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The Journal of Immunology, 2006, 176: 1081-1089.
Copyright © 2006 by The American Association of Immunologists

Identification and Characterization of DC-SCRIPT, a Novel Dendritic Cell-Expressed Member of the Zinc Finger Family of Transcriptional Regulators1

Vassilis Triantis*, Dagmar Eleveld Trancikova*, Maaike W. G. Looman*, Franca C. Hartgers{dagger}, Richard A. J. Janssen{ddagger} and Gosse J. Adema2,*

* Department of Tumor Immunology, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands; {dagger} Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands; and {ddagger} Galádeno, Leiden, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DC) compose a heterogeneous population of cells that hold a leading role in initiating and directing immune responses. Although their function in recognizing, capturing, and presenting Ags is well defined, the molecular mechanisms that control their differentiation and immune functions are still largely unknown. In this study, we report the isolation and characterization of DC-SCRIPT, a novel protein encoded by an 8-kb mRNA that is preferentially expressed in DC. DC-SCRIPT is expressed in multiple DC subsets in vivo, including myeloid DC, plasmacytoid DC, and Langerhans cells. At the protein level, DC-SCRIPT consists of a proline-rich region, 11 C2H2-type zinc fingers, and an acidic region. Localization studies reveal that DC-SCRIPT resides in the nucleus and that nuclear localization is critically dependent on the zinc fingers. The protein displays no transcriptional activation properties according to assorted transactivation assays, but interacts with the corepressor C-terminal binding protein 1. Taken together, our results show that we have isolated a novel DC marker that could be involved in transcriptional repression. In contrast to other DC molecules, DC-SCRIPT identifies all DC subsets tested to date.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DC)3 play a pivotal role in adaptive immunity, because they are the initiators of immune responses. DC are APC that capture Ags in the periphery, process them, and present their peptides in the context of MHC molecules to T cells (1). To activate naive T cells, DC must be activated. Different stimuli such as bacterial or viral products (LPS and dsRNA) (2, 3, 4), cell-to-cell interactions (CD40-CD40L) (5), or soluble factors (TNF-{alpha}) (6) are able to activate DC, a process referred to as DC maturation. Upon maturation, DC undergo several functional and morphological changes, such as up-regulation of costimulatory molecules, enhanced peptide loading of MHC classes I and II, and dendrite formation, and migrate to T cell areas of secondary lymphoid tissue where they can launch immune responses (1).

DC are the most potent APC, and they are involved in immunity and tolerance (7). There are two main subsets described (type I IFN-producing plasmacytoid DC (PDC) and myeloid DC (MDC)) that originate from different hemopoietic lineages, but also a certain subset, Langerhans cells (LC), that resides exclusively at the skin. Distinct subsets may exert diverse functions, such as modulating the type of immune response (8, 9). Even though their functional capacity has been well demonstrated, and DC have already been used in clinical trials, the molecular mechanisms that lie beneath their potency are still, to a great extent, unknown. There have been several DC-specific molecules described, and many of them relate to their immunological capacity, such as DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN), DC-chemokine 1, DC-specific transmembrane protein (DC-STAMP), and langerin (10, 11, 12, 13), yet the molecular basis for their development and unique function remains largely unknown. Surely, DC induction of immune responses or tolerance must be fine-tuned, and a network of transcription factors has been implicated in DC development and immunobiology, including PU.1, SpiB, Id2, and RelB (14, 15, 16, 17). For example, SpiB promotes plasmacytoid DC development while blocking T, B, and NK cell development from hemopoietic precursors. Cross-talk between C/EBP transcription factors and PU.1 is required for myeloid DC development and differentiation. PU.1 is expressed in multiple hemopoietic lineages as well as CD34+ cells, and PU.1-null mice were unable to generate MHC class IIhighCD11c+ MDC in vitro. Id2 is induced by TGF-{beta} and was also shown to orchestrate LC development by repressing B cell genes in DC, whereas RelB, a component of the NF-{kappa}B complex of transcription factors, is a critical regulator of DC differentiation. In mice, the lack of RelB impairs DC derived from bone marrow in both number and function. Thus, a balanced network of transcription factors governs the development and function of DC.

To characterize more genes entangled in DC immunobiology, we previously applied differential display PCR (DD-PCR) to DC. We report the identification of a novel putative transcription repressor expressed in DC, DC-SCRIPT. DC-SCRIPT encodes for a unique protein with a putative DNA binding domain flanked by domains that could be involved in gene regulation. The gene is expressed by several DC subsets, both in vitro and in vivo, suggesting an important function for the protein in the differentiation pathway of DC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Leukocyte preparations

