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 a or b and 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-c response 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 ª 2008 The Authors Journal compilation ª 2008 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 ⁄ EBPb and 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 ª 2008 The Authors Journal compilation ª 2008 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 L S 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 1 2 34 5 6 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 M KH 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 ª 2008 The Authors Journal compilation ª 2008 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 ª 2008 The Authors Journal compilation ª 2008 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 g gg ggt gtg gtc atg ggg tat SP1 ++ E6M1 gtg gTC AGC T gg ggt gtg gtc atg ggg tat + E6M2 gtg gca gag gTA CTC Ctg gtc atg ggg tat ) E6M3 gtg gca gag g gg ggt gAC TCT Ctg ggg tat + ⁄ ) E6M4 gtg gca gag g gg ggt gtg gtc aCA TCA Cat ++ E10 cac agg gca ggc tg g 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 tg g gag CAT GTG aaa ggt + ⁄ ) E10M3 cac agg gca ggc tg g gag ggg GTG CTT ggt + ⁄ ) E14 tga acc gaa tgt tga tgt aag agg cag ggc C ⁄ EBP ++ E14M1 tga acc gaa CTA GCT t gt 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 ª 2008 The Authors Journal compilation ª 2008 FEBS (data not shown). These data indicated that Sp1 can trans-activate the NRAMP1 upstream promoter. Differences in the activity of NRAMP1 promoter upstream elements that depended on the myeloid versus nonmyeloid cellular background were further noted. Cotransfection of Sp1 + plasmid and NRAMP1 promoter fragments of different lengths showed that construct 5E4 was as active as the full-length promoter NR1L in 293T cells (Fig. 3B), although it was inactive during myeloid differentiation in stably transfected HL-60 clones (Fig. 1B). Also, the construct M-2 failed to mediate trans-activation by Sp1 in 293T cells (Fig. 3B), despite containing site E10 (Fig. 1D). How- ever, the E10M0 mutation in the NR1L construct sig- nificantly reduced Sp1-dependent luciferase activity in 293T cells (Fig. 3B), consistent with the prominent role of this site in myeloid cells and derived nuclear extracts (Fig. 2). Overall, a lower level of Sp1-driven expression in 293T cells was detected using the shortest construct NR1S, supporting our proposition that Sp1 interacts with the NRAMP1 promoter upstream of myeloid- specific elements, including site E10. CCAAT-binding factors activate NRAMP1 basal transcription in vivo Cotransfection studies using the Sp1 + plasmid revealed different transcriptional activities of NR1L and NR1S constructs in 293T cells (Fig. 3B), indicating that, despite endogenous factors that function on NRAMP1 proximal elements [8], this system may detect other can- didate transcriptional regulators of NRAMP1. Neither the VDR, PU.1, IRF-4 nor IRF-8 demonstrated activity when cotransfected separately or in various combina- tions with the reporter construct NR1L (data not shown). However, cotransfection of NR1L with a plas- mid expressing the CCAAT displacement protein ⁄ cut homeobox (CDP ⁄ Cut, p200, typical of immature mye- loid cells) inhibited reporter activity (Fig. 3C), consistent with the known repressor activity of full-length CDP. In contrast, a proteolytically processed isoform lacking the inhibitory domain [29,30], CDP ⁄ Cut p110, upregulated NRAMP1 transcription about six-fold as compared to the full-length CDP. Unlike those of Sp1, the effects of CDP isoforms were mediated by the NRAMP1 pro- moter proximal region (Fig. 3C). Also, NRAMP1 trans- cription was stimulated upon cotransfection of NR1S constructs and plasmids expressing C ⁄ EBPs + . The C ⁄ EBP + plasmids upregulated transcription similarly to CDP ⁄ Cut p110, indicating that