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Prepublished online as a Blood First Edition Paper on October 24, 2002; DOI 10.1182/blood-2002-05-1546.
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Blood, 15 March 2003, Vol. 101, No. 6, pp. 2206-2214
HEMATOPOIESIS
Induction of C/EBP activity alters gene expression and
differentiation of human CD34+ cells
Jörg Cammenga,
James
C. Mulloy,
Francisco J. Berguido,
Donal MacGrogan,
Agnes Viale, and
Stephen D. Nimer
From the Laboratory of Molecular Aspects of
Hematopoiesis, Sloan-Kettering Institute, Genomics Core Laboratory and
Division of Hematologic Oncology, Memorial Sloan-Kettering Cancer
Center (MSKCC), New York, NY.
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Abstract |
The CCAAT/enhancer binding protein alpha (C/EBP ) belongs
to a family of transcription factors that are involved in the
differentiation process of numerous tissues, including the liver and
hematopoietic cells. C/EBP / mice show a block in
hematopoietic differentiation, with an accumulation of myeloblasts and
an absence of mature granulocytes, whereas expression of C/EBP in
leukemia cell lines leads to granulocytic differentiation. Recently,
dominant-negative mutations in the C/EBP gene and
down-regulation of C/EBP by AML1-ETO, an AML associated fusion
protein, have been identified in acute myelogenous leukemia (AML). To
better understand the role of C/EBP in the lineage commitment and
differentiation of hematopoietic progenitors, we transduced primary
human CD34+ cells with a retroviral construct that
expresses the C/EBP cDNA fused in-frame with the estrogen receptor
ligand-binding domain. Induction of C/EBP function in primary human
CD34+ cells, by the addition of  -estradiol, leads to
granulocytic differentiation and inhibits erythrocyte differentiation.
Using Affymetrix (Santa Clara, CA) oligonucleotide arrays we
have identified C/EBP target genes in primary human
hematopoietic cells, including granulocyte-specific genes that are
involved in hematopoietic differentiation and inhibitor of
differentiation 1 (Id1), a transcriptional repressor known to interfere with erythrocyte differentiation. Given
the known differences in murine and human promoter regulatory sequences, this inducible system allows the identification of transcription factor target genes in a physiologic, human hematopoietic progenitor cell background.
(Blood. 2003;101:2206-2214)
© 2003 by The American Society of Hematology.
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Introduction |
Pluripotent hematopoietic stem cells have the
potential for self-renewal and for differentiating into mature blood
cells of all lineages.1,2 This is a highly regulated
process and data from genetically modified mice suggest that
transcription factors play an important role in the differentiation
toward specific lineages, by inducing the expression of
lineage-determining genes.3 The transcription factors
C/EBP and PU.1 play a crucial role in hematopoietic differentiation;
C/EBP/ mice lack mature granulocytes and show an
accumulation of myeloblasts in the blood and bone marrow.4
Mice lacking PU.1 show defects in the generation of lymphocytes,
granulocytes, and monocytes, while differentiation toward the
erythrocytic or megakaryocytic lineages is not
affected.5-7
The CCAAT/enhancer binding protein alpha (C/EBP) belongs to
the leucine zipper family of transcription factors, which bind DNA as
homodimers. Expression of C/EBP in a myeloid cell line and in
multipotential avian progenitor cells leads to granulocytic differentiation.8,9 Expression of C/EBP follows the
expression of C/EBP and C/EBP in adipocytes, and plays an
important role in adipocyte differentiation. Similarly, the specific
role of C/EBP in myeloid lineage commitment and in myeloid
differentiation likely reflects the lack of redundancy in some of the
functions of the C/EBP proteins. C/EBP appears to play an important
role in the pathogenesis of human acute myelogenous leukemia (AML): mutations in the C/EBP gene have been identified in AML
of the M2 French-American-British classification (FAB) subtype
that lack the t(8;21),10,11 whereas expression of the
t(8;21)-associated AML1-ETO fusion protein leads to the down-regulation
of C/EBP.12,13 Several of the identified mutations in
the C/EBP gene lead to the expression of a 30-kDa protein
that lacks the amino-terminal transactivation domain. These mutations
are generally monoallelic and the fact that the remaining copy of
C/EBP is unaffected argues for a dominant-negative
function of these mutations.10,11 C/EBP can affect
transcription in a pleiomorphic way. For instance, wild-type C/EBP
can down-regulate c-myc by binding to E2F,14,15 however a
mutant C/EBP that retains its transactivation properties but cannot
bind to E2F does not promote granulocytic
differentiation.16 Thus, the role of C/EBP in
differentiation and growth arrest may be linked yet mechanistically
distinct (for review see McKnight17).