PBMC were obtained by leukapheresis of healthy donors, and adherence for 2 h resulted in a nonadherent PBL fraction. Monocytes were elutriated from PBMC by counterflow centrifugation and were stimulated with 2 µg/ml LPS for 16 h. DC were generated in vitro from adherent monocytes as described previously (18). Purified tonsil B lymphocytes were isolated as described previously (19). Blood DC were isolated from PBMC using the MACS Blood DC isolation kit (CLB). Mature CD11c+ (MDC) blood DC were obtained by culture in RPMI 1640 (Invitrogen Life Technologies) enriched with 10% FCS and 50% (v/v) MCM for 3 days. CD4+/CD11c– (PDC) blood DC were obtained by an additional immunomagnetic depletion of CD11c+ cells with microbeads (Dynal Biotech) and were matured in RPMI 1640 medium enriched with 10% FCS and 100 U/ml IL-3 (Sandoz) for 3 days, followed by 1 µg/ml CD40L for an additional 24 h. LC were isolated as cells that had migrated out of epidermal sheets derived from healthy donors undergoing plastic surgery of breast or abdomen (42 h) in the presence or absence of 500 U/ml GM-CSF (Schering-Plough). LC were enriched using anti-HLA-DR mAbs and MACS (CLB) and were >98% pure as analyzed by FACS.

DD-PCR

DD-PCR was performed as previously described (20). The 3' primers used were the anchored oligo(dT) primers T12MC, T12MA, T12MT, and T12MG, where M represents A, C, G, or T. The 5' primers were randomly designed oligonucleotides of 10 bases. PCR was performed in the presence of [35S]dATP to allow visualization of the products after separation by denaturing PAGE. PCR products that were reproducibly cell specific were eluted from the gel, reamplified by PCR, and cloned into pGEM-T (Promega). The cellular specificity of the clones was determined by RT-PCR, followed by Southern hybridization.

cDNA library screenings

cDNA libraries were prepared as described and screened using the randomly labeled 155-bp DC-SCRIPT fragment from the differential display PCR as a probe (primers, 5'-CCTGCTCATTTAGTCTAAGC-3' and 5'-TTCTGGAAGAATACTCACAGTT-3'). The most 5' end of the DC-SCRIPT cDNA was isolated by preparing a cDNA library with a DC-SCRIPT-specific primer (5'-GTCGCGAGCGGCCGCCCTGCTCATTTAGTCTAAGC-3'), using the SuperScript plasmid system for cDNA synthesis (Invitrogen Life Technologies) and subsequent screening of the library by Southern blot hybridization with a DC-SCRIPT-specific probe (primers used, 5'-CTCAGGGCTTTTCAGAGTAC-3', 5'-TCTGG AAGAATACTCACAGTT-3').

Northern blot analysis

For Northern blot analysis, total RNA was isolated with TRIzol reagent (Invitrogen Life Technologies), resolved on a formaldehyde gel, and transferred to a nylon membrane by capillary blotting. Hybridization was performed overnight at 65°C in Church solution (0.5 M NaHPO4 (pH 7.2), 7% SDS, and 0.5 M EDTA). All membranes were hybridized with a DC-SCRIPT-specific probe containing the most 3' of the open reading frame (ORF) and part of the 3'-untranslated region, obtained by PCR (forward primer, 5'-CTCAGGGCTTTTCAGAGTAC-3'; reverse primer, 5'-TCTGGAAGAATACTCACAGTT-3') and randomly labeled with 32P (T7 QuickPrime Kit; Pharmacia Biotech).

RT-PCR

Total RNA was transcribed into cDNA using an oligo(dT) primer and SuperScript II reverse transcriptase (Invitrogen Life Technologies). Primers for DC-SCRIPT were located in the original DD-PCR product, yielding a specific product of 144 bp (24 cycles; 5'-ACGGTTAGACTAAATGAGCAG-3', 5'-TTCTGGAAGAATACTCACAGTT-3'). As a control for RNA quality, {beta}-actin was amplified (18 cycles; 328 bp; forward and reverse primers, 5'-GCTACGAGCTGCCTGACGG-3' and 5'-CAGGCCAGGATGGATGGAGCC-3'). Southern blot analysis of the PCR products was performed using specific 32P-end-labeled internal oligonucleotides. For semiquantitative PCR analysis, 2.5–5 µg of DNase-treated total RNA was transcribed into cDNA using random hexamers and Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies). PCRs were performed in triplicate according to the TaqMan assay and were run on the ABI/PRISM 7700 Sequence Detector System (Applied Biosystems). The DC-SCRIPT-specific probe was labeled at the 5' end with a FAM fluorescent group and at the 3' end with a TAMRA quencher group. The primers used yield a specific product of 104 bp and surround intron 4, resulting in a product of >3 kb on genomic DNA. The amount of DC-SCRIPT expression was normalized to the housekeeping gene GAPDH and compared with the expression of another housekeeping gene, porphobilinogen deaminase within the same donor. Calculations were performed as described by PerkinElmer.

