of Molecular Biology and Pathology, and§Department of
Cellular Biotechnologies and Hematology, University of Rome ‘‘La Sapienza’’, Piazzale Aldo Moro 5, 00185 Rome, Italy; and†Department of Hematology,
Oncology and Molecular Medicine, Istituto Superiore di Sanita `, 00161 Rome, Italy
Edited by Michael Rosbash, Brandeis University, Waltham, MA, and approved October 25, 2007 (received for review July 25, 2007)
We describe a pathway by which the master transcription factor
PU.1 regulates human monocyte/macrophage differentiation. This
includes miR-424 and the transcriptional factor NFI-A. We show
that PU.1 and these two components are interlinked in a finely
tuned temporal and regulatory circuitry: PU.1 activates the tran-
scription of miR-424, and this up-regulation is involved in stimu-
lating monocyte differentiation
translational repression of NFI-A. In turn, the decrease in NFI-A
levels is important for the activation of differentiation-specific
genes such as M-CSFr. In line with these data, both RNAi against
NFI-A and ectopic expression of miR-424 in precursor cells enhance
monocytic differentiation, whereas the ectopic expression of NFI-A
has an opposite effect. The interplay among these three compo-
nents was demonstrated in myeloid cell lines as well as in human
CD34? differentiation. These data point to the important role of
miR-424 and NFI-A in controlling the monocyte/macrophage dif-
hematopoietic differentiation ? microRNA ? NFI-A ? monocytopoiesis
interacting transcription factors (1). Numerous data indicate that
several such relevant factors are already expressed in the hemato-
poietic stem cell (HSC), although at a low level (2, 3), establishing
the so-called transcriptional priming that would reflect the devel-
opmental potency of the multilineage precursor.
Some of these factors, such as SCL/Tal1 and AML1, are multi-
potent, and their depletion affects the entire blood cell differenti-
ation. On the other hand, other factors have a lineage-specific
expression such as GATA1, C/EBP?, and PU.1. In the case of
myeloid differentiation, the relative levels of PU.1 and C/EBP?
choice (4, 5). Interestingly, these factors have functional intercon-
nections, because C/EBP? and GATA1 can inhibit PU.1 function
(4, 6), whereas increases in PU.1 lead to inhibition of GATA1 (7).
Interestingly, PU.1 not only promotes its own transcription (8) but
also prevents the activation of genes involved in alternative path-
ways (1, 9, 10). Different models involving either antagonistic
cross-regulation (1, 9) or cooperative interplay (10) between pri-
mary lineage-determining transcriptional factors have been pro-
posed for explaining how the initiation and maintenance of lineage
differentiation can be achieved.
In addition to the primary role of transcription factors, a family
recently shown to play a crucial role in hematopoietic lineage
expression of miR-181 in hematopoietic progenitor cells led to an
increased fraction of B-lymphoid cells in both tissue-culture differ-
entiation assays and adult mice (13). Similarly, miR-223 was shown
to increase upon granulocytic differentiation and to promote
maturation of promyelocytic precursors into granulocytes in hu-
ineage specification of hematopoietic multipotential progeni-
tors is a multistep process controlled by a complex system of
mans (14). In other studies, the down-regulation of miR-221 and
-222 was shown to be important for erythropoietic differentiation
of human cord blood CD34? progenitors (15), and the down-
regulation of miR17–5p, 20a, and 106a was required for monocy-
The targets of miRNA translational repression are often repre-
sented by transcription factors playing crucial roles in differentia-
tion. This is the case for MAFB and HOXA1, factors required for
terminal commitment of megakaryocytic cells, that were shown to
be the targets of miR-130 and -10, respectively (17). In some cases,
specific miRNAs and their transcriptional activators happen to be
in regulatory feedback loops in which they control each other (14,
16). These loops may represent regulatory mechanisms allowing
relatively small variations in miRNA concentration to induce
drastic changes in the cellular transcriptional patterns.
