The JI Acurri Cytometers
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bros, M.
Right arrow Articles by Ross, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bros, M.
Right arrow Articles by Ross, R.
The Journal of Immunology, 2003, 171: 1825-1834.
Copyright © 2003 by The American Association of Immunologists

The Human Fascin Gene Promoter Is Highly Active in Mature Dendritic Cells Due to a Stage-Specific Enhancer1

Matthias Bros2, Xiao-Lan Ross, Andrea Pautz, Angelika B. Reske-Kunz and Ralf Ross3

Clinical Research Unit Allergology, Department of Dermatology, Johannes Gutenberg University, Mainz, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC), regarded as the most efficient APCs of the immune system, are capable of activating naive T cells. Thus, DC are primary targets in immunotherapy. However, little is known about gene regulation in DC, and for efficient transcriptional targeting of human DC, a suitable promoter is still missing. Recently, we successfully used the promoter of the murine actin-bundling protein fascin to transcriptionally target DC by DNA vaccination in mice. In this study, we report on isolation of the human fascin promoter and characterization of its regulatory elements. The actively expressed gene was distinguished from a conserved inactive genomic locus and a continuous region of 14 kb covering the gene and 3 kb of 5'-flanking sequences was subcloned, sequenced, and analyzed for regulatory elements. Regulatory sequences were found solely in the 5'-flanking promoter region. The promoter exerted robust activity in DC and a fascin-positive neuronal cell line, but not in the fascin-negative cells tested. Notably, promoter activity in DC markedly increased with maturation of DC. By progressive 5' deletion, we identified a core promoter region, harboring a putative GC box, a composite cAMP responsive element/AP-1 binding site and a TATA box. By internal deletion, we demonstrated functional importance of either regulatory element. Furthermore, we identified a more distal stage-specific enhancer region also containing silencer elements. Taken together, the human fascin promoter allows for transcriptional targeting of mature DC and represents a promising tool for immunotherapy. To our knowledge, this study reports for the first time on promoter activity in human monocyte-derived DC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC),3 the most potent APC of the immune system, are capable of efficiently priming naive T cells (1). DC are of hemopoietic origin and reside in an immature state in nearly all peripheral tissues. As sentinels, they are equipped for effective capture, uptake, and processing of pathogen-derived proteins. Upon activation, DC migrate to the peripheral lymphoid organs, present processed antigenic peptides to T cells, and, thereby, elicit Ag-specific immune responses. Activation-induced maturation of DC is accompanied by fundamental functional changes.

We analyzed maturation of Langerhans cells (LC), i.e., immature DC with sentinel function of the epidermis, into primary stimulatory DC on the mRNA level applying differential screening techniques, namely differential library screening and differential display, and observed that maturation is accompanied by extensive differential gene expression (2, 3). Some differentially expressed genes were analyzed in detail (3, 4, 5) including the murine chemokine CC chemokine ligand 22, which is expressed by mature DC and activated B cells and attracts activated T cells (5, 6). In addition, we demonstrated de novo expression of the actin-bundling protein fascin in maturing LC (7).

Fascin is an evolutionarily highly conserved cytoskeletal protein of 55 kDa containing two actin binding domains that cross-link filamentous actin to hexagonal bundles (8). In addition to DC (9), fascin is expressed by neuronal and glial cells (10) and capillary endothelial cells (11). Perinuclear distribution of fascin in neuronal and glial cells suggests a stabilizing role in cell structure. Moreover, fascin was reported to be necessary for rapid movement of cell extensions of growth cones of neuronal cells and of stress fibers of glial cells (8). Consistently, fascin is involved in cell motility, as has been shown by intracellular treatment with inhibitory anti-fascin Abs (12), and by fascin overexpression (13). By confocal microscopy, we showed that fascin is distributed in mature DC submembraneously, especially including the numerous filopodia-like dendritic cell extensions (7) that were name-giving for DC. Furthermore, we demonstrated that inhibition of fascin expression by antisense oligonucleotide approaches impaired formation of dendrites both in murine LC (7) and human monocyte-derived DC (14).

