Transcription factors Sp1 and C⁄EBP regulate NRAMP1
gene expression
Etienne Richer
1,2
, Carole G. Campion
1
, Basel Dabbas
3
, John H. White
3
and Mathieu F. M. Cellier
1
1 Institut national de la recherche scientifique, INRS-Institut Armand-Frappier, Laval, Canada
2 The Centre for the Study of Host Resistance, McGill University, Montreal, Canada
3 Department of Physiology, McGill University, Montreal, Canada
The natural resistance-associated macrophage protein 1
(Nramp1, also known as solute carrier family 11,
member 1, Slc11A1) confers innate resistance to intra-
cellular parasites in mice [1]. The activity of the Nramp1
transporter in the membrane of phagosomes prevents
growth of ingested microbes and limits their capacity to
produce a lethal infection [2]. Genetic polymorphisms in
the NRAMP1 and vitamin D receptor (VDR) genes
were linked to innate susceptibility to mycobacterial
infections or increased risk of immune diseases [3,4].
Functional analyses of NRAMP1 promoter alleles
suggested a possible impact of polymorphisms on gene
expression and function [5–7].
NRAMP1 expression is restricted to mature myeloid
cells: primary monocytes, macrophages and neutroph-
ils, ranked by increasing mRNA abundance. Transient
transfection studies showed that a DNA fragment
extending 647 bp upstream of the NRAMP1 ATG
enables transcriptional activation in response to VDR
ligands in HL-60 cells, but not in nonmyeloid cells.
HL-60 clones stably transfected with this promoter
fragment showed dose- and time-dependent transcrip-
tional responses to VDR ligands consistent with the
accumulation of endogenous NRAMP1 mRNA
induced during monocytic differentiation [8]. Identifi-
cation of the specific determinants controlling
Keywords
1,25D; innate immunity; myeloid
differentiation; phagocytes; transcriptional
regulation
Correspondence
M. Cellier, INRS-Institut Armand-Frappier,
531, Bd des prairies, Laval, QC H7V 1B7,
Canada
Fax: +1 450 686 5301
Tel: +1 450 687 5010 ext. 4681
E-mail: mathieu.cellier@iaf.inrs.ca
(Received 13 June 2008, revised 24 July
2008, accepted 12 August 2008)
doi:10.1111/j.1742-4658.2008.06640.x
The natural resistance-associated macrophage protein 1 (Nramp1), which
belongs to a conserved family of membrane metal transporters, contributes
to phagocyte-autonomous antimicrobial defense mechanisms. Genetic poly-
morphisms in the human NRAMP1 gene predispose to susceptibility to
infectious or inflammatory diseases. To characterize the transcriptional
mechanisms controlling NRAMP1 expression, we previously showed that a
263 bp region upstream of the ATG drives basal promoter activity, and
that a 325 bp region further upstream confers myeloid specificity and acti-
vation during differentiation of HL-60 cells induced by vitamin D. Herein,
the major transcription start site was mapped in the basal region by S1
protection assay, and two cis-acting elements essential for myeloid transac-
tivation were characterized by in vitro DNase footprinting, electrophoretic
mobility shift experiments, in vivo transfection assays using linker-mutated
constructs, and chromatin immunoprecipitation assays in differentiated
monocytic cells. One distal cis element binds Sp1 and is required for
NRAMP1 myeloid regulation. Another site in the proximal region binds
CCAAT enhancer binding proteins aor band is crucial for transcription.
This study implicates Sp1 and C ⁄EBP factors in regulating the expression
of the NRAMP1 gene in myeloid cells.
Abbreviations
CDP, CCAAT displacement protein; C ⁄EBP, CCAAT enhancer-binding protein; ChIP, chromatin immunoprecipitation; 1,25D, 1a,25-
dihydroxyvitamin D
3;
dsODN, double-stranded oligonucleotide; EB1089, 1(S),3(R)-dihydroxy-20(R)-[5¢-ethyl-5¢-hydroxy-hepta-1¢(E),3¢(E)-dien-1¢-
yl]-9,10-secopregna-5(Z),7(E),10(19)-triene; EMSA, electrophoretic mobility shift assay; IFN-c, interferon-c; IRF, interferon-cresponse factor;
KH1060, 20-epi-22-oxa-24a,26a,27a-tri-homo-1,25-dihydroxyvitamin D
3
; MEF, myeloid Elf-1-like factor; NRAMP1, natural resistance-
associated macrophage protein 1; TSS, transcriptional start site; VDR, vitamin D receptor.