In the present work, we describe a pathway by which the
up-regulation of the lineage-specific factor PU.1 controls human
monocyte/macrophage differentiation. This includes the activation
of miR-424 that synergizes with PU.1 for the activation of terminal
differentiation genes through the repression of NFI-A.
miR-424 Up-Regulation Is Associated with Human Monocyte/Macro-
ferentiation of the human HL60 leukemia cell line (18). To study
the role of miR-424 in this differentiation program, we first
analyzed its levels in human cord blood CD34? cells (19) induced
to differentiate to the monocyte/macrophage-specific unilineage.
Fig. 1A shows an increase from day 6 to day 12 (reaching a 7-fold
accumulation with respect to progenitor cells), indicating that
miR-424 up-regulation occurs in normal cells upon induction to
monocyte/macrophage-specific differentiation. miR-424 expres-
sion was also tested in cells freshly taken from an Acute Promy-
elocytic Leukemia (APL) patient (FAB subtype-M3) induced to
differentiate in vitro to monocytes/macrophages. Fig. 1B indicates
that also in this case, miR-424 is up-regulated (4- to 5-fold) in
response to the differentiation stimulus.
The NB4 cell line, derived from the bone marrow of an APL
patient, is a genuine human promyelocytic cell line (FAB subtype
- M3; ref. 20) that has provided a powerful in vitro model system of
Author contributions: A.R. and M.B. contributed equally to this work; I.B. designed re-
search; A.R., M.B., A.S., O.S., F.G.D.A., M.M., and A.F. performed research; A.S., O.S.,
F.G.D.A., B.M., A.G., and C.P. contributed new reagents/analytic tools; A.R., M.B., A.F., C.P.,
and I.B. analyzed data; and I.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
¶To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
December 11, 2007 ?
vol. 104 ?
no. 50 ?
differentiation: whereas all-trans retinoic acid (ATRA) treatment
induces differentiation to morphologically and functionally mature
granulocytes, TPA induces a monocyte/macrophage phenotype
already after 48 h of treatment. Fig. 1C shows that also in TPA-
treated NB4 cells miR-424 is up-regulated, reaching a 5-fold
accumulation after 48–72 h (Fig. 1C). Similar up-regulation was
found in the HL60 cell line (supporting information (SI) Fig. 6),
derived from a patient with acute promyeloblastic leukemia (21)
and consisting predominantly of promyeloblasts (FAB subtype
-M2). No accumulation was detected in mature B and T lympho-
cytes (SI Fig. 6). In addition, no up-regulation was detected upon
RA treatment of NB4 cells (SI Fig. 6).
The role of miR-424 in monocyte/macrophage differentiation
was analyzed by its ectopic expression in promyelocytic cells.
Overexpression of miR-424 was accomplished by infecting NB4
cells with a lentiviral vector containing a cassette in which the
pri-miRNA is cloned under the U1snRNA regulatory regions
(Lenti-424, Fig. 1D). Forty-eight hours after infection, the miR-424
cells (Fig. 1D). The expression levels of two markers of monocytic/
macrophage differentiation were measured by flow cytometry:
CD11b is a surface marker of granulocytic and monocytic cell
differentiation, whereas CD14 specifically recognizes cells of the
monocytic lineage. Fig. 1E shows that CD11b-positive cells dou-
bled, whereas those positive for CD14 reached a 3-fold increase.
Notably, the ectopic expression of miR-424 also induced morpho-
logical changes in NB4 cells, such as a bluish-gray cytoplasm and a
cytes (Fig. 1F).
Altogether, these data indicate that the artificial increase of
miR-424 levels is able, in the absence of TPA, to induce differen-
tiation of promyelocytic precursors into the monocyte/macrophage
NFI-A Is a Bona Fide Target of miR-424. Among the hundreds of
predicted regulatory targets of miR-424 (22), we noticed the
transcription factor NFI-A, a protein previously shown to be
down-regulated by miR-223 during granulopoiesis (14). Interest-
ingly, a University of California, Santa Cruz (UCSC), genome
target site for miR-424 in the NFI-A 3?UTR is highly conserved
among mammals as well as miR-424 itself (miR-322 in mouse and
rat; SI Fig. 7). After TPA-induced monocytic differentiation, we
observed a strong decrease in the NFI-A levels (Fig. 2A) without a
concomitant decrease of the mRNA levels (Fig. 2B). A similar
TaqMan microRNA assays (Applied Biosystem) on total RNA from: (A) CD34?