Cell lines, transfections, and transductions

Human embryonic kidney (HEK) 293 cells were cultured in DMEM (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FCS (Invitrogen Life Technologies), 10 nM HEPES (pH 7.7; Roche), 0.1 mM MEM nonessential amino acids, and 100 U/ml antibiotic-antimycotic (both from Invitrogen Life Technologies). THP-1 cells were cultured in RPMI 1640 medium (Invitrogen Life Technologies), 10% heat-inactivated FCS, and 100 U/ml antibiotic-antimycotic. 293 HEK cells (8 x 105) were plated in 6-well plates and transfected with 10 µl of Lipofectamine 2000 (Invitrogen Life Technologies) and 1 µg of DNA. Cells were harvested 1 day after transfection. THP-1 cells (4 x 106) were brought to a final volume of 0.8 ml and electroporated with 20 µg of DNA in total. Electroporation took place in a 0.2-cm cuvette (Bio-Rad) at 300 V and 960 µF. Day 6 immature human monocyte-derived DC were transduced with Ad5fib35hDC-SCRIPT-GFP at a multiplicity of infection of 1000, as described previously (Crucell) (21).

Plasmids

FLAG constructs were cloned in the pCATCH vector (22). For the transactivation assays, the pZd-VP16 and pZd plasmids were used as described previously (23), where different regions of DC-SCRIPT were cloned as BamHI-SalI fragments in the pZd vectors. pZ7E4Luc was described previously (24). Yellow fluorescence protein (YFP) fusion proteins were constructed in the pEYFP-C1 vector (BD Clontech), whereas the Myc-His fusion construct of DC-SCRIPT was derived from cloning the full-length ORF into the pcDNA4/TO/myc-His A vector (Invitrogen Life Technologies). Site-directed mutagenesis was performed with the QuikChange Site-Directed Mutagenesis kit (Stratagene), and the mutants were subcloned into the pGBT9 (EcoRI-BamHI) and pEYFP-C1 (BglII-XbaI)) vectors. Full-length C-terminal binding protein 1 (CtBP1) was inserted into the pFLAG-CMV-2 (Sigma-Aldrich) vector as a HindIII-SalI insert.

Yeast two-hybrid system

A yeast two-hybrid system was performed as described previously (25). Briefly, the acidic region of DC-SCRIPT was cloned into pGBT9 (BD Clontech) as an EcoRI-BamHI insert. A DC-derived cDNA library was inserted in pGAD-GH (BD Clontech) in the EcoRI-SalI sites. Bait and prey plasmids were transformed by 1 M sorbitol, 10 mM bicine, and 3% ethylene glycol into yeast strain YGHI. Protein-protein interactions were reported by yeast growth on medium without leucine, tryptophan, or histidine, and expression of {beta}-galactosidase was indicated by blue staining of yeast colonies after replica filter lifting, N2 snap-freezing and incubation for 2–4 h in Z-buffer (60 mM Na2HPO4, 60 mM NaH2PO4, 10 mM KCl, and 1 mM MgSO4) containing 1 mg/ml 5-bromo-4-chloro-3-indolyl-D-galactopyranoside.

Immunoblotting, immunoprecipitations, confocal microscopy, and transactivation assays

Whole-cell lysates were prepared in 1% SDS standard lysis buffer. Equal amounts of protein were separated by SDS-PAGE electrophoresis, and proteins were transferred to a Protran nitrocellulose transfer membrane (Schleicher & Schuell). The following primary Abs were used; mouse anti-GFP (0.04 µg/ml; Roche), mouse anti-Myc (0.2 µg/ml; Invitrogen Life Technologies), M2 mouse anti-FLAG 1 µg/ml (Sigma-Aldrich), mouse anti-EBV mAb, BZLF1 protein, ZEBRA, and clone BZ.1 (DakoCytomation) in combination with a second HRP-conjugated goat anti-mouse IgG (H+L) Ab (0.4 µg/ml; Pierce). Immunoprecipitation was performed in a standard RIPA/1% Triton X-100 buffer for 3 h at 4°C using protein G beads. For immunofluorescent staining, 293 HEK cells or DC were seeded on eight-chamber slides (Nunc) coated with fibronectin (20 µg/ml; Roche) or poly-L-lysine. Cells were fixed with methanol/acetone (1/1) and blocked with 3% BSA (Calbiochem) in PBS. The following Abs were used: M2 mouse anti-FLAG (Sigma-Aldrich), anti-{beta}1 integrin mAb TS2/16, and mouse E-12 anti-CtBP (Santa Cruz Biotechnology). As isotype controls, IgG2a and IgG1 mAbs (BD Biosciences) were used. As secondary Ab, Cy5-conjugated goat anti-mouse IgG, (H+L; Jackson ImmunoResearch Laboratories) and FITC- or Texas Red-conjugated goat anti-mouse IgG (H+L; Molecular Probes) were used. Nuclei were stained with propidium iodide. Slides were mounted with Vectashield (Vector Laboratories) and analyzed by confocal laser-scanning microscopy (Bio-Rad). Transactivation assays were performed using the Dual Luciferase Reporter Assay System (Promega) as proposed by the manufacturer. Luciferase measurements were calibrated using Renilla luciferase.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Differential display PCR identifies a novel DC-specific cDNA