As suggested by its relevance for cell motility in other cell types, fascin may be important for migration of activated DC. Moreover, fascin-dependent dendrites of DC might be involved in formation and maintenance of contact to T cells, because filamentous actin and fascin were found to be focally polarized in DC at the immunologic synapse formed with clustered allogenic Th cells (15), and T cell proliferation was markedly decreased after pretreatment of murine bone marrow-derived DC with fascin-directed antisense oligonucleotides (16).

Recently, we isolated the murine fascin promoter and showed that the promoter allows for transcriptional targeting of mature DC in gene gun immunization of mice. Strong immune responses were elicited against the encoded Ags. Moreover, cellular responses were clearly favored over humoral responses (17).

In this report, we describe isolation of the human fascin gene. Remarkably, two genomic loci were detected; however, only one locus comprises an actively transcribed gene. We searched for regulatory elements in the 5'-flanking promoter region, in exons, introns, and in the 3'-untranslated region. Our results indicate that fascin expression is controlled mainly by cis-acting elements located within a 3-kb promoter region. Besides a proximal core promoter, silencer elements diminished reporter activity in fascin-negative cells. A stage-specific enhancer region conferred strong promoter activity in mature immunostimulatory DC but not in immature DC, which are known to induce nonproliferating, IL-10-producing, negative regulatory T cells (18). Thus, the promoter provides new promising implications for immunotherapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

The human neuroblastoma cell line SHSY-5Y was cultured in a 1/1 mixture of DMEM and Nut Mix F12, the human promonocytic cell line THP-1 in RPMI 1640, and the human keratinocytic cell line HaCaT in DMEM. The culture media (Life Technologies, Karlsruhe, Germany) were supplemented with 5% heat-inactivated FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Life Technologies), 2 mM L-glutamine, and 1 mM sodium pyruvate (Carl Roth, Karlsruhe, Germany). Human DC were generated from PBMC as described (14). Briefly, monocytes were enriched from PBMC by plastic adherence, DC were generated by a 7-day culture in RPMI 1640 supplemented with 3% autologous plasma, 800 U/ml of recombinant human (rh) GM-CSF, and 1000 U/ml rhIL-4, and maturation of DC was induced by 1–3 day cultivation with a cytokine mixture consisting of rhGM-CSF (800 U/ml), rhIL-4 (500 U/ml), rhIL-1{beta} (10 ng/ml), rhIL-6 (1000 U/ml), rhTNF-{alpha} (10 ng/ml), and PGE2 (1 µg/ml). All cytokines were purchased from Strathmann Biotech (Hannover, Germany) except for rhGM-CSF (Sandoz, Nürnberg, Germany). PGE2 was obtained from Pharmacia & Upjohn (Erlangen, Germany). Cells were used for transfection 24 h later.

Immunomagnetic separation of CD83-positive mature DC

Mature DC were purified from DC cultures on day 8 by immunomagnetic separation using the CELLection Pan Mouse IgG kit (Dynal, Hamburg, Germany) as recommended by the manufacturer. Before use, paramagnetic beads were coated with mouse anti-human CD83 mAb (clone HB15e; BD PharMingen, Hamburg, Germany) binding specifically to mature DC. The specificity of Ab-mediated binding of paramagnetic beads to DC was monitored by light microscopy. After magnetic separation, the CD83-positive cell fraction was incubated with DNase I to digest the DNA spacer linking mouse IgG4 Ab to the bead surface. After magnetic separation, CD83-positive mature DC were used for DNA transfection as described.

Flow cytometric analysis

Intracellular fascin staining was performed as described (14). Cells were permeabilized with methanol for 5 min at room temperature, and washed twice with PBS/2% FCS to remove fixative. Anti-human fascin mAb 55K-2 (19) was a generous gift by Dr. F. Matsumura (Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ). Corresponding IgG1 isotype control mAb and dichlorotriazinyl aminofluorescein-conjugated goat anti-mouse IgG Ab were purchased from BD PharMingen.