To evaluate the effects of C/EBP on hematopoietic differentiation
and lineage commitment, we transduced primary human hematopoietic cells
with a bicistronic retroviral vector that expresses C/EBP fused to
the ligand-binding domain of the estrogen receptor (C/EBP-ER) and
also expresses green fluorescence protein (GFP). Induction of C/EBP
activity in human CD34+ cells by -estradiol (-ES)
blocks erythrocyte differentiation (as shown by decreased burst-forming
unit erythroid [BFU-E] formation) and promotes granulocytic
differentiation. We have used this novel system to identify C/EBP
target genes using Affymetrix (Santa Clara, CA) oligonucleotide arrays,
comparing the RNA expression profile of primary human hematopoietic
cells transduced with the empty MIGR1 retroviral vector, with cells
transduced with the MIGR1 C/EBP-ER expression vector. There
were 2 different approaches used to identify "direct" target genes
of C/EBP: a 2-hour time point for RNA collection, to minimize the
possibility of detecting secondarily affected target genes, and an
analysis of the transcriptional profile in the absence of protein
synthesis, which was accomplished by adding cycloheximide (CHX;
since activation of the C/EBP-ER fusion protein is not dependent on
protein synthesis). This approach takes advantage of several powerful
techniques to identify target genes of lineage-specifying transcription
factors, such as C/EBP, and link the expression profile to effects
on the behavior of primary human hematopoietic progenitor cells.
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Materials and methods |
Cell culture
RD18 cells (kindly provided by Dr M. Collins, Imperial College
London, United Kingdom) and 293T cells were grown in Dulbecco modified essential medium with 10% heat-inactivated fetal calf serum
(FCS), 100 U/mL penicillin/streptomycin, and 2 mM
L-glutamine. Charcoal-stripped fetal calf serum (Gemini
Bioproducts, Woodland, CA) and medium without phenol red were used to
eliminate estrogen derivates that would lead to activation of the
C/EBP-ER fusion protein.
Retroviral production by transient transfection or using the
producer cell line RD18
The rat C/EBP-ER cDNA (kindly provided by Dr A. Friedman,
Johns Hopkins University) was cloned into the EcoR1
restriction site of the MIGR1 vector (kindly provided by Dr W. Pear,
University of Pennsylvania). The MIGR1 retrovirus contains the murine
stem cell virus (MSCV) promoter and an internal ribosomal entry site (IRES) element followed by the GFP gene (Figure
1). Retrovirus was made by transient
cotransfection of the MIGR1, pEQ-PAM3(-E) (kindly provided by
Dr E. Vanin, St Jude's Children's Hospital) and pSVampho plasmids
into 293T cells. Retroviral supernatant was collected after 36 and 48 hours. The supernatant was filtered through a 0.45-µm filter to avoid
contamination of the hematopoietic cells with 293T cells.
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| Figure 1.
Expression of C/EBP-ER in primary human
CD34+ cells.
(A) The retroviral vector MIGR1 is shown schematically. The rat
C/EBP cDNA fused in-frame with the ligand-binding domain of the
estrogen receptor (C/EBP-ER) was cloned into the EcoRI
restriction site of MIGR1. (B) Western blot analysis of human
CD34+ cells transduced with MIGR1 or MIGR1 C/EBP-ER
using an -C/EBP antibody (lanes 1-2). Expression of C/EBP-ER
in the producer cell line RD18 was used as a positive control (lane 3).
(Note that the 45-kDa band shown in lanes 2 and 3 is not present in
lane 1 and likely represents a degradation product of the ER fusion
protein.) (C) The experimental design to evaluate the effects of
C/EBP on human hematopoietic progenitor cell behavior and to
identify C/EBP target genes is shown schematically (IFA indicates
immunofluorescence assay).
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Retroviral producer cell lines were generated for the MIGR1 and MIGR1
C/EBP-ER vectors by transducing the RD18 cells with transiently made
retrovirus and selecting clonal green RD18 cells. The multiplicity of
infection (MOI) of the obtained clones was tested by
transduction of U937 cells. Retroviral supernatant from selected RD18
producer cell lines was centrifuged at 27 000 rpm for 90 minutes to concentrate the retrovirus. The retroviral pellet was
resuspended in 0.5 mL Iscoves modified Dulbecco medium (IMDM) with 20%
charcoal-stripped FCS, 100 U/mL penicillin/streptomycin, and 2 mM
L-glutamine and either used immediately or frozen in aliquots at  70°C.