Previously, we applied DD-PCR to identify novel transcripts that are specifically expressed by DC. Immature and mature monocyte-derived DC originating from two healthy donors were compared with a mixture of three monocytic, B, and T cell lines. The full-length cDNA corresponding to the 155-bp initial DD-PCR clone 203 was analyzed in further detail. As shown in Fig. 1A, clone 203 was distinctively present in DC, but not in the monocytic, B, or T cell lines. To confirm DC-specific expression of clone 203, RT-PCR with primers located within this 155-bp fragment was performed on an extensive panel of leukocyte populations and nonleukocytic cell lines (Fig. 1B). The analysis confirmed the preferential expression by DC and revealed that the adherent fraction of PBMC, which mainly consists of monocytes, expressed very low, but detectable, levels of the messenger. However, this might also be due to contamination with peripheral blood DC. Interestingly, the results demonstrated that clone 203 is preferentially expressed by DC. Therefore, we named the clone DC-SCRIPT (DC-Specific tranSCRIPT).



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  		FIGURE 1. DC-SCRIPT is specifically expressed in DC. A, Part of the gel loaded with differential display PCR samples. M, monocytic cell lines; T, T cell lines; B, B cell lines; D, donor’s immature DC; DS, donor’s mature DC. An arrow indicates the DC-specific product DC-SCRIPT. B, RT-PCR analysis of DC-SCRIPT expression. Upper panel, DC-SCRIPT; lower panel, {beta}-actin. DC were cultured for 7 days (DC), then activated with LPS (DC-LPS) or the combination of TNF-{alpha} and an activating Ab to CD40 (DC-act.). Nonadherent PBL and PBMC were stimulated with PHA and rIL-2. Elutriated monocytes were stimulated with LPS (16 h) or GM-CSF (5 days). B cells were isolated from tonsils. BLM is a human melanoma cell line, U2OS is an osteoblastic cell line; U937, Mono-Mac, and THP-1 are monocytic cell lines; EBV is a mixture of three EBV-transformed B cell lines; and Jurkat, CEM, and Peer are T cell lines. C, Northern blot analysis of DC-SCRIPT. Total RNA was isolated from freshly isolated leukocyte populations and cell lines (U937 and Jurkat). All RNA samples were fractionated through a formaldehyde agarose gel, blotted, and hybridized with a specific 32P-labeled DC-SCRIPT probe. DC-SCRIPT mRNA is indicated with an arrow. DC were cultured for 7 days (DC) and activated with LPS (DC+LPS). Adherent and nonadherent cells were stimulated with LPS, PHA, or rIL-2.

 
DC-SCRIPT gene encodes for an 8-kb messenger

Northern blot analysis with several different probes derived from the initial DC-SCRIPT cDNA clone identified a dominant RNA transcript of ~8 kb (Fig. 1C). The 8-kb RNA species was detected in both immature and mature DC (Fig. 1C, lanes 3 and 4), but not in PBMC (lane 5) or activated monocytes with LPS (lane 6). The nonadherent fraction (PBL) did not express any mRNA for DC-SCRIPT. The premonocytic cell line U937 and the T cell line Jurkat also did not express DC-SCRIPT mRNA (lanes 1 and 2, respectively). Tissue blot analysis showed a low level of expression in various tissues, including spleen, kidney, liver, heart, and placenta (data not shown). Possibly, such expression levels can be explained by residual DC in these tissues. Conclusively, DC-SCRIPT is found in DC, but not in other blood cell populations, resting or activated, confirming its preferential expression by DC.

DC-SCRIPT cDNA encodes a novel C2H2 zinc finger motif-containing protein

To obtain the full-length mRNA of DC-SCRIPT, we screened a DC cDNA library using the original 155-bp DD-PCR product as a probe. This resulted in the isolation of a 1.2-kb cDNA without an apparent ORF.

We next generated a DC cDNA library applying a specific primer residing at the 5' end of the 1.2-kb DC-SCRIPT cDNA. Several screening procedures of this library finally resulted in the isolation of a cDNA clone of ~3700 nt. Sequence analysis of this clone revealed the presence of a single 2232-nt-long ORF, starting with the first ATG codon at nt 448, which is in the appropriate sequence context for translation initiation. The protein encoded by this ORF consisted of a proline-rich domain (aa 111–219), followed by 11 C2H2 zinc finger motifs (aa 255–556) and an acidic region (aa 586–690; Fig. 2A). Additional analysis revealed the presence of a putative nuclear localization sequence (NLS) at position 77–80 and several possible phosphorylation sites spanning the entire molecule. In addition, there were two possible N-glycosylation sites in the zinc fingers (394–397 NCSE and 547–550 NLTR), suggesting that the protein can be diversely modified. The zinc fingers of DC-SCRIPT belong to the classical Cys-Cys:His-His subfamily of zinc fingers, as found in FOG-1 (friend of GATA1), transcription factor IIIA, and many other transcription factors. Zinc fingers can mediate protein-DNA, protein-RNA, or even protein-protein interactions in these transcription factors. The Cys-Cys:His-His motif, however, seems to be mainly involved in protein-DNA interactions (26, 27).



