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HEMATOPOIESIS
Identification of target genes and a unique cis element regulated by IRF-8
in developing macrophages
Tomohiko Tamura, Pratima Thotakura, Tetsuya S. Tanaka, Minoru S. H. Ko, and Keiko Ozato
Interferon regulatory factor-8 (IRF-8)/interferon consensus sequence–binding protein (ICSBP) is a transcription factor that
controls myeloid-cell development. Microarray gene expression analysis of Irf8ⴚ/ⴚ myeloid progenitor cells expressing
an IRF-8/estrogen receptor chimera
(which differentiate into macrophages after addition of estradiol) was used to
identify 69 genes altered by IRF-8 during
early differentiation (62 up-regulated and
7 down-regulated). Among them, 4 lysosomal/endosomal enzyme-related genes
(cystatin C, cathepsin C, lysozyme, and
prosaposin) did not require de novo protein synthesis for induction, suggesting
that they were direct targets of IRF-8. We
developed a reporter assay system employing a self-inactivating retrovirus and
analyzed the cystatin C and cathepsin C
promoters. We found that a unique cis
element mediates IRF-8–induced activation of both promoters. Similar elements
were also found in other IRF-8 target
genes with a consensus sequence
(GAAANN[N]GGAA) comprising a core
IRF-binding motif and an Ets-binding motif; this sequence is similar but distinct
from the previously reported Ets/IRF composite element. Chromatin immunoprecipitation assays demonstrated that IRF-8
and the PU.1 Ets transcription factor bind
to this element in vivo. Collectively, these
data indicate that IRF-8 stimulates transcription of target genes through a novel
cis element to specify macrophage differentiation. (Blood. 2005;106:1938-1947)
© 2005 by The American Society of Hematology
Introduction
Transcription factors play a major role in orchestrating the gene
expression programs governing hematopoietic-cell differentiation.1
Dysregulation of such programs can result in hematopoietic
disorders such as leukemia.2
Interferon regulatory factor (IRF)-8/interferon consensus sequence–binding protein (ICSBP) is a hematopoietic transcription factor
belonging to the IRF family.3 A series of studies performed with
Irf-8⫺/⫺ mice have indicated that this factor plays critical roles in
the development and function of immune cells such as macrophages, dendritic cells, and B cells.4-8 Consistent with its developmental role, IRF-8 expression is initiated in hematopoietic progenitors and increases during the development of the above-mentioned
immune cells.7,9
In myeloid progenitor cells, IRF-8 controls lineage selection by
stimulating macrophage differentiation while inhibiting the growth
and differentiation of granulocytes that are another myeloidprogeny cell type.9,10 This finding partially explains why Irf-8⫺/⫺
mice develop a chronic myelogenous leukemia (CML)–like syndrome with expansion of granulocytes and impaired macrophage
development.4,9,11 In addition, IRF-8 was shown to inhibit the
activity of breakpoint cluster region/abelson murine leukemia
(Bcr/Abl), which is the causal oncoprotein of CML, both in vitro
and in vivo.12-14 Expression of IRF-8 is severely impaired in human
patients with CML and acute myelogenous leukemia (AML),
providing additional evidence that IRF-8 is a critical antileukemogenic factor in myeloid cells.15,16 We previously showed that the
c-Myc repressors, B-lymphocyte–induced maturation protein-1
(Blimp-1) and mitogenic Ets transcriptional suppressor (METS),
and the cyclin-dependent kinase inhibitor, cyclin-dependent kinase
inhibitor 2B (CDKN2B/INK4B), are direct target genes of IRF-8 in
differentiating myeloid progenitors.13,17 However, to our knowledge, there is no report on a genome-wide gene expression study to
identify IRF-8–regulated genes in differentiating myeloid cells.
IRF-8 acts either as an activator or a repressor, depending on
interacting factors and target DNA elements.3 For example, IRF-8
interacts with IRF-1 or IRF-2 to repress transcription of genes such
as interferon (IFN)–stimulated gene-15 and 2⬘5⬘-oligoadenylate
synthetase, which both contain an IFN-stimulated response element (ISRE; A/BNGAAANNGAAACT).18-20 It has been suggested
that in immune cells, a subset of ISREs called the Ets/IRF response
element (EIRE; GGAAANNGAAA), is activated by IRF-8 in
cooperation with the IRF-8–interacting Ets transcriptional activator, PU.1.21 Another DNA element activated by IRF-8 and PU.1 is
the Ets/IRF composite element (EICE; GGAANNGAAA), which
is found in various genes such as those encoding immunoglobulin
(Ig) ␬ and ␭, glycoprotein (gp) 91phox, scavenger receptor,
toll-like receptor-4, and interleukin-1␤.22-25 Since PU.1 is
essential for macrophage development,26 IRF-8 may exert its
activity through the EICE and EIRE to stimulate macrophage
differentiation. However, it has been unclear whether IRF-8
regulates endogenous genes through these elements during
macrophage differentiation. Indeed, the molecular mechanism
From the Laboratory of Molecular Growth Regulation, National Institute of Child
Health and Human Development, National Institutes of Health, Bethesda, MD;
and Developmental Genomics and Aging Section, Laboratory of Genetics,
National Institute on Aging, National Institutes of Health, Baltimore, MD.
6, Room 2A01, Laboratory of Molecular Growth Regulation, National Institute
of Child Health and Human Development, National Institutes of Health,
6 Center Dr, MSC 2753, Bethesda, MD 20892-2753; e-mail:
[email protected] or [email protected].
Submitted January 7, 2005; accepted May 15, 2005. Prepublished online as
Blood First Edition Paper, June 9, 2005; DOI 10.1182/blood-2005-01-0080.
The online version of the article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Reprints: Tomohiko Tamura, Building 6, Room 2A05, or Keiko Ozato, Building
© 2005 by The American Society of Hematology
1938
BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
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BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
by which IRF-8 regulates myeloid development is still poorly
understood.