CCAAT-binding factors also bind to the NRAMP1 basal promoter. NRAMP1 transcription is regulated by cooperation between distal and proximal elements Previous transient transfections in immature HL-60 cells showed about three-fold higher NRAMP1 trans- A RLU 4000 6000 8000 * R 0 2000 4000 ND Ctrl ND AS KH Ctrl KH Sp1 KH Sp3 KH AS B 15 20 25 * 0 5 10 Fold induction (RLU) 2 3 4 5 6 7 NR1L NR1S C 0 1 2 Fold induction (RLU) Fig. 3. In vivo transactivation of the NRAMP1 promoter by Sp1 requires the myeloid-specific upstream promoter region. (A) Anti- sense ODNs directed against Sp1, Sp3 or a scrambled anti-Sp1 control ODN were used to treat, for 24 h, HL-60 cells from clone HSRL5, which were previously differentiated for 3 days with KH1060 (KH), and RLU were determined by luminometry. ND, con- trol cells without ODN. (B) NRAMP1 transcriptional activation was measured by luminometry (RLU) in 293T cells transiently cotrans- fected with NR1L promoter constructs either deleted or linker- mutated, together with Sp1 + or control plasmid; results are expressed in Sp1 + fold induction. (C) NRAMP1 transcriptional activa- tion in the presence of trans-acting factors was measured in RLU fold modulation in 293T cells transiently cotransfected with pGL3 constructs carrying long or short NRAMP1 promoter (NR1L or NR1S) and control or expression plasmids encoding various transcription factors, assayed alone or in combination. The mean ± SE of at least three independent experiments is presented in each panel. E. Richer et al. Sp1 and C ⁄ EBP regulate NRAMP1 transcription FEBS Journal 275 (2008) 5074–5089 ª 2008 The Authors Journal compilation ª 2008 FEBS 5079 criptional activity using the NR1L versus the NR1S construct, and upregulation by 1,25D also required the full-length promoter [8]. This suggested that NRAMP1 transcription in myeloid cells involves cooperation between factors bound to distal sites and others acting at proximal sites of the promoter. To address this possibility in 293T cells, the trans-acting activity of combinations of Sp1 and CDP ⁄ Cuts or C ⁄ EBPs was studied by cotransfections with NRAMP1 promoter constructs containing or not containing the upstream myeloid-specific region (Fig. 3C). Significant increases in trans-activation resulted from combining the NR1L construct with Sp1 + and CDP p110 + plasmids, whereas the CDP p200 + construct inhibited luciferase activity (Fig. 3C), demonstrating cross-talk between factors bound to distal or proximal promoter sites. A similar but less pronounced trend was observed when Sp1 and C ⁄ EBP factors were cotranfected in presence of the NR1L construct, some combinations being stimulatory (Sp1 + and C ⁄ EBPa + or b + plasmids) and some inhibitory (Sp1 and C ⁄ EBPe). These effects were specific because they required the NR1L con- struct, and additional cotransfections using C ⁄ EBPa and Sp3 were less efficient than those using Sp1 (data not shown). The results suggest that the func- tion of Sp1 bound to distal sites is subservient to the occupancy of promoter proximal elements by CCAAT-binding or other factors, which may activate or repress transcription. C ⁄ EBPs recognize NRAMP1 proximal promoter site E14 in vitro Assuming a model for NRAMP1 expression that includes a key CCAAT-binding (or other) factor occu- pying a proximal element, which may control tran- scription and integrate the activities of more distal factors (e.g. Sp1), due to intrinsic properties (e.g. CDP) or protein–interactions (e.g. C ⁄ EBPe and Sp1), we sought candidate CCAAT factor-binding sites in the NRAMP1 proximal promoter. The footprint E14 adjacent to the major TSS within the basal transcrip- tion region contains a motif fitting the consensus for the C ⁄ EBP family [12]. Given the prominent role of C ⁄ EBP factors in myeloid development and their activ- ity on the NRAMP1 promoter in 293T cells, we tested C ⁄ EBP binding at site