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  		FIGURE 2. Amino acid sequence of DC-SCRIPT. A, Amino acid sequence of the protein, with the corresponding regions underlined in color. B, Alignment of the region of homology between fZnf1 of F. rubripes and DC-SCRIPT. The region of homology is restricted within the zinc fingers, but not in the flanking regions of the orthologs.

 
Database searches revealed that DC-SCRIPT is identical with the human gene of ZNF366 (REFSEQ accession no. NM_152625.1) originally found to be homologous to the Fugu rubripes gene fZnf1 (28). However, the homology with fZnf1 is restricted solely to the zinc fingers region, which is 93% identical (Fig. 2B). Strikingly, outside the zinc fingers, DC-SCRIPT shares little or no homology with any other given protein, including transcription factors, underlining its unique identity within the family of zinc finger proteins.

Expression pattern of DC-SCRIPT in DC subsets

Using real-time semiquantitative PCR analysis, the expression of DC-SCRIPT in monocyte-derived DC was analyzed in further detail. Upon differentiation into immature DC with GM-CSF and IL-4, DC-SCRIPT is constitutively expressed from days 3–8 (data not shown). Stimuli such as CD40L, either alone or in combination with IFN-{gamma}, did not have a significant effect on the expression level of DC-SCRIPT, whereas another DC-specific gene, DC-STAMP, was down-regulated under these conditions (Fig. 3A). The expression level of DC-SCRIPT was comparable to that of the housekeeping gene PBGD (Fig. 3A).



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  		FIGURE 3. Expression pattern of DC-SCRIPT. A, Quantitative expression of DC-SCRIPT by in vitro cultured DC, freshly isolated blood DC, and LC. The expression of DC-SCRIPT in immature (day 6) and mature monocyte-derived DC (plus CD40L or CD40L and IFN-{gamma}), compared with PBGD and DC-STAMP. B, Freshly isolated blood DC (day 0), blood DC plus MCM (day 3), and CD11c– blood DC plus IL-3/CD40L were studied. C, LC that have migrated out of epidermal sheets in the absence or the presence of GM-CSF. Each graph represents data from one representative donor of two or more.

 
To investigate the expression of DC-SCRIPT by DC subsets in vivo, we isolated peripheral blood DC. Blood DC mainly consist of two defined subsets, CD11c+ MDC and CD11c– PDC. Freshly isolated blood DC as well as cultured and CD40-activated PDC and MDC clearly expressed DC-SCRIPT, as shown in Fig. 3B. DC-STAMP was not expressed in freshly isolated blood DC and was only up-regulated during activation of the MDC, not in activated PDC. LC isolated from epidermal skin layers, were also positive for DC-SCRIPT, with or without the addition of GM-CSF (Fig. 3C). LC did not express DC-STAMP, illustrating once more that DC-SCRIPT is a novel marker expressed by all DC subsets tested to date.

Analysis of DC-SCRIPT protein

To characterize the DC-SCRIPT protein, we generated constructs encoding N- or C-terminal-tagged DC-SCRIPT fusion proteins: FLAG-DC-SCRIPT, DC-SCRIPT-GFP, and DC-SCRIPT-Myc-His. 293 HEK cells were transiently transfected with these constructs, and lysates were analyzed by SDS-PAGE and Western blotting using mAbs directed against the different tags. DC-SCRIPT is estimated to be ~75 kDa. In all cases, however, a specific protein band was detected of a somewhat larger size than the calculated size of the tagged DC-SCRIPT constructs (Fig. 4). Additional analysis demonstrated that the FLAG-DC-SCRIPT fusion protein migrated at the same position in the gel under both denaturing and nondenaturing conditions (data not shown). Possibly the size difference can be explained by the irregular electrophoretic mobility of the acidic C-terminal part of DC-SCRIPT. The electric charge of this region could affect its migration during SDS-PAGE. Alternatively, the difference in size could be attributed to some type of protein modification.



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  		FIGURE 4. Protein analysis of DC-SCRIPT. Western blots were performed on 293 HEK transiently transfected cell lysates. Lysates were analyzed with the corresponding Abs, whereas 293 HEK nontransfected cells were included as negative controls. Arrows indicate the detected products and their sizes. The estimated size of DC-SCRIPT is ~85 kDa.

 
DC-SCRIPT localizes to the nucleus

To investigate the localization of DC-SCRIPT, DC-SCRIPT-YFP and a series of DC-SCRIPT-YFP deletion mutants were constructed (Fig. 5A). These constructs were transfected into 293 HEK cells. Part of the cells were cultured on fibronectin-coated slides and analyzed by confocal laser scanning microscopy, whereas the remainder was used to make lysates and verify protein expression. All DC-SCRIPT-YFP deletion mutants were properly expressed at the protein level (Fig. 5A). Full-length DC-SCRIPT-YFP localized to the nucleus of cells, as shown by simultaneous propidium iodide staining of DNA (Fig. 5B). We noted that both full-length FLAG-DC-SCRIPT and DC-SCRIPT-Myc-His localized to the nucleus, indicating that the nuclear localization is not affected by the tag (data not shown). Interestingly, neither deletion of the N-terminal region containing the putative NLS nor of the C-terminal part of DC-SCRIPT affected its nuclear localization (Fig. 5B). The construct containing the zinc finger region alone could drive the molecule in the nucleus. YFP can spontaneously localize in the nucleus and thus influence the outcome of such experiments. Therefore, we analyzed the localizations of two additional FLAG-tagged constructs (zinc-acidic and acidic regions). Also in this setting, the zinc fingers-containing construct was localized in the nucleus, confirming that this motif can determine the localization of the protein (Fig. 5B).