In this study, we performed complementary DNA (cDNA)
microarray analysis using an in vitro differentiation system in
which Irf-8⫺/⫺ myeloid progenitor cells undergo macrophage
differentiation upon introduction of IRF-8. We identified 69 genes
regulated by IRF-8 in an early phase of differentiation, including 4
novel direct target genes. Furthermore, we identified a unique
target DNA element (GAAANN[N]GGAA) that mediates IRF-8
induction of target genes during macrophage differentiation. This
element consists of a core IRF binding site and an Ets binding site,
with the polarity opposite of that in the EICE and EIRE sequences.
Finally, we showed that IRF-8 binds to this element in vivo,
concomitant with an increased binding of PU.1.
MICROARRAY ANALYSIS OF IRF-8 TARGET GENES
1939
(5⬘-ggagatccttcattacctggag-3⬘ and 5⬘-tgacctcattacagaagccagc-3⬘), and PKC␦
(5⬘-ctgtgctgtgaagatgaaggag-3⬘ and 5⬘-cctgcatttgtagccttgcttg-3⬘). The primers for the other tested genes were as described elsewhere.10 Real-time
fluorescent quantitative PCR was performed in duplicate using the SYBR
green PCR master kit (Applied Biosystems, Foster City, CA) and an ABI
PRISM 7000 sequence detection system (Applied Biosystems). cDNAs
derived from 2 ng of total RNA were used for PCR. The primer sets used
were: cystatin C (5⬘-gcgttggacttcgctgtga-3⬘ and 5⬘-ggctgtggtacgcatcgtt-3⬘);
cathepsin C (5⬘-ccgggcattttaccctcat-3⬘ and 5⬘-ttgaaaaacgcaaaccatttgt-3⬘);
lysozyme M (5⬘-ttagctcagcacgagagcaatt-3⬘ and 5⬘-gctgcgcttcagctcgtt3⬘); and c-fms (5⬘-gatgaggcctgtctcgacttct-3⬘and 5⬘-ctggcctctttgtccagatctt3⬘). The primers for the other examined genes were as described.10 Each
primer sets gave a unique product, and transcript levels were normalized
against the expression levels of glyceraldehyde-3⬘-phosphate dehydrogenase (GAPDH).
Reporter assay
Materials and methods
Cells and retroviral transduction
The establishment and culture conditions of cell line Tot2, a granulocyte
macrophage–colony-stimulating factor (GM-CSF)–dependent myeloid progenitor-cell line derived from an Irf-8⫺/⫺ mouse were described previously.10 Murine stem cell virus (MSCV)–CD8t vectors carrying IRF-8,
IRF-8/estrogen receptor (ER), and ER were constructed by inserting the
relevant cDNAs into the EcoRI site of MSCV-CD8t.27 Other MSCV
retrovirus vectors and transduction procedures for Tot2 cells and bone
marrow lineage–negative (lin⫺) cells were as described previously.9,10,13
Peritoneal macrophages were harvested from mice that were intraperitoneally injected with 3 mL of 3% thioglycollate (Difco, Detroit, MI) 4
days earlier. All animal work conformed to the guidelines of the animal
care and use committee of the National Institute of Child Health and
Human Development. Morphologic differentiation was monitored by
Wright-Giemsa staining of cytospin preparations using Eclipse E400
(Nikon, Melville, NY).
Microarray analysis
Tot2 cells transduced with MSCV-ER-puro or MSCV-IRF-8/ER-puro were
treated with ␤-estradiol for 4 hours, and total RNA was purified using the
Trizol reagent (Invitrogen, Carlsbad, CA). The NIA 15K mouse cDNAs
microarray, probe preparation, and hybridization were as previously
described.28 Briefly, the cDNA probe was prepared from 300 ␮g total RNA
by reverse transcription in the presence of ␣-[33P] deoxycytidine triphosphate (dCTP). Hybridization with the purified probe was carried out
overnight at 65°C with prehybridized nylon membranes spotted with
15 247 mouse developmental cDNA clones. After washing, membranes
were exposed to a phosphoscreen, and the screen was scanned by a
phosphorimager (Storm860; Amersham Pharmacia, Piscataway, NJ). Images were analyzed with the IMAGEQUANT 5.0 software package
(Amersham Pharmacia). The expression levels of each gene were expressed
in arbitrary units after subtraction of the background. Two independent
experiments were used to identify differentially expressed genes with a
statistical significance of P less than .05 by t test.
Semiquantitative and quantitative polymerase chain reactions
with reverse transcription
Reverse transcription (RT) and semiquantitative and quantitative polymerase chain reactions (PCRs) were performed as previously described.13 The
amount of cDNA appropriate for each reaction was determined prior to
actual experiments. cDNA derived from 100 ng of total RNA was used for
amplification of the scavenger receptor (SR), cDNA from 25 ng for c-fms
and protein kinase C (PKC) ␦, and cDNA from 2 ng for the other genes. The
following primers were used: cystatin C (sense 5⬘-gaggcagatgccaatgaggaag-3⬘ and antisense 5⬘-actgcaagaagagtggagccag-3⬘); lysozyme M (5⬘cctgctttctgtcactgctcag-3⬘ and 5⬘-ctccgcagttccgaatatactg-3⬘); cathepsin C
(5⬘-cccgaagcgacattaactgctc-3⬘and 5⬘-ggccacttctccttatcagatc-3⬘), prosaposin
For generation of reporter constructs, we first replaced the neomycin
resistance gene in the self-inactivating retrovirus pSIR (Clontech, Palo
Alto, CA) with a puromycin resistance gene. We then inserted the green
fluorescent protein (GFP) cDNA along with a new multiple cloning site
(MCS) containing XhoI and HindIII sites into the original MCS to generate
retroviral reporter SIRV-GFP. The DNA fragments used for this construction were generated from the appropriate plasmids by restriction enzyme
digestion or PCR using Pfu DNA polymerase (Stratagene, La Jolla, CA).