E14. EMSA using site E14 showed a strong and diffuse band-shift of high mole- cular weight (Fig. 4A). Nuclear factor binding was diminished by the E14M1 and E14M2 mutations, but not by the E14M3 mutation, outside the predicted site (data not shown and Table 1). Competitive EMSA using mutant E14 sites confirmed the results of binding A B C Fig. 4. The NRAMP1 proximal site E14 is bound in vitro by C ⁄ EBPb and C ⁄ EBPa. (A) Nuclear extracts of HL-60 cells differentiated with KH1060 (KH) and activated with IFN-c were incubated with labeled E14 dsODN for EMSA. A specific band-shift (indicated by an open arrow- head) is shown by the absence of competitive EMSA using mutated dsODN (E14M1, C ⁄ EBP mut) as compared to wild-type dsODN. (B, C) EMSA, competitive EMSA and band supershifts (SS) were obtained using nuclear extracts from HL-60 cells differentiated with KH1060 for 5 days (B) or dimethylsulfoxide (DMSO) for 6 days (C), which were incubated with labeled E14 dsODN and additional cold dsODN or in the presence of antibody against C ⁄ EBPa, antibody against C ⁄ EBPb [2 and 6 lg (B) or 3 lg (C)] or control rabbit IgG antibodies, as indicated, and the complexes were resolved by 4% PAGE. Sp1 and C ⁄ EBP regulate NRAMP1 transcription E. Richer et al. 5080 FEBS Journal 275 (2008) 5074–5089 ª 2008 The Authors Journal compilation ª 2008 FEBS experiments, with little competition due to the E14M1 mutation (Fig. 4A). The wild-type C ⁄ EBP dsODN decoy strongly competed with binding, unlike the CDP (Fig. 4A), PU.1 or Sp1 decoys (data not shown). Supershift experiments demonstrated strong binding to site E14 of C ⁄ EBPb and, to a lesser extent, C ⁄ EBPa (Fig. 4B,C). C ⁄ EBPs trans-activate the NRAMP1 proximal region during myeloid differentiation The impact of site E14 on NRAMP1 transcriptional activity was deduced from luciferase activity levels after transient cotransfection of 293T cells using the NR1L mutant constructs E14M1 or E10M0, and the expres- sion plasmids Sp1 + and C ⁄ EBPa + , singly or combined (Fig. 5A). The E14M1 mutation virtually abolished NRAMP1 transcription; minimal effects of added nuclear factors persisted, but were drastically reduced. In comparison, the Sp1-binding site E10M0 mutation limited only the expression levels obtained with Sp1 + plasmid (Figs 3C and 5A). The role of NRAMP1 site E14 was confirmed in myeloid HL-60 cells stably trans- fected with the mutated pSRL construct E14M1. No luciferase expression was detected either prior to or after differentiation (Fig. 5B,C), and genomic DNA PCR fragments indicated promoter construct integrity for all clones tested (data not shown). Such a dramatic effect proved that CCAAT-binding factors are crucial to control of NRAMP1 transcriptional activity. Endogenous Sp1 and C ⁄ EBP factors bind the NRAMP1 promoter in maturing monocytic cells Although myeloid-specific expression is a conserved property among human and mouse NRAMP1 ortho- logs, the factors found to be involved so far differ between species (Fig. 6). The murine promoter con- tains two Inr sequences preceded by a proximal Sp1 site and up to six E-boxes (‘myc-max’) scattered in the upstream region [31,32], and its expression is con- trolled by the macrophage-specific transcription factor IRF-8 [33,34]. To determine whether current species differences reflect divergence [35] or limited knowledge of the mechanisms involved and extent of their conser- vation requires studies detailing gene regulation in vivo. We used chromatin immunoprecipitation (ChIP) assays to test whether C ⁄ EBPs and Sp1 factors were recruited to the endogenous NRAMP1 promoter during mono- cytic differentiation. We studied both C⁄ EBPa and C ⁄ EBPb, and compared HL-60 cells untreated or dif- ferentiated for 24–48 h using the 1,25D genomic agon- ists KH1060 (KH; Fig. 7A) or EB1089 (EB; Fig. 7B). C ⁄ EBPa appeared to be specifically bound to the