cells induced to differentiate to the monocytic lineage (samples taken at the
is used as control. (C) Ten micrograms of RNA, from untreated NB4 cells (lanes
0) or from the same cells treated with TPA for the indicated times, was
analyzed by Northern blot with the probes indicated on the side of each
image. Endogenous spliceosomal U2 snRNA and miR-25 were used as loading
controls. (D Upper) Schematic representation of the lentiviral construct for
miR-424 expression (Lenti-424); (Lower) Northern blot analysis of 10 ?g of
total RNA extracted from NB4 cells infected with the empty vector (lane
Vector) or with Lenti-424 (lane Lenti-424) and incubated for 48 h. miR-424
as fractions with respect to mock-treated cells (Vector), are indicated below
each lane. (E) Percentage of CD11b- or CD14-positive cells in NB4 cells ectopi-
cally expressing either the empty vector or Lenti-424. (F) Morphological
analysis of NB4 cells 7 days after infection with the empty vector or with
Lenti-424. In all histograms, the values represent the means ? SEM from
Role of miR-424 in monocyte/macrophage differentiation. miRNA
the indicated hours in the absence (lanes C) or presence of TPA (lanes TPA),
were analyzed by Western blot with anti-NFI-A antibodies. Signals were
normalized for GAPDH and the values, expressed as fractions with respect to
time 0, are indicated below. (B) qRT-PCR analysis of NFI-A mRNA levels in NB4
cells treated with TPA for the indicated times. The histograms represent the
means ? SEM from triplicates. (C) Western blot analysis of proteins extracted
from cells infected with the empty vector of with the Lenti-424. Signals were
normalized for GAPDH, and the values, expressed as fractions with respect to
of the constructs used in the luciferase assay. The sequences shown below
indicate: the putative miR-424 target site on the wild-type 3?UTR (construct
3?NFI-Awt), its mutated derivative (construct 3?NFI-Amut), and the pairing
regions of miR-424. (E) Northern blot analysis of HeLa cells infected with the
empty vector (lane C) or with Lenti-424 (lane 424). (F) Cells were infected with
with either 3?NFI-Awt (black boxes) or 3?NFI-Amut (white boxes). Renilla
luciferase activity was normalized to Firefly luciferase activity, and then the
forward luciferase-containing constructs were normalized to the mutated
controls. Error bars represent the SEM from triplicates.
www.pnas.org?cgi?doi?10.1073?pnas.0706963104Rosa et al.
decrease in the protein levels was detected also when miR-424
was ectopically expressed in the absence of TPA (Fig. 2C). These
data suggested a functional link between miR-424 and NFI-A
of NFI-A was inserted downstream of a luciferase ORF (3?NFI-
Awt; Fig. 2D). A construct containing a mutated sequence of the
miRNA-binding site, 3?NFI-Amut, was also produced as a control.
The different luciferase constructs were cotransfected into HeLa
cells previously infected with Lenti-424 or with the control vector.
The efficient expression of miR-424 in these cells was analyzed by
Northern blot (Fig. 2E). The measurement of luciferase activity
indicated a specific repression by miR-424 on the wild-type sub-
decrease in luciferase activity was similar to the translational
23). Altogether, these data demonstrated that NFI-A is a target of
NFI-A Counteracts Monocytic Differentiation. RNAi against NFI-A
was performed to analyze its role in monocytic differentiation. The
infection of NB4 cells with a lentiviral construct expressing siRNAs
of the endogenous protein (Fig. 3A). In the presence of TPA, this
resulted in a strong increase of both CD11b and CD14 surface
antigens when compared with mock-treated cells (Fig. 3B). Inter-
estingly, in the same cells, the down-regulation of NFI-A resulted
gene M-CSFr (Fig. 3C), whereas no effect was observed for the
granulocyte-specific marker G-CSFr (data not shown). On the
contrary, the lentiviral-mediated overexpression of a NFI-A deriv-
ative with a 3?UTR devoid of miRNA target sites (Fig. 3D)
counteracted the TPA-induced monocytic differentiation, as eval-
uated by the reduced levels of the CD11b and CD14 antigens (Fig.