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  		FIGURE 5. Localization of DC-SCRIPT. A, Schematic representation and protein expression of the corresponding constructs used for confocal microscopy. Western blots were performed on the lysates from the same transfections as in B and stained with the corresponding Abs. In the case of FLAG fusion constructs, multiple specific bands were observed, which can be attributed to posttranslational modifications. B, Cellular localization of DC-SCRIPT fusion proteins with different tags. Nuclei were stained by propidium iodide (red), whereas YFP autofluorescence is shining in the green channel. The FLAG epitope was visualized by means of FITC-coupled secondary Ab.

 
Transcriptional activity of DC-SCRIPT

To assess any possible transcriptional activity of DC-SCRIPT, we used the ZEBRA transactivation system described previously (23, 24). This system is based on the EBV transcription factor Zebra (BZLF 1). Selected parts of DC-SCRIPT were cloned into a pZd vector as shown in Fig. 6A. Zd encodes for a mutant Zebra protein lacking the transactivation domain of Zebra, but retains the DNA binding and dimerization domain of the native protein. The reporter construct contained seven Zebra-responsive elements upstream of the E4 minimal promoter coupled to luciferase (Fig. 6A). Transfection efficiencies were calibrated by means of a Renilla promoterless construct that had basal activity. The native ZEBRA protein and a fusion protein of the transactivation domain of VP16 to Zd (not shown) were used as positive controls. Western blots were performed to verify expression of the constructs in 293 HEK cells (data not shown). Experiments were repeated in 293 HEK and THP-1 cells, but none of the constructs was able to induce luciferase expression in either 293 HEK or THP-1 cells (Fig. 6B). We also investigated whether lack of transactivation was dependent on cell activation. Therefore, we repeated the experiment in THP-1 cells using the full-length protein of DC-SCRIPT, and cells were activated with 100 ng/ml PMA. PMA is known to activate the NF-{kappa}B pathway (29), and we hypothesized that DC-SCRIPT could be a downstream target of such activation. However, DC-SCRIPT failed to activate transcription in this setting as well (Fig. 6B).



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  		FIGURE 6. Transactivation capacity of DC-SCRIPT. A, Schematic representation of the effector and reporter constructs used during the transactivation assays with the Zebra system. DB, DNA binding domain; DZ, dimerization domain; P, proline-rich region; Zn, zinc fingers; Ac, acidic region; ZB, Zebra-responsive element; TATA, E4 minimal promoter. Besides the constructs, the DC-SCRIPT amino acids fused to the ZEBRA deletion mutant are indicated. B, Effect of DC-SCRIPT on transcription in THP-1 and 293 HEK cells. Results represent fold times luciferase expression. The inactive deletion mutant of ZEBRA Zd has a value of 1. Results were calibrated by use of Renilla luciferase.

 
DC-SCRIPT interacts with the corepressor CtBP1

Because the transactivation assays failed to give a clear picture of DC-SCRIPT’s function, we performed yeast two-hybrid system to identify cooperating molecules that might help illuminate the role of DC-SCRIPT. We used a DC-derived cDNA library to isolate clones that could specifically interact with the acidic region of DC-SCRIPT. Positive colonies arose on days 5 and 8. Sequencing revealed CtBP1 as one of the interacting molecules. CtBP1 was established as a strong binder of DC-SCRIPT (estimated from the {beta}-galactosidase assay) and was represented by five independent colonies in total. CtBP1 is a corepressor that can recruit histone deacetylases at the site of transcription and consequently assist a tighter packing of chromatin and silencing of the locus. CtBP1 binds its interactors via the PXDLS motif, where X can be any amino acid residue. Looking more closely at the protein sequence of DC-SCRIPT, we identified two such motifs within the acidic region that could serve as interaction sites for CtBP1 (Fig. 2A), one at position 590–594 (PFDLS) and one at position 645–649 (PEDLS). To determine whether these motifs were functional, we performed site-directed mutagenesis, mutating alternatively the first, second, or both (L593A and L648A). Using these mutants of the acidic region together with CtBP1 in the yeast two-hybrid system we established that only the first motif is responsible for CtBP1 binding (Fig. 7A). Only the mutant that retained intact the first motif could interact with CtBP1 and be positive in the {beta}-galactosidase assay.