RT-PCR amplification

mRNA was isolated using the QuickPrep Micro mRNA Purification kit as recommended (Amersham Pharmacia Biotech, Uppsala, Sweden). Reverse transcription (RT) reaction was performed as described (20). RNA concentration and efficiency of RT reaction were assessed by RT-PCR using primers specific for the housekeeping gene hypoxanthine phosphoribosyltransferaseas reported (4). Hypoxanthine phosphoribosyltransferase-standardized amounts of cDNA were subjected to PCR with fascin-specific primers. Genomic DNA was prepared from peripheral blood leukocytes (Caucasians) using the QIAamp Blood kit (Qiagen, Hilden, Germany) as recommended or was kindly given to us by Dr. P. Schneider (Institute of Legal Medicine, Mainz, Germany) (Chinese, Japanese (21), anthropoids (22)). All RT and amplification reactions were performed using a Model 480 DNA Thermal Cycler (PerkinElmer, Foster City, CA). The following primers obtained from MWG-Biotech (Ebersberg, Germany) were used for amplification: mFasORF-1/2 (5'-GCC ACC ATG ACC GCC AAC GG-3', 5'-TGT GTC GCG GTC GAT CTC CA-3'), hFasORF-1/2 (5'-AGG CGG GCA AGG CCA CCA AG-3', 5'-ACA TCG AGT GGC GTG ACC GGC-3'), hFasUTR-1/2 (5'-GGC AAG CCT GGC TGT AGT AG-3', 5'-CCA GAG TGA GAT GCA TGT TGG G-3'), hFasINT-1 (5'-CCG TGG TGT TAC CTT GCG TGT GTA G-3'). Denaturation (95°C for 1 min) and extension steps (72°C for 2 min) remained constant. Annealing steps were conducted for 1 min as indicated below. A final extension step (7 min at 72°C) ensured complete double-strand polymerization and was part of each PCR. Templates were heat-denatured (98°C for 5 min) and rapidly cooled on ice before PCR. Reaction mixtures contained 5% (v/v) DMSO.mFasORF-1/2 amplify part of the mouse fascin open reading frame (ORF) (annealing: 2 cycles 70°C, 2 cycles 69°C, 2 cycles 68°C, 30 cycles 67°C) to yield probe mFas-ORF; hFasORF-1/2 amplify part of the human fascin ORF (annealing: 2 cycles 68°C, 2 cycles 67°C, 30 cycles 66°C); hFas untranslated region (UTR)-1/2 amplify part of human fascin cDNA 3' UTR (annealing: 2 cycles 66°C, 2 cycles 65°C, 30 cycles 64°C) to yield probe hFas-UTR. hFasINT-1 was used in combination with hFasORF-1 to amplify part of intron 1/exon 2 (annealing: 2 cycles 68°C, 2 cycles 67°C, 30 cycles 66°C). Probes mFas-ORF and hFas-UTR were labeled during PCR with digoxigenin-11-dUTP as recommended by the supplier (PCR DIG Probe Synthesis kit; Roche Molecular Biochemicals, Mannheim, Germany).