Transduction of human hematopoietic progenitors
Human CD34+ hematopoietic cells were selected using
a magnetic CD34 selection kit system (Miltenyi Biotec, Auburn, CA) from small aliquots of leukapheresis products collected from one healthy donor and one patient undergoing stem/progenitor cell collection after
granulocyte-colony stimulating factor (G-CSF) treatment for the
treatment of a nonhematologic malignancy at Memorial Hospital, following their informed consent. After magnetic selection, more than
95% of the cells expressed the CD34 antigen. An aliquot
containing 5 × 106 CD34+ cells was cultured
for 72 hours in IMDM with 20% heat-inactivated FCS, 100 ng/mL
Flt3-ligand (Flt3-L) and 20 ng/mL granulocytic-macrophage colony
stimulating factor (GM-CSF) (both kindly provided by Immunex, Seattle,
WA); 100 ng/mL of stem cell factor (SCF; kindly provided by
Amgen, Thousand Oaks, CA); 100 ng/mL thrombopoietin (TPO); 100 ng/mL of
interleukin-6 (IL-6) and 50 ng/mL of IL-3 (generous gifts from Kirin
Brewery, Gumma, Japan); 100 U/mL penicillin/streptomycin; and 2 mM
L-glutamine. The stimulated cells were transduced
with retroviral supernatant using the same concentrations of cytokines and 4 µg/mL polybrene. Spinoculation (4 rounds) was performed at 1800 rpm for 45 minutes at room temperature on retronectin-coated plates (TaKaRa Shuzo, Shiga, Japan). The CD34+ cells were
removed from the retronectin-coated plates using 0.02% EDTA
(ethylenediaminetetraacetic acid) in phosphate-buffered saline (PBS)
and expanded in the cytokine mix described above for 2 additional days.
In general, beginning with approximately
5 × 106 cells, we would have approximately
4 × 107 cells (before sorting) for further
analyses (including Western blot, microarray analysis, and in vitro
culture studies). After 48 hours the cells were sorted for GFP
expression and expanded for 2 or 8 hours in IMDM with the addition of
the described human cytokines and 1 µM -estradiol or an
equal volume of ethanol as a control.
Clonogenic assay
The indicated number of hematopoietic cells were resuspended in
0.9% methylcellulose containing 1% bovine serum albumin, 10 µg/mL
bovine pancreatic insulin, 200 µg/mL human transferrin (iron saturated), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin/streptomycin (MethoCultSFBIT; Stem
Cell Technologies, Vancouver, Canada) and 20 ng/mL human IL-6, 20 ng/mL
human IL-3, 20 ng/mL human SCF, 10 ng/mL G-CSF (Amgen) and 6 U/mL human
erythropoietin (EPO) (Amgen). Colonies were evaluated and counted after
12 to 14 days in culture. Cells were collected from the clonogenic
assays by washing with PBS. Cytospins were performed using
8 × 104 cells for immunofluorescence staining or
morphologic analysis after Wright-Giemsa staining.
Flow cytometry
Human hematopoietic progenitor cells transduced with MIGR1 or
MIGR1 C/EBP-ER were washed with PBS, resuspended in 2% FCS/PBS and
stained with CD71-phycoerythrin (PE) antibody (BD Biosciences PharMingen, San Diego, CA) for 45 minutes at 4°C. Stained cells were
sorted for expression of GFP and CD71 (< 102 mean
fluorescence intensity) using a MoFlo (Cytomation, Fort Collins, CO) or
Vantage cell sorter (Becton Dickinson, Clearwater, FL). Cells
collected from the clonogenic assays were washed with PBS, resuspended
in 2% FBS in PBS, and stained with anti-CD11b, CD14, or glycophorin A
PE-conjugated antibodies (BD Biosciences PharMingen). Idiotype controls
were used accordingly. Data were analyzed using the software CellQuest
3.1 (Clearwater, FL).
Immunofluorescence staining and histochemical analysis
Cells were fixed with 2% paraformaldehyde in PBS for 10 minutes
and washed twice with PBS. Fixed cells were incubated with 0.1%
saponin (Sigma Chemical, St Louis, MO) in PBS for 1 hour at room
temperature and washed twice with PBS. Incubation with a 1:150 dilution
of the anti-inhibitor of differentiation 1 (Id1) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in
PBS with 10% FCS was performed for one hour in the dark at room
temperature. The cells were washed 4 times with 0.02% Tween20 (Sigma
Chemical) in PBS. Cells were incubated with an antirabbit antibody
conjugated to Cy3 (Jackson ImmunoResearch Laboratories) for 1 hour at
room temperature and counterstained with DAPI (4,6 diamidino-2-phenylindole), washed once with water, and mounted in
SlowFade (Molecular Probes, Eugene, Oregon). Cells were analyzed using
a fluorescence microscope (Olympus, Melville, NY).
Preparation of labeled cRNA and oligonucleotide array
Total RNA was extracted from cells, which were approximately
50% CD34+ at the end of the retroviral transduction
period, using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the
manufacturer's instruction. Total RNA (5-10 µg) was reverse
transcribed with a cDNA synthesis kit (Gibco BRL/Life Technologies,
Rockville, MD) in the presence of an oligo dT-T7 primer. After
phenol/chloroform extraction and ethanol precipitation, the cDNA pellet
was air dried and resuspended in 12 µL of Rnase-free H2O.
Ten microliters was used for the in vitro transcription
amplification reaction, in the presence of biotinylated nucleotides
(Enzo Diagnostics, Farmingdale, NY). Labeled cRNA (15 µg) was
fragmented by incubation at 95°C for 35 minutes in fragmentation
buffer (40 mM Tris-acetate, pH 8.1, 100 mM KOAc, and 30 mM MgOAc) and
the fragmented cRNA was then hybridized against the Affymetrix
HG_U95Av2 oligonucleotide arrays. The arrays were scanned using a
Hewlett Packard confocal laser scanner and analyzed using MicroArray
Suite 5.0 (Affymetrix), GeneSpring 4.0 (Silicon Genetics, Redwood City,
CA), and in-house analytical tools.