The utilized GFP was a destabilized form known as d2EGFP (Clontech) that
has a half-life of 2 hours. We used the QuikChange site-directed mutagenesis kit (Stratagene) to introduce a point mutation abolishing a HindIII site
within the d2EGFP cDNA without altering the amino acid sequence, thus
enabling us to use the HindIII site for the new MCS. The cystatin C and
cathepsin C promoter regions were PCR amplified using Pfu polymerase
and the genomic DNA of Tot2 cells, and the amplified fragments were
inserted into SIRV-GFP. Mutants were generated by PCR using primers that
introduced the desired mutations and relevant restriction enzyme sites for
ligation. The nucleotide sequences of all constructs containing PCR-derived
DNA fragments were confirmed by sequencing. For reporter assay, cells
were transduced with SIRV-GFP reporter constructs, selected with puromycin, and transduced with MSCV-CD8t vectors. Cells were then either
purified by immunomagnetic cell sorting, or directly stained with anti–
human CD8 conjugated with Cy-Chrome (BD Pharmingen, San Diego,
CA) and analyzed by FACSCalibur (BD Biosciences, Mountain View, CA)
to acquire GFP signals in CD8⫹ cells. The data were analyzed with the
CellQuest software (BD Biosciences).
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assays were performed using the
ChIP assay kit (Upstate Biotechnology, Lake Placid, NY) according to
manufacturer’s instructions with slight modifications. Briefly, 6.5 ⫻ 106
cells were treated with 1% formaldehyde for 30 minutes at room temperature to crosslink proteins and genomic DNA. After washing, cells were
lysed in 650 ␮L sodium dodecyl sulfate (SDS) lysis buffer, and then
sonicated (20 seconds each, 10 times at 15% of maximum power on wet ice
using an XL2007 sonicator; Misonix, Farmingdale, NY) to shear the
genomic DNA into 200- to1000–base pair (bp) fragments. After centrifugation, 100 ␮L supernatant was diluted with 1 mL ChIP Dilution Buffer.
Chromatin was precleared with 80 ␮L of protein A/G agarose–25% slurry
(Santa Cruz Biotechnology, Santa Cruz, CA) supplemented with salmon
sperm DNA (200 ␮g/mL; Invitrogen) for 30 minutes at 4°C. Precleared
chromatin was incubated with 5 ␮g of normal goat immunoglobulin G
(IgG), normal rabbit IgG, goat anti–IRF-8 antibody (C-19; Santa Cruz
Biotechnology) or rabbit anti-PU.1 antibody (T-21; Santa Cruz Biotechnology) overnight at 4°C with rotation. Protein A/G–25% slurry with salmon
sperm DNA (60 ␮L) was added and samples were rotated for 1 hour at 4°C
to collect the antibody-chromatin complexes. Agarose-antibody-chromatin
complexes were then washed twice with each of the following in order: low
salt wash buffer, high salt wash buffer, LiCl wash buffer, and 10 mM
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1940
TAMURA et al
tris(hydroxymethyl)aminomethane (Tris)–HCl (pH 8), 1 mM ethylenediamine-tetraacetic acid (EDTA). After elution of the chromatin complexes,
the crosslink was reversed, and RNA and proteins were digested with
RNase and proteinase K, respectively. DNA was then recovered by
phenol/chloroform extraction followed by ethanol precipitation, and resuspended in 100 ␮L of 5 mM Tris, pH 8. Each sample (5 ␮L) was used for
quantification of the specific region of genomic DNA (50-150 bp) by
duplicate real-time PCR amplifications. Input DNA (0.1%) was used for
normalization. The primers used for PCR were: cystatin C promoter
(5⬘-gcaatgaccaacttctctggtg-3⬘ and 5⬘-cttaccagttcctcttctgtgc-3⬘); cathepsin C
promoter (5⬘-ggagtcagaaatgcaggaaagtg-3⬘ and 5⬘-gggttgacaagagtcttaaagtctactg-3⬘); HPRT promoter (5⬘-gggcctaaatcttgaggaatcac-3⬘ and 5⬘-gtctcccagaggattcccagata-3⬘), and SIRV-IECS-Ld40 (5⬘-tgaagtcgatgccgcttttc-3⬘ and
5⬘-aaccagcgtccgcagatct-3⬘).
Results
cDNA microarray analysis of genes regulated by IRF-8
To begin understanding the mechanism by which IRF-8 controls
myeloid-cell development, we sought to identify IRF-8–regulated
genes. We had previously established an in vitro macrophage
differentiation system in which introduction of IRF-8 drives
Irf-8⫺/⫺ myeloid progenitor Tot2 cells to fully differentiate into
macrophages.10 To examine early changes caused by IRF-8, we
employed an estrogen-inducible IRF-8/ER chimera (Figure 1A).13
In the absence of ␤-estradiol, IRF-8/ER–transduced Tot2 cells
remained undifferentiated. However, after 24 hours of estradiol
treatment, IRF-8/ER cells showed an intermediate stage of differentiation as judged by Wright-Giemsa staining (Figure 1B). In
contrast, cells transduced with ER alone did not show any sign of
differentiation in the absence or presence of estradiol. RT-PCR
revealed that estradiol treatment rapidly induced expression of 2
macrophage-specific genes, c-fms (CSF-1/M-CSF receptor) and
scavenger receptor (SR), only in IRF-8/ER–transduced cells (Figure 1C). These results confirmed that the estrogen-inducible form
of IRF-8 induced rapid differentiation in these cells. The differenti-
BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
ated cells were growth arrested, and showed the property of
functional macrophages as assessed by their ability to express
IL-12p40 and inducible nitric oxide synthase (iNOS) mRNAs in
response to IFN␥ and lipopolysaccharide (LPS; Figure 1C). Fc␥
receptor I (Fc␥RI) expression was detected in the differentiated
cells and was further induced by the immune stimuli.
IRF-8/ER– and ER-transduced Tot2 cells treated with estradiol
for 4 hours were then subjected to a genome-wide expression study
using the NIA 15K mouse developmental cDNA microarray.28 We
identified 69 genes (including 23 known/annotated genes) as
displaying more than 2-fold differences between IRF-8/ER and ER
cells (Tables 1, 2; also Supplemental Table S1, available online by
clicking on the Supplemental Table link at the top of the article on
the Blood website). Notably, the majority of the altered genes (62
genes) showed up-regulation, while only 7 genes were downregulated by IRF-8/ER, suggesting that IRF-8 may act mainly as a
transcriptional activator in this system. The microarray data were
confirmed by semiquantitative RT-PCR analysis of 7 selected genes
(Figure 2).