Isolation and characterization of human genomic fascin clones

A human genomic library derived from PBMC (library no. 704) (23) was obtained from the Resource Center/Primary Database, (Heidelberg, Germany). The library was screened by hybridization with probe mFas-ORF. Within the range of the probe, mouse and human fascin cDNA show 91% sequence identity at the nucleotide level (GenBank accession nos. U03057, HSU09873, and L33726). Probe hFas-UTR was used to identify genomic restriction fragments containing 3' parts of the gene. Hybridization reactions and signal detection were performed as reported (24). PAC clones selected following library screening were used for DNA preparation (NucleoBond Plasmid kit; Clontech Laboratories, Palo Alto, CA). Hybridization with murine fascin cDNA probe was verified by Southern blot analysis. Fragments were subcloned into pZErO-2.1 (Invitrogen, Groningen, The Netherlands). Nucleotide sequences were determined by cycle sequencing using the ABI PRISM Dye Termination Cycle Sequencing Ready Reaction kit as recommended and analyzed on a PE 373A DNA sequencer (Applied Biosystems, Foster City, CA). GC-rich DNA templates were linearized, heat-denatured (98°C for 5 min), and mixed with 5% (v/v) DMSO before sequencing. Potential transcription factor binding sites were identified by MatInspector version 2.2 (http://transfac.gbf.de; core similarity: 0.75, matrix similarity: 0.85). Transcription factors mentioned in the text are named according to the first corresponding publication.

Reporter gene constructs

For luciferase reporter assays, appropriate fragments of the fascin gene and of retroposon {psi}hFP1 were subcloned into the promoterless luciferase expression vector pGL3-Basic (Promega, Heidelberg, Germany). Nested deletions of promoter fragments were conducted using the double-stranded Nested Deletion kit (Amersham Pharmacia Biotech). To normalize absolute firefly luciferase reporter expression values for differences in transfection efficiency and cell recovery, renilla luciferase expression construct pRL-CMV was cotransfected. To exclude differences in coreporter activity due to potentially cytokine-mediated stimulation of the CMV promoter (25) and to allow for comparison of luciferase activity between cell types, activity of pRL-CMV was compared with pGL3-EF1{alpha}1. To generate pGL3-EF1{alpha}1, the human EF1{alpha}1 promoter (26), which is regulated mainly by GC box binding transcription factors, was used to drive constitutive firefly luciferase expression by insertion of the EF1{alpha}1 promoter comprising ~200 bp of the upstream flanking region, exon 1, intron 1, and the first 10 bp of exon 2 of the gene, derived from plasmid pEF-Bos lacZ (27), into pGL3-Basic.

For enhanced green fluorescent protein (EGFP) reporter gene assays, EGFP ORF, originally derived from pCMV{beta} (Clontech Laboratories) was inserted into pZErO-2.1. The bacterial ccdB selection cassette and the lacZ promoter of pZErO-2.1 were deleted, generating promoterless pEGFP. The 3.1-kb fascin promoter was inserted to drive the expression of EGFP (phFascin-EGFP). In promoter studies, pEGFP-N1 (Clontech Laboratories) served as positive control, and promoterless pEGFP was used as negative control. To allow for verification of comparable transfection efficiency, EGFP expression constructs were cotransfected with a renilla luciferase reporter construct.

Transfection

Transfection of human monocytes and monocyte-derived DC was performed by biolistic gene transfer using the helium-driven PDS-1000/He system (Bio-Rad, Hercules, CA) as described (28). For transfection of monocytes, 3 x 106 PBMC were seeded into 60-mm culture plates (Corning Costar), and plates were incubated for 1 h at 37°C to allow for adherence of monocytes. Nonadherent leukocytes were rinsed off and plates were placed into the biolistic chamber for particle bombardment. The distance between the macrocarrier holder and the plate was 6 cm. A blasting pressure of 450 pounds per square inch (psi) was chosen based on optimization experiments.

For transfection of DC, 5 x 105 cells were placed into a Transwell (24-mm diameter, 3-µm pore size; Corning Costar, Corning, NY) and excess medium was removed. Biolistic transfection was conducted at a blasting pressure of 900 psi.

In some experiments, DC were transfected using the Nucleofector System (Amaxa, Köln, Germany) according to the recommendations of the manufacturer. Briefly, 1 x 106 DC were resuspended in 100 µl of human Dendritic Cell Nucleofector Solution (Amaxa) and mixed with 5 µg of plasmid DNA. Electroporation was performed using the Nucleofector program U-02. Afterward, cells were cultured for 24–48 h in the DC maturation mixture (see Cell culture) in 12-well plates.