The default parameters were used for the statistical algorithm and for
probe set scaling using Microarray Suite 5.0 (with a target intensity
of 500). The data were then filtered so that the absolute value of the
fold change was more than 1.5 and the "fold change" P
value was less than .005. Additionally, we removed genes that were
scored as absent in experimental and baseline files (both
numerator and denominator of the fold change), as well as genes scored
as increasing but absent in the experimental file (numerator of the
fold change). We chose a conservative procedure for combining the
duplicate data (intersecting the filtered list for each replicate based
on a list of genes that passed the filtering criteria for both replicates).
Real time reverse transcriptase-polymerase chain reaction
(RT-PCR) analysis
To quantify the expression of the Id1, c-myc, and calgranulin
mRNAs, PCR amplification was carried out using the 7700 Sequence detector (PE Applied Biosystems, Norwalk, CT) and the PCR products detected using SYBR Green I chemistry. Each cDNA was
quantified relative to glyceraldehyde phosphate dehydrogenase
(GAPDH), which served as the reference gene transcript. The
following primer sequences were used: Id1, forward primer
5'-GGACGAGCAGCAGGTAAACG-3' and reverse primer
5'-TGCTCACCTTGCGGTTCTG-3'; c-myc, forward primer 5'-GCTGCTTAGACGCTGGATTTTT-3' and reverse primer
5'-TCGAGGTCATAGTTCCTGTTGGT-3'; calgranulin, forward primer
5'-TTCCATGCCGTCTACAGGGA-3' and reverse primer
5'-TCCAACTCTTTGAACCAGACGTC-3'; GAPDH, forward primer
5'-ATTGGGCGCCTGGTCAC-3' and reverse primer
5'-AAGATGTAAACCATGTAGTTGAGGTCA-3'. All primers were designed using
PrimerExpress 1.0 software (PE Applied Biosystems), and the
default TaqMan parameters. Primer sets spanning intron-exon junctions
were chosen to prevent amplification of any possible residual,
contaminating genomic DNA in the cDNA sample. The reaction mixture
consisted of 1X SYBR Green PCR master mix (PE Applied Biosystems), 0.1 µL of each primer (100 ng/µL) and oligo-dT-generated cDNA
in a final volume of 25 µL. Amplification conditions were as follows:
50°C for 2 minutes, 95°C for 10 minutes, then 40 cycles at
95°C for 15 seconds and 60°C for 1 minute. A negative control (lacking the cDNA template) was included in every assay and the size of
the PCR products was confirmed by agarose gel electrophoresis. The
comparative threshold cycle (CT) method was used for
analysis after a validation experiment was performed for all
primer sets.
Western blot analysis
Cells were washed with PBS and either frozen as a pellet or
lysed directly with protein lysis buffer (1% Triton X-100, 5 mM EDTA,
50 mM HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5, 50 mM NaCl, 10 mM Na Pyrophosphate, and 50 mM
NaF) and protease inhibitors (Roche, Indianapolis, IN). Cell lysates were sonicated and centrifugated for 10 minutes. Protein concentrations were measured using the Bradford method (Bio-Rad Laboratories, Hercules, CA). The indicated amounts of protein were mixed with an
equal volume of 2 × sodium dodecyl sulfate (SDS) loading buffer and
boiled for 10 minutes. Proteins were electrophoretically separated on a
8% to 16% SDS polyacrylamide gel electrophoresis (PAGE) gel (Bio-Rad Laboratories) and blotted on a polyvinylidene fluoride (PVDF)
membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The
membranes were blocked with 5% nonfat dry milk in Tris-buffered saline
(TBS) with 0.1% Tween20, and incubated first with the anti-C/EBP primary antibody (Santa Cruz Biotechnology) at a 1:200 dilution overnight at 4°C, washed 4 times for 15 minutes with 0.1% Tween20 in
TBS followed by the secondary antibody (conjugated to horseradish peroxidase) for one hour at room temperature. After being washed, the
blot was developed using the enhanced chemiluminescence (ECL) Western blotting detection reagent (Amersham Pharmacia Biotech) and
exposed to film (Amersham Pharmacia Biotech).
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Results |
C/EBP blocks erythrocyte differentiation and enhances neutrophil
maturation of human hematopoietic progenitors
To define the effects of C/EBP on the differentiation and
lineage commitment of primary hematopoietic stem/progenitor cells, we
expressed the rat C/EBP-ER fusion cDNA (which has cross-species activity and is capable of differentiating a human leukemia cell line13) in human CD34+ cells by retroviral
gene transfer (Figure 1A).