Somewhat unexpectedly, the genes showing the strongest
induction at this early time point were those related to lysosomal/
endosomal enzymes (Table 1). The cystatin C gene (induced
⬃10-fold) encodes an endogenous inhibitor of the lysozomal
cysteine proteases called cathepsins.29 We also observed significant
induction of 2 cathepsins, cathepsin C/dipeptidyl-peptidase I and
cathepsin L, as well as lysozyme M, a principal myeloid protease
that has bactericidal activity,30,31 and prosaposin, which encodes a
precursor of the sphingolipid activator proteins called saposins.32
Six of the more moderately up-regulated genes were related to
signal transduction, including kinases and molecules involved in
cyclic adenosine monophosphate (cAMP)/G-protein signaling.
Notably, 1 of the up-regulated genes encodes PKC␦, which was
previously shown to render myeloid progenitor 32Dcl.3 cells to
respond to phorbol ester by differentiating into monocytic cells.33
There were 3 growth-related genes including c-Myc; this was
strongly repressed by IRF-8, consistent with our previous finding.13
Figure 1. Macrophage differentiation by estradiol-inducible IRF-8/ER chimera in Irf-8ⴚ/ⴚ myeloid progenitor cells. (A) Diagram of the IRF-8/hormone-binding domain of
the estrogen receptor (IRF-8/ER). (B) Wright-Giemsa–stained Irf-8⫺/⫺ myeloid progenitor Tot2 cells transduced with a retrovirus carrying ER or IRF-8/ER (original
magnification, ⫻ 1000). Cells were either left untreated or treated with 1 ␮M ␤-estradiol (Est) for 24 hours. (C) Semiquantitative RT-PCR analysis for macrophage
differentiation-related genes. RNA from cells treated with 1 ␮M ␤-estradiol for the indicated time was subjected to semiquantitative RT-PCR for c-fms and scavenger receptor
(SR) expression. Cells treated with 1 ␮M ␤-estradiol for 3 days were also analyzed for expression of IL-12p40, inducible nitric oxide synthase (iNOS), and Fc␥ receptor I (Fc␥RI)
transcripts. ␤-actin was used as the loading control.
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BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
MICROARRAY ANALYSIS OF IRF-8 TARGET GENES
1941
Table 1. List of annotated genes that displayed more than 2-fold changes following IRF-8 induction
Unigene ID
Gene name (gene symbol)
Clone name
Fold change,
IRF-8/control
Endosomal/lysozomal enzymerelated genes
Mm.4263
Cystatin C (Cst3)
H3033F11
Mm.45436
Lysozyme M (Lyzs)
H3054F05
10.6
Mm.180056
Cathepsin C (Ctsc)
H3037F12, H3055G02
Mm.277498
Prosaposin (Psap)
H3151D11
5.3
Mm.930
Cathepsin L (Ctsl)
H3028F03
2.1
Mm.191749
Phosphodiesterase 4A, cAMP specific (Pde4a)
H3037G12
6.0
Mm.28262
Regulator of G-protein signaling 2 (Rgs2)
H3053A12
4.0
Mm.317331
v-yes-1 oncogene homolog (Lyn)
H3001B08
3.5
Mm.2314
Protein kinase C, delta (Prkcd)
H3059B03
3.2
Mm.275839
Janus kinase 2 (Jak2)
H3067C12
2.4
Mm.2444
Myelocytomatosis oncogene (Myc)
H3076D10, H3089H11
Mm.333406
Cyclin D2 (Ccnd2)
H3026D04
2.1
Mm.217318
Microtubule-associated protein 4 (Mtap4)
H3058E07
2.1
Mm.3532
Thymosin, beta 10 (Tmsb10)
H3001H10
3.8
Mm.240839
Tropomyosin 3, gamma (Tpm3)
H3022F08
3.2
Mm.6384
Nebulin-related anchoring protein (Nrap)
H3020G05
2.4
10.6
7.6, 7.6
Signal transduction-related
genes
Cell growth-related genes
0.38, 0.34
Cytoskeleton-related genes
Others
Mm.295578
Tripartite motif protein 30 (Trim30/Rpt1)
H3063C08
3.1
Mm.66056
Membrane bound C2 domain containing protein (Mbc2)
H3094D04
2.6
Mm.290906
Nuclear antigen Sp100 (Sp100)
H3052B09
2.5
Mm.16373
Histocompatibility 2, class II, locus DMa (H2-DMa)
H3109E03
2.3
Mm.193021
Rho GTPase activating protein 17 (Arhgap17)
H3100H08
2.6
Mm.248327
C-type lectin, superfamily member 9 (Clecsf9)
H3053D08
2.0
Mm.253061
Ankyrin repeat and SOCS box-containing protein 13 (Asb13)
H3051A11
2.0
Tot2 cells transduced with ER (control) or IRF-8/ER were treated with ␤-estradiol for 4 hours and analyzed with the NIA 15K cDNA microarray. Two independent
experiments were performed to identify genes differentially expressed between the two samples with a statistical significance of P below .05 by t test.
Other up-regulated genes included a major histocompatibility
complex II gene central to the antigen presenting capability of
macrophages, and a C-type lectin that acts as a pattern recognition
receptor responsible for sensing foreign glycoproteins. The biologic significance of the noted changes in some of those genes, such
as the cAMP/G-protein–related genes, is unclear at present and will
require further investigation.