5074 FEBS Journal 275 (2008) 5074–5089 ª2008 The Authors Journal compilation ª2008 FEBS
NRAMP1 expression will shed light on the regulatory
cis-acting elements and trans factors involved during
myelopoiesis and immune responses, and further
increase our understanding of the possible influence of
NRAMP1 promoter genetic polymorphisms in human
susceptibility to diseases, including infections by intra-
cellular parasites.
Vitamin D agonists have profound effects on the
immune system, specifically stimulating innate antimi-
crobial defenses and macrophage maturation [9,10].
Several major transcription factors known to regulate
myelopoiesis [11–13] also control genes expressed dur-
ing differentiation induced by the hormonal form of
vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), includ-
ing the VDR [14], Sp1 [15] and CCAAT enhancer
binding proteins (C ⁄EBPs) [16]. Sp1 regulates genes
associated with innate immunity in cooperation with
other tissue-specific or ‘terminal differentiation’-specific
nuclear factors [17]. Sp1 is thus often associated with
Ets-related transcription factors, e.g. myeloid Elf-1-like
factor (MEF) [18,19]. Members of the C ⁄EBP family
are important nuclear factors that cooperate with
others, including Sp1, to regulate myeloid genes
[11,12]. C ⁄EBP factors have prominent roles in myelo-
poiesis [20], and several isoforms, e.g. C ⁄EBPa,
C⁄EBPband C ⁄EBPe, are differentially regulated dur-
ing myelo-monocytic differentiation [16,21–23].
An NRAMP1 promoter-proximal region starting
263 bp upstream of the ATG is sufficient for maximal
transcription reporter activity in nonmyeloid cell lines,
whereas the more distal region (264–588 bp upstream
of the NRAMP1 ATG) is required for maximal pro-
moter activity and for responsiveness to 1,25D in HL-
60 cells but not in Jurkat T-cells [8]. The data suggest
that the NRAMP1 promoter comprises a proximal
region binding a basal transcription complex (core
promoter) and a more distal, myeloid-specific region
(upstream promoter). To test this hypothesis, we
mapped the NRAMP1 transcriptional start site in dif-
ferentiated HL-60 cells, located basal and myeloid-spe-
cific cis-acting sites, and identified important
transcription factors controlling NRAMP1 expression
in myeloid cells.
Results
Delineating NRAMP1 cis-acting elements in HL-60
cells undergoing differentiation
Primer extension mapping previously revealed several
NRAMP1 transcriptional start sites (TSSs) in different
cell types [24,25]. NRAMP1 TSSs in HL-60 cells were
thus located by an S1 nuclease protection assay
(Fig. 1A). Two major protected fragments were
obtained, 10 or 38 bp shorter than the specific probe
used, the latter being more abundant (hereafter
denominated )28 and +1, respectively). The absence
of a larger fragment lacking only the control 5¢
synthetic 7-mer (Supporting information Table S1)
excluded other start sites upstream of the probe. The
results indicated heterogeneity of NRAMP1 TSSs in a
single cell type, suggesting that the NRAMP1 pro-
moter fragment NR1S may bind a basal transcription
complex close to the +1 or )28 site (Fig. 1D).
Myeloid-specific cis elements were mapped by assay-
ing the transcriptional activity of nested deletions of a
promoter fragment extending 647 bp upstream of the
NRAMP1 ATG in transfected HL-60 cells differenti-
ated with 1,25D or dimethylsulfoxide. We used stable
transfections based on previous data obtained with
HL-60 transfected clones, which showed luciferase
reporter activity that paralleled the accumulation of
endogenous NRAMP1 mRNA in response to differen-
tiation [8]. The clone HSRL5 was used as a positive
control to characterize the activity of clones represent-
ing deletion constructs (Fig. 1B; 5E3, 5E4, M-1, and
data not shown). Some activity persisted with con-
struct 5E3, and little remained with further deletions.
Similar results were obtained with 1,25D and dimethyl-
sulfoxide (Fig. 1B), implying that the NRAMP1 region
upstream of )365 contributes to myeloid regulation.