Transduced cells were sorted for GFP expression and the levels of
C/EBP-ER and the endogenous C/EBP protein were evaluated by
Western blot analysis, using an anti-C/EBP antibody. Expression of
the 72-kDa C/EBP-ER protein was readily detectable, whereas the
42-kDa wild-type C/EBP protein was not detectable in these immature
hematopoietic cells (Figure 1B). The C/EBP-ER fusion protein
translocates from the cytoplasm to the nucleus in the presence of
-estradiol, which leads to its activation (Pabst et
al13 and data not shown). Hematopoietic progenitors
transduced with MIGR1 C/EBP-ER (or MIGR1 as a control) were sorted
for GFP expression and plated into clonogenic assays with or without
-estradiol (shown schematically in Figure 1C). The addition of
-estradiol to the C/EBP-ER-transduced cells led to significantly
reduced numbers of colony-forming unit erythrocyte (CFU-E) and BFU-E
(Figure 2A), demonstrating a strong
inhibitory effect of C/EBP on erythrocyte differentiation.
The decrease in colony-forming unit granulocyte, erythrocyte,
megakaryocyte and monocyte (CFU-GEMMs), coupled with a minimal increase
in colony-forming unit granulocyte macrophage (CFU-GMs), may reflect
inhibition of the erythroid cells in the CFU-GEMM leading to the
misidentification of CFU-GEMM as CFU-GM (or it could represent a
stimulatory effect of C/EBP on the granulocytic lineage).
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| Figure 2.
C/EBP inhibits erythrocyte differentiation and leads to granulocytic
differentiation.
(A) Hematopoietic cells transduced with MIGR1 or MIGR1 C/EBP-ER were
sorted for expression of GFP and plated in clonogenic assays in
triplicate with or without -estradiol. Colonies were scored and
counted 12 to 14 days later. One of 2 representative experiments is
shown. (B) Transduced hematopoietic cells were sorted for GFP and CD71
expression higher than 102 mean fluorescence intensity
(CD71bright) and plated in clonogenic assays in duplicate.
Shown is 1 of 3 independent experiments. (C) Cells were
collected from the clonogenic assays (see Figure 2B) and analyzed for
surface marker expression (CD11b, CD14, and glycophorin A) by flow
cytometry. Cytospins were performed and stained with Wright-Giemsa
(last panels on the right; original magnification, × 600).
Cells transduced with MIGR1 and treated with ethanol or -estradiol
showed the same phenotype as cells transduced with C/EBP-ER and
treated with ethanol (data not shown).
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To evaluate whether C/EBP expression could alter the phenotype of
cells already displaying erythroid characteristics we sorted the
transduced hematopoietic cells for expression of GFP and for high
expression of the CD71 antigen (CD71bright) and plated
these cells into clonogenic assays with or without -estradiol. The
CD71 antigen (transferrin receptor) is an activation marker and is also
highly expressed on BFU-E cells.18 Most of the
CD71bright cells transduced with control vector (with or
without -estradiol) formed erythrocyte colonies (BFU-E/CFU-E) or
mixed colonies (CFU-GEMM), while cells expressing active C/EBP
protein showed a dramatic reduction of erythrocyte colonies, as well as
a significant reduction of CFU-GEMM and increased numbers of CFU-GM
(Figure 2B). No such effect was seen in the absence of -estradiol.
The CFU-GM colonies expressing the active C/EBP protein were
smaller, yet less compact, which implies an accelerated differentiation
and earlier acquisition of mobility (data not shown).
The GFP+/CD71bright cells from the clonogenic
assays were collected, stained for lineage-specific cell surface
markers, and analyzed by flow cytometry. The progenitor cells
transduced with the control vector or those that contained the inactive
form of C/EBP showed high-level expression of the erythrocyte
surface marker glycophorin A, while cells that contained the active
C/EBP protein predominantly expressed the differentiation marker
CD11b, but not CD14 (Figure 2C). Cytospins revealed that the cells
expressing C/EBP-ER protein in the presence of -estradiol were
predominantly granulocytes, while the cells plated in ethanol showed
predominantly an erythroblastic morphology. Cells expressing only GFP,
in the presence or absence of -estradiol, had a phenotype
indistinguishable from cells expressing C/EBP-ER in the absence of
-estradiol (data not shown).
Identification of C/EBP target genes in primary human
hematopoietic cells
To identify C/EBP target genes involved in hematopoietic
differentiation and commitment we transduced hematopoietic progenitor cells with control or C/EBP-ER-expressing retrovirus, sorted the
cells for GFP expression, and grew them in the presence of -estradiol for 8 hours. RNA was prepared and the transcriptional profiles of the GFP+ cells were analyzed using Affymetrix
HU95A oligonucleotide arrays (which contain probe sets that can analyze
the level of expression of 12 000 transcripts; see the
www.affymetrix.com website for more information). Genes whose level of
expression changed at least 1.5-fold 8 hours after the addition of
-estradiol, in each of 3 independent experiments, were considered
up- or down-regulated by C/EBP. Although the probes on the
oligonucleotide arrays generally hybridize to the highly specific 3'
untranslated region of each gene, the probe for estrogen
receptor expression hybridizes to sequences in the coding
region. Because we used a C/EBP-ER fusion protein, estrogen receptor
expression was up-regulated in all experiments, serving as an
(unexpected) internal control, but not as a target gene of C/EBP. A
list of C/EBP-regulated genes is shown in Table
1.