Initial testing of fascin promoter 5' deletion constructs in THP-1 and SHSY-5Y was performed by biolistic transfection as well. THP-1 suspension cells (106) were transfected as described for DC. Adherent SHSY-5Y (106 cells) were seeded into 60-mm culture plates on the day before transfection, and cells were transfected with a blasting pressure of 450 psi. For all biolistic transfections, 4.5 pmol of test construct and 0.5 pmol of coreporter were used.

In some experiments, cell lines were transfected by lipofection using GenePorter (Gene Therapy Systems, San Diego, CA). At the day before transfection, 5 x 105 cells of adherent SHSY-5Y or HaCaT were seeded into 24-well cluster plates. Cells were transfected with 950 ng of the largest test construct, and equimolar amounts of the shorter reporter constructs, respectively. Fifty nanograms of coreporter were used. Degraded herring sperm DNA (Roche Molecular Biochemicals) was added up to 1 µg of total DNA to equalize different amounts of plasmid DNA. DNA was complexed with cell type-dependent amount of GenePorter reagent as determined in prior optimization experiments.

Detection of reporter genes

Cells transfected with luciferase constructs were cultured for 24–48 h, harvested, washed in PBS, and lysed in Passive Lysis Buffer (Promega). Cell lysates were analyzed sequentially for firefly and renilla luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) in a Turner luminometer TD-20/20 (Turner Design, Sunnyvale, CA) as recommended by the manufacturer. Reporter activities are calculated as the ratio of firefly luciferase test construct activity divided by activity of the cotransfected renilla luciferase reporter construct.

EGFP expression was monitored 24 or 48 h after transfection by fluorescence microscopy (IX70; Olympus, Melville, NY). After visual inspection, cells were lysed, and assayed for renilla luciferase expression as described to determine transfection efficiency.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The human genome contains two conserved fascin loci, the actively transcribed gene and a nontranscribed, inactive, intronless retroposon

Screening of a representative human genomic PAC library with the murine fascin cDNA-specific probe mFas-ORF under both stringent and relaxed hybridization conditions identified 16 clones. Eight of these clones proved to be positive in Southern blots with both an ORF-(mFas-ORF) and a 3'-UTR-(hFas-UTR) specific probe. Four clones each showed one of two clearly distinguishable banding patterns, raising the possibility of two gene loci for fascin. One PAC clone of each group was chosen for further characterization. Several restriction enzymes were used to create independent lines of overlapping subclones, which were used for sequence analysis.

PAC clone RPCI704C24766Q3/4 contains a fascin gene locus that was sequenced completely (accession no. AY044229). The gene spans a genomic region of ~14 kb and consists of five exons (Fig. 1a, upper panel). The first exon encompasses the short 5'-UTR and about half of the translated sequence. Separated by large intron 1 (9.5 kb) the following short exons 2–4 are clustered within 0.8 kb and are all of roughly the same size, encoding in total ~30% of the ORF. Exon 5 is located 1.2 kb further downstream and contains the remaining coding part and the 3'-UTR of the gene. Most exon/intron splice junctions exhibit the consensus dinucleotide sequences and the surrounding consensus elements. As an exception, the 5' splice donor of exon 4 represents a sequence variation (GC instead of GT). Sequence comparison with the recently cloned mouse fascin gene (GenBank accession no. U90355) revealed conservation of the overall genomic organization with respect to number of exons, localization of exon splice sites, and lengths of intronic sequences. In contrast to the human gene, all exon/intron boundaries of the murine gene carry the consensus dinucleotide sequences.