NRAMP1 promoter and to be more abundant in growing than in differentiating cells. In contrast, asso- ciation of C ⁄ EBPb with the NRAMP1 promoter was detected in differentiating cells (Fig. 7A,B). Assays A 6 1 2 3 4 5 B Fold induction (RLU) 0 1 RLUs C 10 4 10 5 No treatment KH 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 DMSO HSRL -5 E14M1 -2 E14M1 -4 Fig. 5. The proximal cis element E14 is essential for NRAMP1 basal transactivation. (A) Basal transcription levels in 293T cells transiently cotransfected with linker mutations of the NRAMP1 pro- moter construct NR1L (E10M0 or E14M1) and with carrier DNA or expression plasmids encoding Sp1, C ⁄ EBPa or both. RLU fold induction was determined by comparison with the wild-type NR1L construct alone. The data presented are the mean ± SE of at least three independent experiments. (B, C) Luciferase activity (RLU) of HL-60 clones stably transfected with NRAMP1 long promoter con- structs containing the mutation E14M1 and compared to HSRL5, either untreated (B) or differentiated with KH1060 (KH) or dimethyl- sulfoxide (DMSO) for 4 days (C). E. Richer et al. Sp1 and C ⁄ EBP regulate NRAMP1 transcription FEBS Journal 275 (2008) 5074–5089 ª 2008 The Authors Journal compilation ª 2008 FEBS 5081 targeting Sp1 and using HL-60 cells differentiated with 1,25D showed a weaker specific signal (Fig. 7C), which was not seen when cells were treated for 24 h or less (data not shown); other assays using different condi- tions (specific antibodies against Sp1, oligonucleotide primer pairs) confirmed Sp1 binding to the NRAMP1 A B C Fig. 6. NRAMP1 distal and proximal promoter features. (A) The four cis elements identified in this study within the 647 bp NRAMP1 promoter are indicated (E2, E6, E10 and E14); the dsODNs that were used in EMSA, and the 6 bp linker mutations abrogating trans-acting factor binding in vitro, are indicated by overlining and bold letters, respectively. E2, E6, E10 and a fifth element, E3, located between the deletion boundaries 5E3 and 5E4, are required for NRAMP1 promoter trans-activation in vivo during HL-60 differentiation, whereas E14 is essential for NRAMP1 promoter activity. The major TSS (+1) and another TSS upstream ()28) are boxed, and the 63-mer probe used for S1 nuclease mapping is indicated. The polymorphic CA repeat is shaded, and CpG dinucleotides, which are mainly clustered in the basal proxi- mal promoter region, are in bold italics. The initiation codon is indicated by ATG (Met). (B) Alignment of the promoter sequences of NRAMP1 (top) and the cattle and mouse counterparts (respectively, middle and bottom) shows the absence of conservation of the five sites important for NRAMP1 regulation that were identified in this study. (C) Schematic representation of the cis-acting elements that were identified in the promoter regions of the human NRAMP1 and mouse orthologs. Sp1 and C ⁄ EBP regulate NRAMP1 transcription E. Richer et al. 5082 FEBS Journal 275 (2008) 5074–5089 ª 2008 The Authors Journal compilation ª 2008 FEBS promoter after monocytic differentiation (Fig. 7D). These in vivo data established that C ⁄ EBP and Sp1 factors regulate NRAMP1 expression during myeloid differentiation. Discussion This study identified cis-acting sequences in the NRAMP1 promoter and transcription factors binding to them, providing a mechanistic basis for the restric- tion of NRAMP1 gene expression to mature myeloid cells. The results also provide novel, valuable knowl- edge with which to interpret human genetic polymor- phisms, including allelic variation at the NRAMP1 promoter and in genes encoding the DNA-binding factors that regulate its transcription, in relation to disease resistance. The NRAMP1 promoter exhibits several characteris- tic myeloid properties: it is compact, lacks canonical TATA, initiator sequences or CCAAT boxes, and shows heterogeneous TSSs [36]. The NRAMP1 pro- moter