Identification of direct C/EBP target genes
An advantage of using the C/EBP-ER construct is that
activation of C/EBP function occurs independently of protein
translation because the fusion protein is present but is inactive in
the absence of ligand, because of its cytoplasmic location. The
C/EBP-ER protein translocates to the nucleus and becomes functional
upon the addition of -estradiol.13 Therefore, we used
cycloheximide (CHX), which inhibits protein synthesis, to
confirm our identification of "direct" C/EBP target genes.
Transduced, GFP-sorted hematopoietic cells were treated with 5 µg/mL
CHX 30 minutes before exposure to -estradiol and the transcriptional
profile was analyzed as described in the preceding paragraph.
It is well known that CHX can alter RNA levels for some genes by
affecting proteins that control RNA stability (Kannan et
al19). Therefore, as an alternative approach to confirming
the identity of direct C/EBP target genes, we prepared RNA after a
brief (2 hour) exposure to -estradiol alone; these potential direct
target genes are shown in Table 2. Genes
identified as "direct" targets of C/EBP based on the triplicate
experiments (following an 8-hour treatment with -estradiol) that
were also found after either a short exposure to -estradiol or to
CHX are shown in the seventh and eighth columns of Table 1.
Identification of C/EBP target genes in erythroid
progenitor cells
The most dramatic effect of C/EBP expression on
hematopoietic colony growth was the inhibition of BFU-E and CFU-E
formation (Figure 2). Therefore, to identify genes involved in the
inhibition of erythrocyte differentiation by C/EBP we transduced
human CD34+ cells with MIGR1 or MIGR1 C/EBP-ER, sorted
for GFP+/CD71bright-expressing cells, and
incubated the cells with -estradiol for 8 hours before preparing RNA
for transcriptional profiling. A list of genes up- or down-regulated is
shown in Table 3. C/EBP target genes
identified in these CD71bright cells, which were also
identified as "direct" target genes in Table 2, are listed in the
sixth and seventh columns of Table 3.
Target genes commonly regulated by C/EBP
The transcriptional repressor Id1 and adipophilin
were up-regulated after the expression of C/EBP in all experiments,
although a higher fold induction was seen in the erythroid precursors. Both genes were also identified as "direct" C/EBP target genes according to the experiments using either CHX (to inhibit translation) or the early time point (Tables 1 and 3). No genes were significantly and consistently down-regulated by C/EBP in both the multipotent and
the erythroid progenitors. Given the described impairment in IL-6R and
G-CSFR expression in C/EBP/ mice, we checked whether
changes in the expression of these genes were also found in our
experiments: G-CSFR expression was low and remained low despite
induction of C/EBP activity. Unfortunately, probe sets for detecting
IL-6R mRNA are not present on the U95A chip.
To validate the C/EBP target genes identified by the oligonucleotide
arrays, we also analyzed changes in gene expression using real-time
RT-PCR to quantify the levels of Id1, calgranulin B, and c-myc mRNAs.
Using RNA from 2 of the 3 triplicate experiments described in Table
1, we performed the real time PCR assays in triplicate.
Similar levels of Id1 and calgranulin up-regulation were seen comparing
real-time PCR with the microarray data (6.7- and 4.6-fold for each
gene); down-regulation of c-myc was also seen in both of these experiments.
Id1 protein is up-regulated in primary hematopoietic cells
after C/EBP activation
Id1 mRNA was up-regulated after the expression of C/EBP in all
experiments, and especially in the erythroid precursors. To evaluate
whether the level of Id1 protein correlated with Id1 mRNA, we performed
immunofluorescence staining on cytospins obtained from GFP-sorted cells
after 16 hours of -estradiol treatment. Cells expressing the active
form of C/EBP showed an increase in Id1 protein, with a nuclear
localization pattern (Figure 3A). Western
blot analysis showed high-level expression of Id1 protein in cells
expressing the active C/EBP protein, compared with the low Id1
expression seen in cells transduced with the empty MIGR1 vector or in
cells expressing the inactive form of C/EBP (Figure 3B).
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| Figure 3.
Id1 protein levels are increased after expression of
active C/EBP.
(A) Human CD34+ cells transduced with MIGR1 or MIGR1
C/EBP-ER, were sorted for the expression of GFP and treated with
-estradiol (+) or ethanol () for 16 hours. Immunofluorescence was
performed using an -Id1 antibody and counter stained with DAPI to
identify the nucleus (original magnification, × 600). (B)
Cells sorted for GFP expression and treated for 16 hours with
-estradiol or ethanol were analyzed for expression of Id1 protein by
Western blot analysis using an -Id1 antibody. The control lane
contains cell lysate from 3T3 cells. The level of -tubulin
expression is indicated, to control for equal protein
loading.