3 F and G), the M-CSFr mRNA expression levels (Fig. 3H) and
with LNA oligonucleotides against miR-424 resulted in increase of
NFI-A levels as well as in reduction of the differentiation markers
(SI Fig. 8).
These results show that NFI-A down-regulation is important for
monocytic differentiation, therefore indicating that one of the
pathway by which miR-424 promotes monocytopoiesis is through
PU.1 Is Responsible for miR-424 Activation. Two transcriptional start
sites for the pri-miR-424 were identified, by 5? RACE on RNA
nucleotides upstream of the premiRNA 5? end (Fig. 4A; F. Pagano,
personal communication). The region upstream to these sites was
analyzed with the ChIP Mapper software (24) for hematopoietic
transcription factor-binding sites, and one canonical PU.1-binding
genome browser analysis revealed that this site is embedded in a
(1) and, in NB4 cells (Fig. 4B) as well as in HL60 (SI Fig. 6D),
undergoes activation upon TPA treatment. The protein reaches a
maximum level at 24 h and decreases afterward. Interestingly, the
up-regulation of PU.1 precedes miR-424 activation (compare Figs.
1C and 4B and SI Fig. 6 B and D).
To investigate whether PU.1 physically interacts in vivo with the
miR-424 promoter, we performed a ChIP assay on uninduced and
TPA-treated cells. DNA from the PU.1 immunoprecipitates was
amplified with a couple of PCR primers (prom/1) located in the
promoter region surrounding the putative PU.1-binding site (Fig.
4A). Specific PU.1 interaction was detected after TPA induction
(Fig. 4C, miR-424). The specificity of PU.1 occupancy in the
miR-424 promoter was demonstrated by the absence of immuno-
precipitation with oligos corresponding to an unrelated genomic
region (Fig. 4C, UR). As a positive control, PU.1 immunoprecipi-
tation was tested on the high-affinity PU.1-binding site present in
the ?14-kb URE3? region of the PU.1 promoter (26). Fig. 4C
(URE3?) shows that the PU.1 factor interacts efficiently with this
region, and that its binding is induced by TPA, as observed for
miR-424. Moreover, EMSA performed with three oligos, corre-
sponding to regions prom/1, prom/2 (an unrelated miR-424 up-
region that corresponds to the one obtained with the positive
URE3? control (SI Fig. 10).
in vivo to the miR-424 promoter.
tion, we also analyzed the miRNA expression levels in cells where
PU.1 was knocked-down by RNAi. Fig. 4D shows that a 50%
reduction in the PU.1 levels were obtained in cells infected with a
In these cells, miR-424 does not undergo up-regulation after the
TPA-treatment (Fig. 4E, compare lanes TPA?).
Promoter fusion constructs were used to further correlate the
binding of PU.1 with miR-424 transcriptional activity. Two thou-
the empty lentivector (Vector) or with a lentiviral construct expressing siRNAs
?g analyzed by Western blot with anti-NFI-A and control anti-GAPDH anti-
bodies. Signals were normalized for GAPDH and the values, expressed as
lane. (B) CD11b- or CD14-positive cells were analyzed by FACS; the values
indicate the fold induction of TPA-induced positive vs. untreated cells. (C)
Expression levels of M-CSFr mRNA measured by qRT-PCR in NB4 cells infected
with the siNFI-A lentiviral construct or with an empty vector. The values
indicate the fold induction of TPA-induced cells vs. untreated ones. (D) Sche-
matic representation of Lenti-HA-NFIA and Western analysis with an anti-HA
antibody of its ectopic expression in NB4 cells. (E–H) NB4 cells were infected
with the empty lentiviral vector (Vector) or with the Lenti-HA-NFIA. After the
infection, half of the culture was treated with (?TPA) and half without TPA
(?TPA). Cells were analyzed by FACS for CD11b (F) and CD14 (G) expression,
by qRT-PCR for M-CSFr expression (H), and by Wright–Giemsa staining for
morphology (E). For each image, the histograms represent the means ? SEM
NFI-A knockdown and overexpression. NB4 cells were infected with
Rosa et al.