is divided into two regions: (a) the proximal core region, which spans the TSS and is presumed to bind the basic RNA polymerase II-dependent mac- hinery; and (b) the 5¢-upstream region required for developmental regulation of expression. Three upstream cis-acting elements (E2, E6 and E10) mediate differentiation-dependent NRAMP1 tran- scription induced by 1,25D, as well as by dimethylsulf- oxide (E10 only). Binding of Sp1 to site E10 was demonstrated using in vitro and in vivo assays, includ- ing in the context of chromatinized DNA in HL-60 monocytic cells. The key role of Sp1 in the control of myeloid gene expression has been recently reviewed [17]. Although Sp1 is a ubiquitous factor, it contrib- utes to myeloid-restricted expression by rendering promoters more accessible by various mechanisms, including demethylation of Sp1-binding sites (‘GC box’) [37], local chromatin structure rearrangement [38,39], interactions with other nuclear factors, and ⁄ or post-translational modifications [17]. C ⁄ EBP factors also have prominent roles in myeloid cell development [40]. These factors trans- activated NRAMP1 through the proximal site E14; C ⁄ EBPa and C ⁄ EBPb bound this site in vitro and the NRAMP1 proximal promoter in vivo, as they do with other myeloid genes [41,42]. Higher levels of C ⁄ EBPb binding in HL-60 monocytic cells are consistent with upregulation of this factor in mature cells [16]. The combined action of Sp1 and C ⁄ EBPa is essential for maximal myeloid expression of LF and CD11c [43,44], and some C ⁄ EBP sites were found to be crucial for gene activation by Sp1. Synergistic trans-activation by Sp1 and C ⁄ EBPb was also reported in hepatoma cells [45,46]. Interestingly, both C ⁄ EBPb and Sp1 can recruit Mediator, a multiprotein complex acting as molecular bridge between enhancer-bound activators and the core transcriptional machinery [40]. Thus, it appears possible that NRAMP1 expression results from direct cooperation between C ⁄ EBPb located on a proximal element and Sp1 bound to more distal sites. KH (h) HL 60 0 24 0 24 0 24 0 24 A 200 bp 300 bp HL-60 B 200 bp 300 bp 200 bp 300 bp (-111/+163) (+1924/+2139) Untreated HL-60 24 h EB HL 200 bp 300 bp 48 h EB HL-60 HL-60 C (-204/+73) D 48 h VitD HL-60 300 bp 200 b p 300 bp 72 h EB HL-60 (-392/-93) (+1924/+2139) Fig. 7. In vivo recruitment of C ⁄ EBPb and Sp1 on the proximal and distal parts of the NRAMP1 promoter during HL-60 differentiation induced by 1,25D (VitD) analogs. ChIP assays were performed using antibodies against C ⁄ EBP (A, B) or Sp1 (C, D) as indicated in Experi- mental procedures. C ⁄ EBPa and C ⁄ EBPb binding were assayed in resting HL-60 cells and in cells preincubated for 24 h with the 1,25D agonist KH1060 (KH) (A). Cells untreated or differentiated for 24– 72 h using the 1,25D agonist EB1089 (EB) were assayed for C ⁄ EBPs (B) and Sp1 (D); NRAMP1 exon 3 PCR amplification was used to control DNA fragment size. Control normal rabbit IgG was used as specificity control (A, C). Numbers in parentheses indicate NRAMP1 gene base pair coordinates relative to the major TSS. E. Richer et al. Sp1 and C ⁄ EBP regulate NRAMP1 transcription FEBS Journal 275 (2008) 5074–5089 ª 2008 The Authors Journal compilation ª 2008 FEBS 5083 [...].. .Sp1 and C ⁄ EBP regulate NRAMP1 transcription E Richer et al Despite prominent roles of Sp1 and C ⁄ EBP factors in the regulation of the myeloid NRAMP1 promoter, other cis elements identified in this study suggest that additional factors participate in this regulation, e.g CDP, a factor that is downregulated during myelomonocytic development, which... little anti-Sp3 antisense inhibition of NRAMP1 expression in HL-60 cells (Fig 3A) and similar results for ChIP assays using antibodies directed at Sp1 ⁄ Sp3 or Sp1 only (Fig 7C,D)] NRAMP1 transcriptional activation presumably requires local activation of chromatin-modifying activities, recruitment of RNA polymerase II and associated factors, and stimulation of the basic transcriptional preinitiation complex... 