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  		FIGURE 7. DC-SCRIPT interacts with CtBP1. A, Yeast transformed with wild-type and deletion mutants of the acidic region together with CtBP1 ({beta}-galactosidase assay). Ac, acidic region wild type; Ac/mut1, acidic region with the first binding motif mutated; Ac/mut2, acidic region with the second binding motif mutated; Ac/double mut, acidic region with both binding motifs mutated. B, 293 cells transfected with wild-type and deletion mutants of the acidic region together with CtBP1. CtBP1 interacts with DC-SCRIPT only if the first motif is intact, whereas the second motif does not influence the interaction of the two proteins.

 
DC-SCRIPT binds CtBP1 in vivo

To confirm DC-SCRIPT binding to CtBP1 in a mammalian system, we performed coimmunoprecipitations in 293 HEK cells. 293 HEK cells were transfected with FLAG-CtBP1 and YFP-fusion DC-SCRIPT constructs (Fig. 7B). Transfection with YFP together with FLAG-CtBP1 was used as a control. Another negative control was another region of DC-SCRIPT that normally should not bind to CtBP1 (zinc fingers). The same mutations that were tested in the yeast two-hybrid system were examined in 293 HEK cells for their ability to bind CtBP1. The results demonstrated that DC-SCRIPT could specifically precipitate CtBP1. Moreover, when we mutated the first site, DC-SCRIPT could no longer immunoprecipitate CtBP1. However, altering the second site did not have any affect on the binding of CtBP1. Consequently, when both sites were mutated, then CtBP1 could not interact with DC-SCRIPT. In accordance with the yeast two-hybrid results, DC-SCRIPT can bind CtBP1 in 293 HEK cells, and the motif at positions 590–594 is responsible for bringing together the two proteins.

DC-SCRIPT colocalizes with CtBP1 in DC

To study DC-SCRIPT in its native cellular environment, we generated immature DC from PBMC with GM-CSF and IL-4 and transduced them with an adenovirus encoding for DC-SCRIPT-GFP. Transduced DC were attached on poly-L-lysine slides fixed and stained with Ab directed against endogenous CtBP1. As shown in Fig. 8A, DC-SCRIPT-GFP was confined to the nucleus, whereas DC transduced with GFP alone gave an overall staining pattern in DC. Costaining of the DC for endogenous CtBP1 revealed that CtBP1 also localized to the DC’s nucleus (Fig. 8B). Colocalization was obvious in all cases, suggesting that DC-SCRIPT and CtBP1 could interact in DC.



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  		FIGURE 8. DC-SCRIPT and CtBP1 colocalize in the nucleus of DC. A, DC were transduced by means of an adenovirus encoding for DC-SCRIPT-GFP or GFP alone (green) and were plated on poly-L-lysine-coated slides. An anti-{beta}1 integrin Ab was used to stain the membrane (red). B, DC were prepared as described in A. Endogenous CtBP1 was visualized by means of an anti-CtBP1 Ab (blue), whereas the nucleus was stained red with propidium iodide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
DC are the instructors of adaptive immune responses by guiding naive T cells against Ags that were captured in the periphery. Today DC are used in clinical trials against various types of cancer (30), but as yet little is known regarding the molecular signature of a DC. To date, various markers with expression restricted to DC have been described with variable functions. DC-SIGN, a C-type lectin involved in pathogen recognition (31, 32, 33, 34); DC-chemokine 1, a chemokine recruiting naive T cells to germinal centers (11); langerin, another type II lectin (13); and DC-STAMP, a multiple membrane-spanning receptor that resides in the endoplasmic reticulum with an as yet unknown function (35), are some of these markers. However, none of these markers is expressed by all types of DC. For example, DC-SIGN is also expressed by macrophages under certain conditions (34), and although DC-STAMP is absent from LC (Fig. 3A), langerin is restricted to these cells (13).

In this article, we describe a novel DC marker, DC-SCRIPT. DC-SCRIPT is preferentially expressed by DC among the leukocyte populations tested. Expression of the messenger is an early hallmark of DC differentiation from monocytes. These findings suggest that DC-SCRIPT is essential for DC function and immunobiology. DC-SCRIPT is present at the mRNA level in various DC subsets, with an enduring expression throughout the DC life cycle. During activation, DC-SCRIPT is not significantly down- or up-regulated regardless of the stimulus applied. Other crucial proteins engaged in numerous DC subset differentiation and development also portray the same expression pattern while retaining steady levels during activation. SpiB and PU.1, for example, seem to govern plasmacytoid DC and LC development, respectively, and their expression is kept stable upon activation (15, 36).