December 11, 2007 ?
vol. 104 ?
no. 50 ?
sand base pairs of the miR-424 upstream region, or a mutated
version deleted of the PU.1-binding site, were fused to the coding
After transfection in NB4 cells, half of the culture was treated with
TPA for 48 h, and half was maintained without the inducer for the
same time. Fig. 4F shows that, compared with the WT promoter,
the ?PU.1 construct produced half the levels of miR-126 transcrip-
that PU.1 interacts in vivo with the miR-424 promoter and is
necessary for at least 50% up-regulation of miR-424 upon TPA
Role of the PU.1/miR-424/NFI-A Circuitry in Normal Hematopoiesis.
during normal hematopoiesis, specifically in the monocytic/
macrophage unilineage differentiation of human cord blood
CD34? precursor cells. In this in vitro system, ?95% purified
human cord blood CD34? progenitors undergo a gradual, syn-
chronized, and selective differentiation maturation through the
monocyte/macrophage lineage (19). The cultures were monitored
along a 2-week period; differentiation was evaluated by morpho-
logical and immunophenotype analysis, particularly for the appear-
ance and rise of CD14?cells. The percentage of CD14?cells is
miRNA progressively accumulates during differentiation, parallel-
ing the CD14 marker (Fig. 5A Middle). Western analysis (Fig. 5A
Bottom) showed that the PU.1 protein is produced very early after
induction and is persistently expressed, perfectly paralleling miR-
424 accumulation. At variance with PU.1 and miR-424, the NFI-A
protein is already present in CD34?cells, and its levels strongly
This is in agreement with what was observed in the cell lines: in a
24-h interval after TPA treatment, whereas miR-424 doubles,
NFI-A undergoes almost a 3-fold reduction (compare 24 and 48 h
in Figs. 1 and 2).
To prove the physiological relevance of the circuitry described in
cell lines, CD34?progenitor cells induced to differentiate toward
the monocyte/macrophage lineage were infected with the lentiviral
constructs to knockdown PU.1 (siPU.1) or to overexpress either
miR-424 (424) or the miR-resistant form of NFI-A (HA-NFIA).
After GFP sorting, the differentiation status of the transduced cells
was assessed by cell growth, CD14 expression, and morphology.
When compared with cells infected with the empty vector, the
overexpression of miR-424 resulted in an increase of the differen-
tiation parameters (Fig. 5B). On the contrary, both the enforced
expression of the miR-resistant form of NFI-A or the knockdown
of PU.1 counteracted differentiation (Fig. 5C). To have a CD14-
independent evaluation of the effect on differentiation of siPU.1
and HA-NFI-A, we have quantified the morphological data, prov-
ing that the effect of both constructs on counteracting differenti-
ation is relevant and similar (50% reduction) in both cases (Right).
In conclusion, these experiments demonstrated that the altered
expression of PU.1, miR-424, and NFI-A has similar outcomes on
the monocyte/macrophage differentiation program of promyelo-
cytic cell lines as well as human CD34? progenitors.
In this study, we identified a PU.1-dependent regulatory pathway,
required to commit promyelocytic blasts to the monocyte/
macrophage lineage, which consists of the specific up-regulation of
miR-424. The PU.1 factor was shown to interact with the miR-424
promoter and to be responsible for at least 50% of its activation
the myeloid precursors was shown to promote monocytic differen-
tiation in the absence of the inducer. Therefore, miR-424 appears
to have a high hierarchical position among the factors regulating
this hematopoietic lineage.