To locate NRAMP1 promoter cis-acting sites, in vitro
DNase 1 footprint experiments were conducted on both
strands, revealing 14 protected areas (e.g. E2, E6, and
E10; Fig. 1C). Few clear differences were observed in
the patterns obtained with different cell types. Double-
stranded oligonucleotides (dsODNs, 30 bp) overlapping
the protected sites were used in electrophoretic mobility
shift assays (EMSAs) to assess transcription factor-
binding activities in vitro. Three DNase-protected sites
confirmed by specific EMSA are indicated relative to
NRAMP1 deletion construct ends (Fig. 1D). To
characterize candidate cis-acting elements, consensus
binding sites for known transcription factors were
tested as decoys for inhibition of nuclear extract
binding to NRAMP1 dsODNs, and linker mutations
were designed to delineate NRAMP1-specific cis-acting
sites using in vitro EMSAs and in vivo stable trans-
fection assays.
Sp1 binds NRAMP1 promoter site E10 and
influences protein binding at site E6
The cytosine-rich site E10 located between the bound-
aries of fragments M-2 and S (Fig. 1D) was detected
as a faint footprint that was similar in intensity and
E. Richer et al. Sp1 and C ⁄EBP regulate NRAMP1 transcription
FEBS Journal 275 (2008) 5074–5089 ª2008 The Authors Journal compilation ª2008 FEBS 5075
70 b
AB
2
8
+1
luc
M-1 -296 lucluc
53 b
7
1
3
2
luc
luc
5E4
5E3
-365
-398
lucluc
lucluc
1
(-28)
25 b
0
5
2
4
luc
SRL
-498 lucluc
Fold induction (RLU)
78
(+1)
C
149bp
117bp
278bp 414bp
427bp
Myeloid specific region
+1
Basal region -28
D
249bp
ATG
S1 probe
Luc
E2 E6
LS
M-1 M-2
5E3 5E4
CA
ACGT 1 2 3 4 5
E6
E10
E2
ACGT 1 2 3 4 5 ACGT 1 2 3 4 5
E10
123456
KH
DMSO
Fig. 1. Organization and activity of the NRAMP1 promoter. (A) Identification of the major TSS by S1 protection assay (+1, located 147 bp
upstream of the ATG), and several minor TSSs either adjacent or 28 bp upstream, using HL-60 cells differentiated for 3 days with the 1,25D
genomic analog KH1060 (KH). (B) NRAMP1 transcriptional activity in HL-60 clones, identified by numbers, which were stably transfected
with promoter constructs of the indicated length relative to the +1 site. Transcriptional activation was measured by luminometry and
expressed as relative luciferase (luc) units (RLU) fold induction between cells untreated and cells treated for either 3 days with 1,25D or
5 days with dimethylsulfoxide (DMSO); the mean ± standard error (SE) of at least three independent experiments is presented. (C) DNase
digestion footprints of three putative cis elements, E10, E6 and E2, that bind nuclear extracts from various cell types: lane 2, HL-60 cells;
lane 3, HL-60 cells treated with 10
)8
MKH for 4 days and activated with IFN-c; lane 4, HL-60 cells treated for 6 days with 1.25% dimethyl-
sulfoxide; lane 5, Jurkat lymphoid T-cells; lane 1, control with no extracts. A sequencing ladder run was used to locate the protected sites,
indicated by a vertical bar. (D) Schematic representation of the luciferase reporter constructs showing deletions in the upstream region of
the NRAMP1 promoter, which is required for myeloid regulation, and locations of the three protected sites E2, E6 and E10, as well as the
polymorphic CA repeat adjacent to the downstream E6 site.
Sp1 and C ⁄EBP regulate NRAMP1 transcription E. Richer et al.
5076 FEBS Journal 275 (2008) 5074–5089 ª2008 The Authors Journal compilation ª2008 FEBS
quality between different extracts (Fig. 1C, left). Unla-
beled probe E10 in excess competed with this band-
shift, but not the mutant probe E10M0 (Fig. 2A, left).