Identification of direct target genes of IRF-8
The rapid and strong induction of lysosomal/endosomal enzyme
and related genes observed in our microarray and RT-PCR analyses
led us to examine whether IRF-8 directly regulates the expression
of these genes. To distinguish direct and indirect targets, we
examined the effect of a protein synthesis inhibitor, cycloheximide
(CHX), on the observed induction. If the gene induction occurs
directly, estradiol should induce transcript expression in the
presence of CHX, whereas CXH should abolish induction if the
induction is indirect (ie, mediated by one or more proteins newly
induced by IRF-8/ER). Accordingly, CHX was added 10 minutes
prior to addition of estradiol. Real-time RT-PCR analysis demonstrated that the induction of the cystatin C, cathepsin C, lysozyme
M, and prosaposin genes was not abrogated in the presence of CHX
(Figure 3A), but rather increased over that treated with estradiol
alone, indicating that these 4 genes are direct targets of IRF-8. In
contrast, CHX inhibited the up-regulation of c-fms, indicating that
this gene is an indirect target. We also observed that the suppression
of c-Myc was inhibited by CHX (data not shown), in agreement
with our previous findings that c-Myc is repressed through action of
other transcription factors, Blimp-1 and METS.13
Consistent with the IRF-8–dependent induction of these
genes, expression of cystatin C, cathepsin C, and prosaposin
transcripts was lower in Irf-8⫺/⫺ cells than that in wild-type cells
(data not shown). To confirm that IRF-8 is responsible for the
induction of the target genes in freshly isolated bone marrow
cells, we transduced IRF-8 or control vector into Irf-8⫺/⫺ lin⫺
cells and cultured them in the presence of stem-cell factor (SCF)
and M-CSF (Figure 3B). Consistent with the data with Tot2
cells, expression of the cystatin C, cathepsin C, and prosaposin
genes was significantly induced in IRF-8–transduced bone
marrow cells during the culture, whereas low or no upregulation of these genes was observed in control vector–
transduced cells. Expression of the lysozyme M gene was
induced both in IRF-8–transduced and control cells. This is
presumably because Irf-8⫺/⫺ bone marrow progenitor cells
differentiate into granulocytes that also express lysozyme M.
Together, IRF-8 directly induces transcription factors such as
Blimp-1 and METS as well as macrophage proteases and related
genes. These genes are in turn likely to affect subsequent gene
expression patterns and cellular functions (Figure 3C).
Analysis of the cystatin C gene promoter
We next sought to identify the target DNA element through which
IRF-8 activates transcription of the isolated target genes. We cloned
the approximately 1 kilobase (kb) genomic sequence upstream of
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BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
TAMURA et al
Table 2. List of unannotated genes that displayed more than 2-fold
changes after IRF-8 induction
Clone name
Fold change, IRF-8/control
H3062F07
0.05
H3065B03
0.11
H3065D05
0.38
H3089G03
0.43
H3014D08
0.43
H3060D07
0.49
H3059D03
5.2
H3033D11
3.9
H3051B12
3.8
H3051B09
3.8
H3054G06
3.1
H3020F06
3.1
H3037E11
3.0
H3085F09
3.0
H3053A11
3.0
H3085B04
2.9
H3051F11
2.8
H3001H12
2.8
H3094H01
2.8
H3044E04
2.8
H3051H11
2.8
H3051H12
2.6
H3110B06
2.6
H3037C12
2.5
H3066F01
2.5
H3055F02
2.5
H3033F10
2.4
H3047C03
2.4
H3048G11
2.3
H3054F07
2.3
H3057G12
2.3
H3061E07
2.2
H3051E10
2.2
H3018H11
2.1
H3008A03
2.1
H3048C04
2.1
H3047C04
2.1
H3089B06
2.1
H3055G04
2.1
H3158H05
2.1
H3003B10
2.0
H3053B12
2.0
H3053D06
2.0
H3056F06
2.0
H3152C05
2.0
H3065F02
2.0
the cystatin C coding region, and analyzed the promoter activity
using a novel reporter construct, SIRV-GFP (Figure 4A).
SIRV is a self-inactivating retrovirus containing a 176-bp
deletion in the 3⬘ long terminal repeat (LTR) that removes the
enhancer sequence. Following reverse transcription, the 3⬘ LTR is
copied and replaces the 5⬘ LTR, resulting in inactivation of the 5⬘
LTR promoter. This enables us to use this vector for our reporter
assay. We inserted a destabilized form of GFP (d2EGFP) into SIRV
as a reporter, and placed the promoter sequences upstream of GFP.
With this system the activity of chromatin-integrated promoters can
be monitored in live cells as GFP signals via flow cytometry. In a
preliminary experiment, introduction of SIRV-GFP containing
the IFN␥ activation site (GAS) along with the Ld40 minimal
promoter allowed cells to express GFP in response to IFN␥,
indicating that this promoter-reporter system functioned properly (data not shown).
Tot2 cells expressing ER alone or IRF-8/ER were transduced
with empty SIRV-GFP or SIRV-GFP containing the cystatin C
promoter (SIRV-CysC-GFP), and were then treated with estradiol for 13 hours. The GFP signals in empty SIRV-GFP–
transduced ER and IRF-8/ER cells were very low (comparable
to the autofluorescence of untransduced Tot2 cells) and were not
altered by estradiol treatment, confirming that the LTRs were
inactivated in this system (Figure 4B-C). In the absence of
estradiol, SIRV-CysC-GFP cells showed modest levels of GFP
signals, reflecting the basal promoter activity in undifferentiated
Tot2 cells. Following estradiol treatment, GFP expression
increased approximately 4-fold in IRF-8/ER cells but not in ER
cells (Figure 4B-C), indicating that IRF-8 activated transcription of the cystatin C promoter–driven reporter.
The 1-kb cystatin C promoter did not contain the typical
EICE sequence, but an EIRE was located at ⫺617 (element-1;
Figure 4A). We chose 2 additional DNA sequences (elements
⫺2 and ⫺3) as candidate target elements for IRF-8; these
elements consisted of an IRF-binding motif (GAAA) followed
by an Ets-binding motif (GGAA) without spacing nucleotides
(element-2) or with 2 spacer nucleotides (element-3). We
introduced mutations in all 3 elements and examined the ability
of IRF-8 to activate the reporter gene. The mutation in
element-3 (Mut-3) severely diminished IRF-8–induced activation of the promoter, whereas the mutations in element-1 and
element-2 (Mut-1 and Mut-2) had no effect (Figure 4B-C),
indicating that element-3 is important for the IRF-8 response.