Among the numerous putative targets of miR-424, the NFI-A
in TPA-treated promyelocytic cells enhanced monocytic differen-
tiation, whereas the ectopic expression of a miR-resistant form of
NFI-A or the LNA-mediated depletion of miR-424 produced the
opposite effect. These results indicated that NFI-A down-
regulation is indeed required for progression to monocytic differ-
entiation, similarly to what is shown in granulopoiesis (14). There-
differentiation in both myeloid lineages. Notably, miR-223 and
-424, both repressors of NFI-A, are activated by the master regu-
latory factors of the corresponding lineages, C/EBP? and PU.1,
miR-424 genomic region and of the constructs containing the wild-type (WT) or
mutant (?PU.1) miR-424 promoter fused to the premiR-126 coding region. The
transcriptional start sites (TSS) are indicated by open arrows. The sequence and
location of the PU.1-binding site are indicated by the dashed box, whereas the
protein was analyzed by Western blot with anti-PU.1 antibody. Signals were
0, are indicated below. (C) Chromatin from cells at different times of induction
submitted to qPCR with prom/1 oligos (miR-424). Oligonucleotides correspond-
ing to the ?14-kb URE3? region of the PU.1 promoter were used as positive
control (URE3?). An unrelated genomic region (UR) was amplified as a negative
experiments. (D) NB4 cells were infected with a lentiviral construct expressing
was analyzed by Western blot analysis with anti-PU.1. Signals were normalized
for GAPDH, and the values, expressed as fractions with respect to mock-treated
were normalized for U2 snRNA hybridization, and the values, expressed as
fractions with respect to TPA-minus samples, are indicated below. (F) NB4 cells
were normalized against the U6 snRNA. The histograms represent the means ?
SEM from triplicates.
PU.1 binds the miR-424 promoter. (A) Schematic representation of the
www.pnas.org?cgi?doi?10.1073?pnas.0706963104Rosa et al.
respectively. These conclusions were subsequently validated in a
CD34?hematopoietic progenitors (19). In unilineage monocyte/
macrophage culture, miR-424 activation paralleled the expression
profile of PU.1, whereas NFI-A displayed an inverse correlation
with respect to miR-424, similarly to what is observed in APL cell
circuitry identified in cell lines indeed regulates normal monocytic
In conclusion, our data show that miR-424 synergizes with PU.1
in inducing the gene expression pattern required for monocyte/
macrophage differentiation and in establishing strong transcrip-
tional commitments. In addition, they point to an important
function of NFI-A in the differentiation commitment of two
myeloid-specific pathways (granulocyte and monocyte/macro-
levels of this factor.
Reagents. TPA and RA were purchased from Sigma and used for
the indicated times after concentration (TPA, 1.6 nM and RA,
Cell Cultures. Primary cells were obtained at the time of diagnosis
from the bone marrow of a patient with acute promyelocytic
leukemia carrying the PML/RAR? fusion (FAB classification,
and NB4 and HL60 cell lines were cultured as described (14).
Monocytic (Mo) unilineage cultures were performed according
Constructs. The psiU-424 plasmid was generated by cloning a
fragment containing the premiR-424 (from ?120 to ?160 bp
relative to the 5?-end of mature miR-424) into the psiUx plasmid
(27). The constructs for RNAi were also raised in psiUx; they
transcribe a short hairpin that produces the accumulation of
siRNAs against NFI-A (14) or against PU.1 target sequence
(5?-GTCCGTATGTAAATCAGATCT-3?). The psiU-derived ex-
pression cassettes were subcloned into the 3?-LTR of the lentiviral
vector pRRLcPPT.hPGK.EGFP.WPRE, as described (14). The
miR-resistant form of NFI-A was obtained by RT-PCR amplifica-
5?-ATGTATTCTCCGCTCTGTCT-3? and reverse: 5?-TTATC-
CCAGGTACCAGGACT-3?. NFI-A was HA-tagged at the N
pCCL.sin.cPPT.PGK.mCMV.GFP.WPRE lentiviral vector (28).
Infective particles were produced according to ref. 29.