Mutants E10M1–3 competed slightly better, but less
than wild-type E10 (Table 1; data not shown), indicat-
ing specificity in nuclear factor binding to the E10
motif. Moreover, a wild-type Sp1 dsODN decoy
strongly competed with E10 probe binding, and an
antibody against Sp1 induced a specific band supershift
(Fig. 2A, right). Finally, two stably transfected HL-60
clones revealed that NR1L mutant E10M0 abolished
differentiation-induced transcription (Fig. 2C), imply-
ing that Sp1 binding to NRAMP1 site E10 is impor-
tant for gene transcription in vivo during myeloid
differentiation.
Another cytosine-rich site downstream of the poly-
morphic CA dinucleotide repeat (E6; Fig. 1D) was
protected with all nuclear extracts tested (Fig. 1C,
center). E6 binding specificity was demonstrated by
competitive EMSA and abrogated by the mutation
E6M2 (Fig. 2B, Table 1). Although the E6 motif was
predicted to bind Sp1, inclusion in EMSAs of an
antibody against Sp1 reduced band-shift intensity
only slightly. Little binding competition occurred
with excess unlabeled wild-type Sp1 dsODN decoy,
albeit reproducibly (Fig. 2B). Similar weak competi-
tion was associated with a wild-type dsODN decoy
for the Ets family member MEF (Fig. 2B), but not
for PU.1, AP-1, Stat and PU.1-IRF factors (data not
shown), contrasting with the strong competition by
wild-type E10 dsODN (Fig. 2B). These data indicate
that Sp1 contributes to interactions with NRAMP1
promoter site E6, and suggest MEF as a potential
binding partner. The role of site E6 in vivo was studied
using clones stably transfected with the NR1L mutant
construct E6M2, which lost transcriptional response to
1,25D (Fig. 2C), but conserved some activity induced
with dimethylsulfoxide. Similar responses were observed
with PCR mutant clones having a 2 bp CA deletion
(promoter allele 9 [6], data not shown), indicating that
the E6M2 mutation limits NRAMP1 transcriptional
activation in vivo in response to 1,25D (Fig. 2C).
A
B
C
Fig. 2. The distal cis elements E2, E6 and E10 are required for NRAMP1 promoter
activation during myeloid differentiation induced with 1,25D. (A, B) EMSA using
dsODNs covering sites E10 (A) or E6 (B) and nuclear extracts from HL-60 cells
treated with KH1060 (KH) and IFN-c. To characterize the specific band-shifts indi-
cated by an open arrowhead, a 50-fold excess of either cold dsODN or, as indi-
cated, a corresponding inactive linker mutated dsODN (E10M0 and E6M2, Table
1), or specific dsODN decoys and their mutated inactive counterparts [Sp1 and
mutated (mt) Sp1, MEF and MEF mt] were used in competitive EMSA. NS, non-
specific. Band supershifts (SS) were obtained using an antibody against Sp1. Com-
plexes were resolved by 6% PAGE. (C) HL-60 clones stably transfected with
NR1L promoter constructs, identified by numbers and containing sites inactivated
by linker mutagenesis (E2M2, E6M2 and E10M0), were used to measure RLU
fold induction between untreated cells and cells treated for 3 days with KH1060,
producing monocyte-like cells, or with dimethylsulfoxide (DMSO) to generate neu-
trophils. The mean ± SE of at least three independent experiments is presented.
E. Richer et al. Sp1 and C ⁄EBP regulate NRAMP1 transcription
FEBS Journal 275 (2008) 5074–5089 ª2008 The Authors Journal compilation ª2008 FEBS 5077
The 5¢-region upstream of )365 is important for
NRAMP1 myeloid expression
The region spanning )498 to )365 (constructs SRL
and 5E4) is required to regulate the promoter (Fig. 1B),
and encompasses site E2 (Fig. 1D), detected as a weak
DNase footprint that appeared to be better protected
with extracts from differentiated HL-60 cells (Fig. 1C
right, lanes 3 and 4). An excess of cold mutant dsODN
competed for protection of site E2 specifically (E2M1
but not E2M2) (Table 1, and data not shown). Another
dsODN overlapping this site, E2.2, competed with E2
and E2M1 but not E2M2 (data not shown). Sequence
analyses suggested that E2 might be a composite site
for an interferon-c(IFN-c) response factor (IRF),
which would mediate NRAMP1 upregulation in mature
phagocytes exposed to IFN-c. However, neither
dsODN decoys for PU-IRF, GAS (IFN-c-activating
sequence) and Stats (signal transducers and activators
of transcription, data not shown) nor any other factor
tested competed with E2 binding (data not shown). To
examine the impact of the E2M2 mutation in vivo,
three independent stably transfected HL-60 clones were
obtained, and all showed abrogation of NRAMP1 tran-
scriptional activity in response to 1,25D (Fig. 2C). The
E2M2 and E6M2 mutations seemed to preserve the
response to dimethylsulfoxide in vivo. In comparison,
the deletion 5E3 downstream of the E2M2 mutation
had a less severe, more variable impact on NRAMP1
gene activation (Fig. 1B,D). These data establish a
regulatory role in myeloid cells for the NRAMP1 pro-
moter region upstream of )365, including site E2.