Analysis of the cathepsin C promoter
We next analyzed the cathepsin C promoter in the same manner. As
shown in Figure 5, IRF-8/ER plus estradiol activated transcription
through the 1-kb genomic sequence upstream of the open reading
frame, whereas ER alone did not. We found that the cathepsin C
promoter contained a DNA element (element-1) very similar to the
important element-3 identified in the cystatin C promoter (Figure
5A). In addition, a typical EICE was found (element-2). In
mutational experiments shown in Figure 5B, we found that a
mutation in element-1 (Mut-1), but not that in element-2 (Mut-2)
diminished IRF-8/ER–induced activation of the cathepsin C promoter, indicating that this element is required for induction of
promoter activity by IRF-8. Thus, similar DNA elements mediate
IRF-8–induced transcription from both cystatin C and cathepsin C
promoters, indicating that these elements may represent a common
Figure 2. RT-PCR confirmation of IRF-8–inducible genes identified by microarray analysis. Tot2 cells transduced with ER or IRF-8/ER were treated with
␤-estradiol for the indicated time and analyzed for gene expression by semiquantitative RT-PCR.
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MICROARRAY ANALYSIS OF IRF-8 TARGET GENES
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and CDKN2B), looking for similar DNA elements. Indeed, we
found that all of them except one gene (METS) contain a similar
element in their promoter regions with the consensus sequence
GAAANN(N)GGAA (Table 3). We designated this element the
IRF-Ets composite sequence (IECS). In most cases, the element
is located within approximately 1 kb upstream of the start
codon. The IECS is distinct from the previously reported EICE
and EIRE sequences, as the IRF-binding motif in the IECS is
located 5⬘ of the Ets-binding, whereas the reverse is true in the
latter 2 elements.
The IECS mediates IRF-8 activation of transcription
To examine whether the IECS is sufficient to confer IRF-8–induced
activation of transcription, we constructed SIRV-IECS-Ld40-GFP,
in which GFP transcription is driven by 3 copies of the IECS from
the cystatin C gene followed by minimal promoter Ld40 (Figure
6A). SIRV-Ld40-GFP lacking IECS was used as a control. Ld40
alone had essentially no promoter activity in ER and IRF-8/ER
cells before and after estradiol treatment (Figure 6B). IECS-Ld40
showed a modest basal transcriptional activity in untreated ER and
IRF-8/ER cells. Importantly, transcription via the IECS was
dramatically induced by estradiol treatment only in IRF-8/ER cells
but not in ER cells.
We also examined whether wild-type (nonchimeric) IRF-8
can activate the IECS. Tot2 cells were transduced with SIRVLd40-GFP or SIRV-IECS-Ld40-GFP, and then with empty
MSCV or MSCV-IRF-8. Promoter activity was measured on day
2 of transduction, when cells did not yet show any morphologic
changes. IRF-8 clearly activated transcription through the IECS
under these conditions (Figure 6C). Taken together, our results
show that the IECS is sufficient to mediate IRF-8 activation of
transcription in differentiating myeloid progenitor cells.
IRF-8 binds to target promoters and the IECS in vivo
Figure 3. Direct activation of lysosomal/endosomal enzyme-related genes by
IRF-8. (A) Effect of cycloheximide (CHX) on IRF-8/ER–mediated induction of the
indicated genes. IRF-8/ER–transduced Tot2 cells were treated with ␤estradiol (Est) and/or CHX (10 ␮g/mL). CHX was added 10 minutes before addition of
␤-estradiol. Transcript levels were quantified in duplicate by real-time RT-PCR. Data
were normalized by GAPDH levels and shown as values relative to those in untreated
cells (mean ⫾ standard deviation). (B) Expression of the indicated genes in
IRF-8–transduced bone marrow lin⫺ cells. Irf-8⫺/⫺ bone marrow lin⫺ cells were
cultured in the presence of SCF, IL-6, and IL-3 for 1 day and were transduced with
MSCV–IRF-8 or control MSCV retrovirus on the following 2 days. Next day, cells were
washed and reinoculated in the presence of SCF and M-CSF. Cells were harvested 2
and 7 days after addition of M-CSF. Transcript levels were determined as in panel A.
(C) A model for transcriptional pathways activated by IRF-8. IRF-8 activates
transcription regulators and macrophage proteases. IRF-8–induced transcription
factors regulate their target genes, including those that regulate cell growth. The
proteases are critical for the functionality of macrophages, and might also influence
transcription factor activity.
target DNA sequence for IRF-8 during an early stage of macrophage differentiation. It should be noted, however, that the residual
induction of the Mut-1 cathepsin C promoter (1.9-fold) suggests
that there may be an additional element targeted by IRF-8.
We scanned the genomic sequence of 5 other genes directly
activated by IRF-8 (lysosome M, prosaposin, Blimp-1, METS,
We next analyzed whether IRF-8 binds to endogenous target
gene promoters containing the IECS. ChIP assays were performed in MSCV-IRF-8–transduced Tot2 cells, and specific
PCR primers were used to amplify 50 to 150 nucleotides
spanning the IECSs in the cystatin C and cathepsin C promoters.
Real-time PCR analysis demonstrated that in IRF-8–transduced
cells, both promoter fragments were significantly immunoprecipitated by anti–IRF-8 antibody, compared with the low levels
of background signals generated by control IgG (Figure 7A, top
2 of the left panels). As expected, the ChIP signals generated by
IRF-8 antibody in control MSCV-transduced cells were low,
similar to background levels. We also used an anti-PU.1
antibody, which revealed that PU.1 bound to both promoters
even in control MSCV-transduced cells. Interestingly, IRF-8
transduction increased PU.1 binding to the promoters approximately 3-fold (Figure 7A, top 2 of the right panels). Binding of
IRF-8 and PU.1 to the irrelevant, but active hypoxanthine
phosphoribosyltransferase (HPRT) promoter was also tested
(Figure 7A, bottom panels). The ChIP signals generated by
IRF-8 antibody were as low as background levels. PU.1
antibody showed only slightly elevated binding over control
antibody in both control and IRF-8–transduced cells. These
results suggest that IRF-8 specifically binds to endogenous
promoters containing IECSs in vivo, and that IRF-8 binding is
concomitant with enhanced binding of PU.1. We also confirmed
the binding of endogenous IRF-8 protein to the promoter region
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TAMURA et al
BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
Figure 4. Analysis of the cystatin C promoter. (A) Diagram of the self-inactivating retrovirus reporter (SIRV-GFP) carrying the cystatin C promoter. Both LTRs are inactivated
and have no promoter activity. ⌿⫹, the extended viral packaging signal; MCS, multiple cloning site; H4P-Puror; the puromycin resistant gene driven by the histone H4 promoter.