Apart from its distinct expression pattern, DC-SCRIPT bares 11 Cys:Cys-His:His Zn fingers, a proline-rich domain, and an acidic region. Therefore, it seems to represent a transcription factor, but beyond the zinc fingers, it shares no homology with proteins. The N-terminal part of the protein contains a classical NLS. Such sequences are described to be necessary for nuclear import, and the major types of NLSs fall into two categories. The first type consists of a single cluster of basic amino acids, for example, PKKKRKV in the SV40 large T Ag NLS (37). The second type is the bipartite NLS, composed of two clusters of basic amino acids separated by eight to 16 random residues, i.e., KRPAATKKAGQAKKKK as found in nucleoplasmin (38). DC-SCRIPT contains a typical NLS (RKRK), albeit this does not seem to be essential for translocation of the protein in the nucleus, because DC-SCRIPT lacking the NLS still localizes in the nucleus. Our results show that the zinc finger domain alone can facilitate nuclear import of the whole molecule (Fig. 5B). However, no NLS-like stretch is present in this region, suggesting that there must be another mechanism for targeting the protein into the nucleus. Indeed, it has been shown that proteins such as pancreatic transcription factor-1 are carried in the nucleus via protein-protein interactions, rather than using an inherent NLS. In such a scenario, the protein uses a "Trojan horse" that takes the complex in the nucleus (39). The DNA binding domain encompasses 11 zinc fingers of the classical Cys-Cys:His-His type. Zinc fingers come in many types and forms, serving different functions. They can be DNA binding mediators or binding platforms for other proteins, such as RING fingers (26), or even bind themselves to RNA (27). In addition, there are examples of proteins with NLSs embedded in their DNA binding domain, such as Wilms’ tumor 1, JAZ, mouse orphan receptor (TR2), and erythroid Kruppel-like factor/Kruppel-like factor 1 (37, 40, 41). In these cases, the tertiary structure of the zinc fingers or even residues within some of the zinc fingers are sufficient for nuclear localization of the protein. Once the protein is in the nucleus, the zinc fingers retain it within by anchoring the protein to other nuclear targets or DNA. Indeed, DC-SCRIPT has basic residues within its zinc fingers that together can act as a less well-defined NLS, similar to erythroid Kruppel-like factor/Kruppel-like factor 1, but it can be assumed as well that some of the zinc fingers are responsible for nuclear import and retention, whereas others are binding DNA or interact with other proteins, comparable to CCTC binding factor, another 11-zinc finger protein (42). Therefore, DC-SCRIPT may use different combinations of zinc fingers to exert different functions.

We also investigated whether DC-SCRIPT has an effect on transcriptional activation. However, no such effect could be observed. Therefore, we applied the yeast two-hybrid system to identify cooperating molecules that could hint about the role of DC-SCRIPT. One of the molecules produced was CtBP1. CtBP1 is a corepressor that recognizes the PXDLS motif as an anchoring site on other proteins (43). DC-SCRIPT bares two such sites in its acidic region, and by site-directed mutagenesis, we pinpointed the exact site of the interaction. CtBP1 cooperators can be DNA binding proteins that use CtBP1 as an intermediate to finally recruit histone deacetylaces and block transcription of their binding locus (44, 45, 46). We have evidence that DC-SCRIPT is a DNA-binding protein, and cooperation with CtBP1 can represent a repression mechanism for DC-SCRIPT. That could explain the lack of transcriptional activation of the constructs tested in Fig. 6. Interestingly enough, CtBP1 has also been implicated in hemopoiesis by interacting with members of the Ikaros family of proteins (47).

Taken together, our results show that DC-SCRIPT is the first marker for DC that has been found in all DC subsets tested to date and probably represents a transcriptional repressor. Nevertheless, its role in DC immunobiology remains evasive. Future studies including knockout mice are essential to answer this question.


   Acknowledgments
 
We thank M. B. M. Teunissen (University Medical Center, Amsterdam, The Netherlands) for providing us with LC populations and A. A. C. Lemckert and M. J. E. Havenga (Crucell Holland, Leiden, The Netherlands) for providing the adenoviruses used in this study.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
V. Triantis, M. W. G. Looman, and G. J. Adema are with the University Medical Center in Nijmegen, The Netherlands, and are among the inventors of a patent with the provisional title of "Dendritic cell polypeptide DC-SCRIPT and application in immune modulation".


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants 901-10-092 and 912-02-034 from the Dutch Foundation for Scientific Research. Back

2 Address correspondence and reprint requests to Dr. Gosse J. Adema, Department of Tumor Immunology 187, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. For express deliveries: Geert Grooteplein Zuid 28, 6525 GA Nijmegen, The Netherlands. E-mail: g.adema{at}ncmls.ru.nl Back

3 Abbreviations used in this paper: DC, dendritic cell; DD-PCR, differential display PCR; HEK, human embryonic kidney; LC, Langerhans cell; MDC, myeloid DC; NLS, nuclear localization sequence; PDC, plasmacytoid DC; ORF, open reading frame; YFP, yellow fluorescence protein; DC-SIGN, DC-specific ICAM-3 grabbing nonintegrin; DC-STAMP, DC-specific transmembrane protein; CtBP1, C-terminal binding protein 1. Back

Received for publication April 27, 2005. Accepted for publication October 31, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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V. Triantis, V. Moulin, M. W. G. Looman, F. C. Hartgers, R. A. J. Janssen, and G. J. Adema
Molecular characterization of the murine homologue of the DC-derived protein DC-SCRIPT
J. Leukoc. Biol., May 1, 2006; 79(5): 1083 - 1091.
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