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Discussion |
C/EBP/ mice lack mature granulocytes and show
an accumulation of myeloblasts, providing strong evidence that this
transcription factor is involved in hematopoietic
differentiation.4 C/EBP is specifically up-regulated
during granulocytic differentiation,20 and overexpression
of C/EBP leads to differentiation of myeloid leukemia cell
lines4,8,13 and to eosinophil/neutrophil differentiation of multipotential avian hematopoietic progenitor
cells.9
We used retroviral transduction of human CD34+
hematopoietic cells to evaluate the role of C/EBP in hematopoietic
differentiation and commitment of primary human progenitor cells, and
to identify target genes of C/EBP involved in these processes.
Following the expression of functional C/EBP protein, we observed a
dramatic inhibition of erythrocyte differentiation, with a decrease in BFU-E, CFU-E, and CFU-GEMM formation. We also observed increased CFU-GM
colony formation using purified erythrocytic progenitor cells
(CD71bright) following induction of C/EBP. To
identify the genes involved in this process we analyzed the
transcriptional profiles of GFP+ or
GFP+/CD71bright-sorted cells following
transduction with either the MIGR1 or MIGR1 C/EBP-ER retrovirus.
Using this approach, we have identified a number of C/EBP target
genes, many of which are linked to C/EBP for the first time.
Previous studies, which compared myeloblasts from
C/EBP mice with granulocytes from wild-type
littermates, may have identified many differentiation-specific genes
that are not necessarily regulated by C/EBP4,21 Other
genes, involved in the lineage commitment of primitive progenitor
cells, may have been missed because C/EBP was inducibly expressed in
lineage-restricted leukemia cell lines.8,15
C/EBP target genes that are involved in granulocytic
differentiation include calgranulin A and calgranulin B. Calgranulin A
(MRP-8) is induced during myeloid differentiation, and it was recently
identified as a C/EBP target gene.15,22 We have
confirmed its up-regulation using both Affymetrix microarrays and
quantitative (real-time) RT-PCR. The MRP-8 promoter has 5 potential
C/EBP binding sites and it has been successfully used to express
oncogenic fusion proteins in transgenic mouse models of myeloid
leukemia.23-25 Having identified it as a "direct"
C/EBP target gene (using cycloheximide), the potential regulation of
its promoter by C/EBP could make its use problematic for expressing
fusion proteins, such as AML1-ETO, or dominant-negative C/EBP
proteins in transgenic mice.13,23
We observed C/EBP-induced down-regulation of c-myc mRNA in primary
CD34+ hematopoietic cells, arguing for its key role in
granulocytic differentiation and confirming previous observations that
C/EBP expression in a leukemia cell line leads to c-myc
down-regulation.15 Down-regulation of c-myc appears to be
mediated through repression of E2F-dependent
transcription15 (depicted in Figure
4B) and was required for granulocytic
differentiation in that system.15 Similarly, knock-in mice
that lack wild-type C/EBP and express only C/EBP mutants that do
not suppress E2F-dependent transcription, lack granulocytic
differentiation.16 This demonstrates that down-regulation of c-myc by C/EBP is required for the
differentiation of c-myc-expressing leukemia cells and also that its
down-regulation plays a pivotal role in the differentiation of
multipotential progenitor cells. Inhibitory effects of C/EBP on the
cell cycle (mediated via suppressing E2F, c-myc, and other
proteins) may explain our observation that C/EBP expression
leads to smaller colonies and seems to accelerate myeloid
differentiation in clonogenic assays.
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| Figure 4.
Models for the role of C/EBP in hematopoietic
differentiation and lineage commitment.
(A) Known effects of Id1 expression on erythroid differentiation and
the complex of GATA-1, TAL1 (SCL), Lmo2, and Ldb1 are shown. (B)
Different mechanisms of "direct" target gene activation/repression
by C/EBP (for review see Loken et
al18).
|
|
C/EBP expression in primary human hematopoietic cells also induced
the expression of genes involved in the acute-phase response to
bacterial infection or inflammation, such as those regulated by
tumor necrosis factor (TNF; TSG-6, TSG-14, IL-6,
COX-2), and exodus, those induced by IL-1 (IEX-1, IL-6, and IL-1RA),
and several cytokine genes directly (pro-IL-1 and IL-6) (shown in Table 4). These cytokines also promote
the proliferation and/or survival of early myeloid cells. Several of
the C/EBP target genes are also regulated by nuclear factor
(NF)- B (eg, IL-6, IEX-1, and COX-2), and C/EBP and NF-B may
cooperatively regulate the transcription of these genes. This is of
interest because C/EBP/ mice lack the acute-phase
response to inflammation and other members of the C/EBP family cannot
substitute for this function.26 Mature granulocytes are
required for the nonspecific host defense against bacterial infections,
and bacterial products induce a rapid and persistent increase in
granulocyte numbers in the peripheral blood. Besides regulating
acute-phase response genes, C/EBP expression leads to a faster
maturation of granulocytes, which may also contribute to this response.