Sequences and mutations in element-1 to element-3 are shown. The numbers indicate the nucleotide positions relative to the start codon. (B) Cystatin C promoter activity in live
cells, as monitored by flow cytometry of GFP signals. Tot2 cells were transduced with SIRV-GFP carrying wild-type (WT) or mutant (Mut-1 to Mut-3) promoters, and then with
MSCV-ER or MSCV–IRF-8/ER. Cells were either left untreated or treated with 1 ␮M ␤-estradiol (Est) for 13 hours. (C) Quantification of the promoter activity measured in panel
B. The activity is shown as mean fluorescent intensity (MFI) of GFP signals.
of both cystatin C and cathepsin C genes in peritoneal macrophages (Figure 7B). As expected, PU.1 binding was also
detected on both promoters.
Finally, we examined whether IRF-8 and PU.1 bind to the IECS
itself in vivo (Figure 7C). Tot2 cells were transduced with
SIRV-IECS-Ld40-GFP, and then with MSCV–IRF-8. ChIP assays
were performed using the same antibodies, and the presence of the
IECS in immunoprecipitated chromatin was determined using PCR
primers designed to specifically amplify the element. IRF-8
binding to the IECS was clearly detected in IRF-8–transduced Tot2
cells. PU.1 bound to the IECS even in the absence of IRF-8, but the
binding was enhanced 4-fold in the presence of transduced IRF-8.
Collectively, these results indicate that IRF-8 binds to the IECS
concomitant with enhanced binding of PU.1, leading to stimulated
transcription of IRF-8 target genes.
Discussion
Here, we used cDNA microarray analysis to identify genes that are
rapidly induced or repressed by IRF-8 in differentiating myeloid
progenitor cells. Most of these genes have not previously been
described as IRF-8–regulated genes. Furthermore, we identified a
unique target DNA element, the IECS, through which IRF-8
activates transcription.
We found that IRF-8 directly induces expression of genes
encoding multiple lysosomal/endosomal enzymes (cathepsin C and
L, lysozyme M, and prosaposin) as well as their inhibitor (cystatin
C). This suggests that IRF-8 regulates not only “regulatory” genes
such as those encoding transcription factors and signaling molecules mediating expression of other genes, but also “functional”
genes that operate macrophage functions (Figure 3C). The latter
enzymes might also have “regulatory” roles in addition to their
involvement in end-stage protein breakdown. For example, cathepsin L was recently reported to be capable of cleaving a transcription
factor, resulting in activation of transcription.34 Other cathepsins
were demonstrated to degrade signal transduction molecules such
as epidermal growth factor receptor and internalized insulin.35,36
Thus, it is possible that lysosomal/endosomal enzymes identified as
IRF-8 targets in this study may also contribute to regulation of gene
expression. As a related issue, Irf-8⫺/⫺ macrophages are shown to
terminate CSF-1/M-CSF signaling prematurely due to increased
CSF-1 receptor ubiquitination, which may be caused by impaired
Table 3. The IRF-Ets composite sequence (IECS) present in multiple
IRF-8 target genes
Figure 5. Analysis of the cathepsin C promoter. (A) Diagram of SIRV-GFP reporter
carrying the cathepsin C promoter. Sequences and mutations of element-1 and
element-2 are shown on right. The numbers indicate the nucleotide positions relative
to the start codon. (B) Cathepsin C promoter activity in live cells monitored by flow
cytometry. The promoter activity was analyzed as in Figure 4B. Values represent fold
changes in MFI caused by estradiol treatment.
Cystatin C (⫺414 ⬃ ⫺404)
tcatGAAAcagGGAActtg
Cathepsin C (⫺810 ⬃ ⫺801)
tagaGAAAga GGAAgtag
Lysozyme M (⫺496 ⬃ ⫺487)
tcaaGAAAag GGAAaaga
Prosaposin (⫺3681 ⬃ ⫺3690)
actaGAAAag GGAAgagg
Blimp-1 (⫺873 ⬃ ⫺863)
ggtcGAAAggaGGAAgtta
CDKN2B/INK4B (⫺1212 ⬃ ⫺1203)
cagcGAAAta GGAAgaga
Numbers indicate nucleotide positions in relation to the ATG start codon.
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MICROARRAY ANALYSIS OF IRF-8 TARGET GENES
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The SIRV-GFP system-based promoter analysis of the cystatin
C and cathepsin C genes allowed us to identify a common DNA
element that is responsible for IRF-8 activation of these 2 direct
target genes. This element, the IECS, consists of an IRF-binding
motif followed by an Ets-binding motif; the order of the 2 motifs is
reversed relative to the previously reported EICE and EIRE
Figure 6. IRF-8 activation of transcription through the IECS. (A) Diagram of the
SIRV reporter driven by the IECS. (B) Activation of transcription through the IECS by
IRF-8/ER. Tot2 cells were transduced with SIRVIECS-Ld40-GFP or SIRV-IECS-Ld40GFP, and then with MSCV-ER or MSCV–IRF-8/ER. Cells were either left untreated or
were treated with 1 ␮M ␤-estradiol (Est) for 13 hours. Values represent fold changes
in MFI induced by estradiol treatment. (C) Activation of transcription through the IECS
by wild-type IRF-8. Tot2 cells were transduced with SIRV-Ld40-GFP or SIRV-IECSLd40-GFP and then with empty MSCV or MSCV–IRF-8. Cells were analyzed on day
2 after transduction of MSCVs. Values represent fold changes in MFI induced by
IRF-8.
expression of cathepsin B and accumulation of the ubiquitin ligase
c-casitas B-lineage lymphoma (c-Cbl).37 However, it should be
noted that regulation of CSF-1 signaling is not a sole mechanism of
IRF-8–induced macrophage differentiation, since CSF-1 does not
play major roles in Tot2-cell differentiation.10,13
We previously reported that the Blimp-1 and METS genes are
direct targets of IRF-8.13 Blimp-1 and METS suppress transcription
of growth-promoting genes such as c-Myc, and are also implicated
in macrophage differentiation.38,39 Consistent with this, our microarray analysis detected c-Myc as a gene down-regulated by IRF-8.