100 lL containing 1.5 lL of GenePORTER and 400 ng of total DNA: 50 ng of pRLTK control luciferase plasmid, 150 ng of reporter plasmid, and 200 ng of cotransfected plasmids (pBluescriptKS+ control and ⁄ or transcription factorexpressing vectors, Sp1 [73], Sp3 [74], VDR [75], CDP and CDP878–1505 [76], C ⁄ EBPa, C ⁄ EBPb and C ⁄ EBPe [77], and PU.1, IRF4 and IRF8 [78] The GenePORTER ⁄ DNA mixture was... pairs E2M2 and E6M2, and E10M0 and E14M1, were combined with RVP3 or LucIR to PCR-amplify overlapping fragments (95 °C for 45 s; four cycles of 95 °C for 45 s, 48 °C for 60 s, and 72 °C for 120 s; 22 cycles of 95 °C for 45 s, 54 °C for 60 s, and 72 °C for 120 s; 72 °C for 10 min) using Pfu polymerase (Stratagene) PCR products (145 and 651 bp, E2M2; 299 and 497 bp, E6M2; 429 and 367 bp, E10M0; 613 and 233... subsequent to the divergence of mice and humans, facilitated by dissemination throughout the genome on mobile elements [68] In summary, we conclude that Sp1 and C ⁄ EBP factors directly contribute to the control of NRAMP1 promoter activity during monocytic maturation induced by 1,25D, suggesting that C ⁄ EBPb could recruit the basal transcription machinery, and Sp1 activate transcription from a more distal... functions for Nramp1 and Nramp2 [65], and only Nramp2 orthologs share clusters of transcription factor-binding sites 5084 that are conserved among rodents and humans [66] Assuming that 1,25D regulated immune response during wound repair to prevent autoimmunity in early tetrapods, and because 1,25D metabolism depends on sunlight [67], divergent regulation of antimicrobial functions between diurnal and nocturnal... fragments using the primers LucIR and RVP3 (95 °C for 45 s; four cycles of 95 °C for 45 s, 48 °C for 60 s, and 72 °C for 120 s; 22 cycles of 95 °C for 45 s, 54 °C for 60 s, and 72 °C for 120 s; 72 °C for 10 min) Gel-purified products were endo- FEBS Journal 275 (2008) 5074–5089 ª 2008 The Authors Journal compilation ª 2008 FEBS 5085 Sp1 and C ⁄ EBP regulate NRAMP1 transcription E Richer et al digested... or upstream activator sites [48], and which represses transcription by competing for binding site occupancy [12,49] Sp3 is another potential candidate factor, because it is closely related to Sp1, it also binds to ‘GC boxes’, and it may antagonize Sp1 effects to regulate target elements [50,51] However, in vivo evidence so far has shown less pronounced effects than Sp1 [reduced trans-activation in cotransfection... FEBS Sp1 and C ⁄ EBP regulate NRAMP1 transcription E Richer et al 7 Searle S & Blackwell JM (1999) Evidence for a functional repeat polymorphism in the promoter of the human NRAMP1 gene that correlates with autoimmune versus infectious disease susceptibility J Med Genet 36, 295–299 8 Roig EA, Richer E, Canonne-Hergaux F, Gros P & Cellier MF (2002) Regulation of NRAMP1 gene expression by 1alpha,25-dihydroxy-vitamin... (1997) Transcription factors, normal myeloid development, and leukemia Blood 90, 489–519 Kao WY, Briggs JA, Kinney MC, Jensen RA & Briggs RC (1997) Structure and function analysis of the human myeloid cell nuclear differentiation antigen promoter: evidence for the role of Sp1 and not of c-Myb or PU.1 in myelomonocytic lineage-specific expression J Cell Biochem 65, 231–244 Noti JD (1997) Sp3 mediates transcriptional . that C ⁄ EBP and Sp1 factors regulate NRAMP1 expression during myeloid differentiation. Discussion This study identified cis-acting sequences in the NRAMP1 promoter and transcription factors binding to. 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. observed when Sp1 and C ⁄ EBP factors were cotranfected in presence of the NR1L construct, some combinations being stimulatory (Sp1 + and C ⁄ EBPa + or b + plasmids) and some inhibitory (Sp1 and C ⁄