Sp1 recognizes myeloid-specific sites and
transactivates the NRAMP1 promoter in vivo
A role for the transcription factor Sp1 in the regula-
tion of NRAMP1 activation in mature myeloid cells
was tested by using an antisense phosphorothioate
ODN that inhibits Sp1 expression and observing the
effect on pSRL-driven luciferase activity in HSRL5
cells differentiated with 1,25D (Fig. 3A). A modest
reduction in reporter activity was noted with the
anti-Sp1 ODN that was statistically significant as
compared to scrambled anti-Spl control ODN (AS),
suggesting that in monocytic HL-60 cells Sp1 could
contribute to the upregulation of NRAMP1 trans-
cription.
To show trans-activation of the NRAMP1 promoter
by Sp1, 293T epithelial cells were transiently cotrans-
fected using NRAMP1 promoter luciferase constructs
and pCMV vectors expressing similar levels of the Sp1
family members Sp1 and Sp3 [26–28]. As previous
studies in 293T cells revealed similar activities for the
constructs NR1L and NR1S in the absence of cotrans-
fected transcription factor [8], and because Sp1 is
known to interact with distal or proximal parts of the
promoters that it regulates [15–19], we compared lucif-
erase activity levels obtained in the presence of Sp
factors of NR1S and NR1L as well as of other con-
structs of intermediate length. Sp1 increased the tran-
scriptional activity of constructs NR1L, 5E4 and M-1
about two-fold to three-fold as compared to the NR1S
construct (Fig. 3B), and experiments using NR1L and
Sp3
+
plasmids resulted in a lower level of activation
Table 1. Specific electromobility shifts.
Motif Sequence (5¢-to-3¢)
#
Predicted site Decoy
E2 ttc ctc tgt ggc cct caa agg gaa act gaa IRF +
E2M1 ttc ctc tgt ggA GTC GCa agg gaa act gaa +
E2M2 ttc ctc tgt ggc cct caa TCA CTG act gaa’ )
E2.2 ctc aaa ggg aaa ctg aag cct tga gga cat IRF +
E6 gtg gca gag ggg ggt gtg gtc atg ggg tat SP1 ++
E6M1 gtg gTC AGC Tgg ggt gtg gtc atg ggg tat +
E6M2 gtg gca gag gTA CTC Ctg gtc atg ggg tat )
E6M3 gtg gca gag ggg ggt gAC TCT Ctg ggg tat +⁄)
E6M4 gtg gca gag ggg ggt gtg gtc aCA TCA Cat ++
E10 cac agg gca ggc tgg gag ggg aac aaa ggt SP1 PU.1 ++
E10M0 cac agg gca CTA GCA gag ggg aac aaa ggt )
E10M1 cac agg gca ggc GCA TGC ggg aac aaa ggt +⁄)
E10M2 cac agg gca ggc tgg gag CAT GTG aaa ggt +⁄)
E10M3 cac agg gca ggc tgg gag ggg GTG CTT ggt +⁄)
E14 tga acc gaa tgt tga tgt aag agg cag ggc C ⁄EBP ++
E14M1 tga acc gaa CTA GCT tgt aag agg cag ggc +⁄)
E14M2 tga acc gaa tgt tga CAG TCA agg cag ggc +⁄)
E14M3 tga acc gaa tgt tga tgt aag CTA GTC ggc ++
#
Upper case letters indicate linker-mutations; nucleotides fitting the indicated transcription factor predicted sites are underlined.
Sp1 and C ⁄EBP regulate NRAMP1 transcription E. Richer et al.
5078 FEBS Journal 275 (2008) 5074–5089 ª2008 The Authors Journal compilation ª2008 FEBS