We observed up-regulation of cyclin A1 and cyclin D3 expression
following C/EBP activation (in both of the CD71bright
experiments and 2 of the 3 GFP experiments), but their role in the
observed phenotypic changes is unclear. The identification of cyclin A1
as a C/EBP target gene is perhaps surprising because cyclin A1 is
highly expressed in leukemic cell lines and expression of cyclin A1
leads to leukemia in mice.27,28 Furthermore,
cyclin A1 is induced after expression of the oncogenic fusion proteins PML/RAR and promyelocytic leukemia zinc finger/retinoic acid receptor gene (PLZF/RAR) and the transcription factor
c-myb.29,30
We identified 2 genes that could help explain the phenotype of
the C/EBP/ mice, namely adipophilin and glycogen
phosphorylase. Glycogen phosphorylase is important for glycogenolysis,
while adipophilin is involved in lipid storage. These proteins
are relevant because C/EBP/ mice have no white fat,
and they die shortly after birth from severe hypoglycemia due to
defects in energy homeostasis.31,32 Adipophilin is
expressed in monocytes/macrophages but its role in hematopoietic cell
commitment and differentiation needs further investigation.33,34
C/EBP is a key transcription factor involved in hematopoiesis
that can enhance commitment toward a specific hematopoietic lineage but
also block differentiation toward the "competing" lineage. Such
effects can occur either via protein-protein interactions with other
lineage-directing transcription factors or through direct binding to
the promoter of target genes.35-38 A major phenotypic effect of C/EBP expression in human CD34+ cells was the
block in erythrocytic differentiation. A direct C/EBP target gene,
which was strongly up-regulated in erythroid precursors, is the
transcriptional repressor Id1. Id1 is a helix-loop-helix (HLH) protein,
which lacks the basic region required for DNA binding but
heterodimerizes with and represses other HLH transcription factors.39 Erythroid differentiation is associated with
down-regulation of the Id1 protein, whereas constitutive expression of
Id1 leads to a block in erythrocyte differentiation even in the
presence of transcription factors that promote erythrocyte-specific
gene expression.40 The HLH proteins E47 and Tal-1 (SCL)
associate with the zinc finger transcription factor GATA-1 and the LIM
proteins Lmo2 and Ldb1 in erythroid cells,41 and this
complex is disrupted by Id1.42 The Id1 promoter contains 7 potential C/EBP binding sites,43 and we have shown that
Id1 mRNA and protein levels are increased in cells expressing active
C/EBP. We propose a molecular mechanism for C/EBP in lineage
commitment that involves up-regulating Id1 expression (and other
proteins) that interfere with the erythrocyte differentiation
transcriptional program, and at the same time activating genes
important in neutrophilic differentiation (Figure 4A). In preliminary
experiments, overexpressing murine Id1 in human hematopoietic
progenitor cells did not lead to a significant block in erythrocyte
differentiation in clonogenic assays (data not shown). It is likely
that other genes must cooperate with Id1 to shift the differentiation
program away from the erythroid lineage. It is also possible that the
level of Id1 expression must be within a certain range to generate a
detectable block in differentiation. Similar to the effects of C/EBP
on granulocytic versus erythroid differentiation, constitutive
expression of PU.1 in murine hematopoietic progenitor cells blocks
T-cell development without impairing macrophage development. The block
in T-cell differentiation is accompanied by, among other things, an
increase in Id2, which has been shown to inhibit T-cell
differentiation.44,45
Unlike prior studies, we have used primary human hematopoietic
cells to identify C/EBP target genes, representing the first attempt
to identify target genes of a transcription factor in the physiologic
cellular background. The use of multipotential human hematopoietic
progenitor cells and the brief period of exposure to active C/EBP
(achieved using the C/EBP-ER fusion protein) allow us to detect
changes in gene expression that are not due to changes in
differentiation but rather reflect a change in hematopoietic lineage commitment.
| |
Acknowledgments |
We would like to thank Dr Alan Friedman for providing the
C/EBP-ER cDNA; KIRIN Brewery (Gumma, Japan), Amgen (Thousand Oaks, CA), and Immunex (Seattle, WA) for generously providing us with human
cytokines; Dr Malcolm Moore, Dr Robert Benezra, and Paola De
Candia (Sloan-Kettering Institute, New York, NY) for providing technical and scientific advice; and Diane Domingo for flow cytometry. The RD18 cell line was a generous gift from Dr Mary Collins.
The following reagent was obtained through the AIDS Research and
Reference Reagent Program, Division of AIDS, National Institute of
Allergy and Infections Diseases (NIAID), NIH from Dr Nathaniel Landau and Dr Dan Littman: SV-A-MLV-env.
| |
Footnotes |
Submitted June 4, 2002; accepted October 16, 2002.
Prepublished
online as Blood |
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Abstract
Introduction