?2,000 to ?25 bp (WT) relative to the pre-miR-424 was cloned
a deletion corresponding to the PU.1 consensus site. Twenty
micrograms of each construct were transfected by electroporation
into NB4 cells. After 3 hours, cells were divided in two and one
aliquot treated with TPA for 48 h. Total RNA was analyzed by the
TaqMan MicroRNA Assay (Applied Biosystems).
miRNA Knockdown. Locked nucleic acid (LNA) oligonucleotides,
FITC-labeled by the manufacturer (Exiqon), were individually
transfected by Lipofectamine 2000 (Invitrogen) into NB4 cells as
RNA Analysis. Total RNA, extracted using TRIzol reagent (Invitro-
gen) according to the manufacturer’s instructions, was analyzed by
Northern blot as described (14). The following oligonucleotides
were used as probes: ?-223, 5?-GGGGTATTTGACAAACT-
GACA-3?; ?-424, 5?-TTCAAAACATGAATTGCTGCTG-3?;
NFI-A in normal hematopoiesis. (A)
tion of human CD34? cells. Samples
were collected at the indicated days
after induction. (Top) FACS analysis of
CD14?cells; (Middle) TaqMan mi-
croRNA assays of miR-424 (Applied
Biosystems). The values are normal-
represent standard errors from tripli-
cates. (Bottom) Western blot analysis
ies against the indicated proteins. (B)
CD34? cells, induced to differentiate
toward the monocyte/macrophage
lineage, were infected with Lenti-424
and sorted for the GFP marker carried
by the vector. Growth in hematopoi-
medium. (Left), CD14 expression (Cen-
7 (Right) are shown. (C) CD34? cells,
induced to differentiate toward the
monocyte/macrophage lineage, were
infected with Lenti-HA-NFIA (Upper)
or Lenti-siPU.1 (Lower) and sorted for
the GFP marker carried by the vector.
(Left) Growth in HPC M culture; (Cen-
ter) CD14? expression at day 10
phological analyses at day 10; repre-
sentative fields are shown together
all histograms, the values represent
the means ? SEM from three separate experiments.*, P ? 0.01 compared with control.
Role of PU.1, miR-424, and
Rosa et al.
December 11, 2007 ?
vol. 104 ?
no. 50 ?
U2 snRNA was detected with oligo U2R, 5?-GGGTGCACCGT-
Where indicated, miRNA quantification was performed by real-
time PCR in ABI PRISM 7900 Sequence Detection System (Ap-
plied Biosystems). Delta-delta Ct values were normalized with
those obtained from the amplification of the endogenous U6
snRNA. All reactions were performed in triplicate.
mRNA quantification was performed by real-time PCR with
iTaq SYBR green supermix (BioRad), according to the manufac-
turer’s instructions. The following oligonucleotides were used:
M-CSFr up, 5?-TCCAAAACACGGGGACCTATC-3? and down,
5?-TCCTCGAACACGACCACCT-3?. The NFI-A transcript was
detected with TaqMan oligonucleotides HS 00325656.m1 (Applied
Biosystems). Delta-delta Ct values were normalized with those
obtained from the amplification of the endogenous GAPDH
mRNA with Quantitect Primer Assay (Qiagen). All reactions were
performed in triplicate.
Immunoblot Analysis. Fifty micrograms of proteins were fraction-
ated by electrophoresis on 10% SDS polyacrylamide gel, electro-
blotted onto nitrocellulose membrane (Protran, S&S), and reacted
with anti-NFI-A (Abnova), anti-PU.1 (Cell Signaling Technology),
anti-GAPDH (Abcam), or anti-HA (Santa Cruz Biotechnology)
antibodies. Immunoreactivity was determined by using the ECL
method (Amersham) according to the manufacturer’s instructions.