We also identified the c-fms gene as another “indirect” target but in
this case up-regulated by IRF-8. These data suggest that regulatory
genes directly induced by IRF-8 cause a number of subsequent
changes in gene expression. Since Blimp-1 and METS are repressors, there may be 1 or more yet-unidentified transcriptional
activators that mediate IRF-8 induction of indirect target genes.
Such activators may be additional direct targets of IRF-8, and are
likely to cooperate with other IRF-8 targets to orchestrate the
molecular program for macrophage differentiation (Figure 3C).
In this work, we developed a reporter system (SIRV-GFP) using
a self-inactivating retrovirus. Unlike the traditional reporter assay
in which reporter constructs and effectors are transiently overexpressed, our system requires low copies of both reporters and
effectors. Moreover, the reporter gene is integrated into the genome
unlike transiently transfected plasmids, providing a more physiologic system of promoter analysis.
Figure 7. Binding of IRF-8 to target gene promoters and to the IECS in vivo. (A)
Binding of IRF-8 and PU.1 to endogenous target gene promoters detected by
chromatin immunoprecipitation (ChIP) assay. Tot2 cells were transduced with empty
MSCV or MSCV–IRF-8, and were analyzed on day 3. Crosslinked, sheared
chromatin was precipitated by goat anti–IRF-8, rabbit anti-PU.1 antibody, or control
IgG (normal goat IgG for IRF-8 ChIP and normal rabbit IgG for PU.1 ChIP).
Precipitated DNA was analyzed in duplicate by real-time PCR to detect genomic DNA
from the cystatin C and cathepsin C promoter regions using primers that amplified the
IECS region of each promoter. Levels of precipitated promoter DNA (ChIP signals)
are shown as values relative to 0.1% input DNA (mean ⫾ standard deviation). The
HPRT promoter was analyzed as a control irrelevant to IRF-8 and PU.1. (B) Binding
of endogenous IRF-8 and PU.1 to the target gene promoters detected by ChIP assay.
Peritoneal macrophages from wild-type mice were analyzed as in panel A. (C) ChIP
assay for binding of IRF-8 and PU.1 to the IECS in vivo. Tot2 cells were transduced
with SIRV-IECS-Ld40-GFP, and then with MSCV or MSCV–IRF-8. Cells were
analyzed on day 3 after transduction of MSCVs. The IECS DNA was detected using
primers designed to amplify the IECS sequence in SIRV.
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BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
TAMURA et al
sequences. The IECS was sufficient to mediate IRF-8 activation of
transcription. Furthermore, IRF-8 bound to endogenous target
promoters containing the IECS, and to the IECS itself in vivo.
Notably, we observed that IRF-8 enhanced PU.1 binding to this
element, whereas lower levels of PU.1 binding to the irrelevant
HPRT promoter were not enhanced by IRF-8. Previous work
showed that PU.1 transcript levels do not change during the early
stages of differentiation in Tot2 cells.10 Therefore, the enhanced
PU.1 binding may be due to stabilization of PU.1-DNA binding by
physical interaction between IRF-8 and PU.1. It is also possible
that IRF-8 may change the chromatin to a state more accessible to
PU.1. Since PU.1 levels can affect lineage selection among
macrophages, granulocytes, and B cells,40-42 IRF-8 enhancement of
PU.1 binding may be a key event in IRF-8–mediated macrophage
differentiation.
Similar elements were found in 6 out of 7 IRF-8 target gene
promoters, further supporting the notion that the IECS is a common
target DNA element for IRF-8. In contrast, the EIRE and EICE
elements identified in the cystatin C and cathepsin C promoters,
respectively, did not contribute to IRF-8 activation of these
promoters. In addition, we found that 8 of 66 previously reported
PU.1 binding sequences (those found in Fc␥RI, myeloperoxidase,
p47phox, IL-7R, TAL1/SCL, Epstein-Barr virus LMP/TP2, lymphotropic papovovirus, and simian virus 40) are similar to the
IECS.43,44 This implies that IRF-8 and PU.1 may cooperate to
mediate transcription from these promoters, as well.
Despite clear IRF-8 binding to the IECS detected by our ChIP
assay, we did not detect significant binding of IRF-8 to an IECS
oligonucleotide by electrophoretic mobility shift assay (data not
shown). The discrepancy between in vivo and in vitro binding
assays suggests that IRF-8 may act on chromatinized templates, but
not on naked DNA. Given that histones are known to play central
roles in regulating transcription, we speculate that IRF-8 binding to
the IECS might require an interaction between histones and the
protein complex composed of IRF-8, PU.1, and possibly other
proteins. Future work will be required to identify such an interaction.
In conclusion, our study describes identification of target genes
activated/repressed by IRF-8, and shows a new target element by
which IRF-8 regulates transcription in myeloid progenitor cells.
These findings add to our broad understanding of the molecular
mechanism by which specific transcription factors direct myeloid
cell differentiation.
Acknowledgments
We thank Kevin Becker for microarray production, and Ben-Zion
Levi, Hee Jeong Kong, Akira Nishiyama, and Tomonori Uno for
valuable help.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2005 106: 1938-1947
doi:10.1182/blood-2005-01-0080 originally published online
June 9, 2005
Identification of target genes and a unique cis element regulated by
IRF-8 in developing macrophages
Tomohiko Tamura, Pratima Thotakura, Tetsuya S. Tanaka, Minoru S. H. Ko and Keiko Ozato
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