Chromatin Immunoprecipitation Assay. DNA/protein cross-linking
was obtained by incubating the cells for 20 min at 37°C in 1%
formaldehyde. After sonication, chromatin was immunoprecipi-
tated overnight with 10 ?l of anti-PU.1 antibody (Cell Signaling
Real-time PCRs of genomic regions containing the putative
PU.1-binding site were performed in triplicate by using iTaq
SYBR green supermix (BioRad) with oligos prom/1a (5?-
TACATCGTGTGTTTGGGGTG-3?) and prom/1b (5?-ACGC-
CTCTTCCTCTGTTCATAC-3?). Control amplifications were
performed with oligos URE3?a (5?-TGGCTCTGGTCT-
CAACTCTG-3?) and URE3?b (5?-GCAGGAAAGAG-
GAAGGC-3?) and URa (5?-CCAGCTGATTGAGAATG-
CAGA-3?) and URb (5?-AAGGACACTAGGTGGTTGAGA-
3?). The relative occupancy of the immunoprecipitated factor at
a locus is estimated by using the comparative threshold method
(30): 2ˆ(Ctmock–Ctspecific), where Ctmock and Ctspecific are
from mock and specific immunoprecipitations.
EMSA for in Vitro DNA Binding. Nuclear extract from NB4 cells
treated with TPA, 1.6 nM, for 24 h was prepared as described (25).
The following oligonucleotides were annealed, labeled with
[?-P32]ATP by the use of Klenow enzyme, and incubated with 30
?g of NB4 extracts in 10 mM Hepes (pH 7.8), 50 mM KCl, 1 mM
at 0°C.: prom/1fw (5?-GATCATGAACAGAGGAAGAGGCG-
TATTC-3?), prom/1rev (5?-GATCGAATACGCCTCTTCCTCT-
GTTCAT-3?); prom/2fw (5?-GATCGCTTGAAACAGGAAG-
GAGCGACTT-3?), prom/2rev (5?-GATCAAGTCGCTCCTT-
CTGCCGATGTGGA-3?), URE3?rev (5?-GATCTCCACATCG-
GCAGCAGCAAGGCCGG-3?). Reaction mixtures were sepa-
rated with 6% polyacrylamide gels in 0.5? TBE buffer at 4°C.
Luciferase Assays. The pRL-TK-3?NFI plasmid (14) was used to
generate the 3?NFI-Amut plasmid containing a mutated site for
miR-424. Five hundred nanograms of pRL-TK derivative (Rr-luc)
and 50 ng of pGL3 control vector (Pp-luc; Promega) were cotrans-
vector. Cells were harvested 24 h posttransfection and assayed with
Dual Luciferase Assay (Promega) according to the manufacturer’s
Immunophenotyping and Morphological Analysis. Cell differentia-
tion was evaluated by immunofluorescence staining by using pri-
mary mouse anti-human CD11b and mouse anti-human CD14 cell
surface myeloid-specific antigens (eBiosciences) and a secondary
anti-mouse IgG R-phycoerythrin conjugate (Immunological Sci-
by a FACScan flow cytometer (Becton Dickinson) by using CellFit
software (BD Biosciences) for data acquisition and analysis. Mor-
phology was evaluated in conventional light-field microscopy of
Wright–Giemsa-stained cytospins at a magnification of ?400.
We thank Dr. M. Cavaldesi for FACS analysis and Prof. A. Santoni, Dr.
M. Cippitelli, and Dr. D. Milana (Department of Experimental Medi-
We also thank Dr. L. Naldini (University Vita Salute “San Raffaele,”
Milan) for providing lentiviral vectors, T. Incitti and M. L. De Marchis
for helping with plasmid construction, M. Arceci for technical assistance,
and A. Zito for graphics. This work was partially supported by grants
from Associazione Italiana per la Ricerca sul Cancro (AIRC) and
AIRC-ROC, Sixth Research Framework Program of the European
Union; Project RIGHT (Grant LSHB-CT-2004 005276), and SIROCCO
(Grant LSHG-CT-2006–037900); Ministero dell’Universita ´ e della
Ricerca Scientifica e Technologica (Grants FIRB-p.n. RBNE015MPB
and RBNE01KXC9); Progetti di Ricerca di Interesse Nazionale and
‘‘Centro di Eccellenza Biologia e Medicina Molecolare;’’ and the
Italy–US Oncology Program on MicroRNAs (C.P.).
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