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 1. The human genome contains an actively transcribed fascin gene and an evolutionarily conserved fascin retroposon. a, Organization of the fascin gene (upper panel) and the fascin-derived retroposon {Psi}fascin (lower panel). Both loci were sequenced completely (GenBank database, fascin gene: accession no. AY044229; {Psi}fascin: accession no. AY044230). Exons of the gene and corresponding parts of {Psi}fascin are indicated by boxes (UTR in white, ORF in black). Relevant restriction sites used for subcloning are shown above the genomic regions: E, EcoRI; H, HindIII; Hc, HincII; P, PstI; S, SacI; Ss (in {Psi}fascin), SspI. Subcloned fragments used for further characterization are shown below the loci. b, Prevalence of {Psi}fascin in primates was analyzed by PCR. Primers hFasORF-1 and hFasORF-2 were used to amplify a 304-bp fragment of the retroposon ({psi}). A 385-bp fragment of the fascin gene (g) was amplified in parallel using primers hFasINT-1 and hFasORF-2. PAC clone RPCIP704C24766Q3/4 harboring the fascin gene (hF) and clone RPCIP704L131089Q4 spanning {Psi}fascin ({psi}hF) were used as control templates. Of the numerous human genomic templates tested, two samples (#1, #2) derived from Caucasians, Chinese, and Japanese, respectively, are shown. Anthropoids chimpanzee (chimp), gorilla (gorilla), and orangutan (orang) were tested as well. Lane N, Negative control (no template). Lane M, Molecular weight marker ({lambda} DNA, HindIII digest + {phi}X174 DNA, HaeIII digest). PCR products were separated on a 1.4% agarose gel and visualized by ethidium bromide staining. c, Fascin expression in monocytes (MO, upper panel) and mature DC on day 9 of culture (DC, lower panel) was analyzed by intracellular immunofluorescence, monitored by cytofluorometry. Thick lines, anti-fascin mAb 55K-2; thin lines, isotype control. d, A 3.1-kb genomic fragment, 5'-flanking and overlapping with the first exon of the fascin gene (phF3.1), was subcloned into the vector pGL3-Basic to drive the expression of the reporter gene photinus luciferase. pGL3-Basic (Basic) served as a negative control. Promoter activity of phF3.1 was evaluated following transient transfection of fascin-negative monocytes (MO) and fascin-positive monocyte-derived DC. To normalize for transfection efficiency, expression vector pRL-CMV, coding for renilla luciferase under control of the CMV promoter, was cotransfected, and renilla luciferase activity was measured separately in the same samples by the Dual-Luciferase Reporter Assay System (Promega). Data represent mean ± SD of two experiments performed in triplicate. e, Promoter activity of phF3.1 and a corresponding reporter construct derived from {Psi}fascin (p{psi}hF) was tested by dual luciferase assays (see above) in the neuroblastoma cell line SHSY-5Y. pRL-CMV was used as coreporter. Data represent mean ± SD of two experiments performed in duplicate. The empty reporter vector pGL3-Basic (Basic) served as negative control.

 
The second genomic fascin locus is represented in clone RPCIP704L131089Q4. Alignment of the sequence derived from the genomic locus (accession no. AY044229) revealed sequence identity of 95% to the human fascin cDNA and lack of intervening intronic sequences (Fig. 1a, lower panel). A premature stop codon (GAG->TAG) restricts the length of the ORF to a coding potential for the first 53 amino acids. Three amino acids are exchanged compared with the sequenced fascin cDNA. Because the fascin-related genomic locus is both intronless and highly conserved as compared with the fascin cDNA, it probably represents a genomic full-length cDNA copy and was named {Psi}fascin. Two imperfect direct repeats of 11 bp flank both the first conserved base of {Psi}fascin and the last adenine residue of a putative truncated poly(A) stretch and, probably, represent the original target site of the retrotransposition event, duplicated in the course of genomic integration of the fascin cDNA. The genomic sequence flanking {Psi}fascin is characterized by a high AT content between 70–80%, and a high frequency of homomeric A and T stretches.

We sought to confirm prevalence of the two genomic loci using a PCR-based approach. A product specific for {Psi}fascin was amplified from genomic DNA with primers hFasORF-1 and hFasORF-2, binding in exons 1 and 3, respectively. Both primer binding sites are present in the fascin gene as well, but amplification is prevented by intervening large intron 1. As an internal control a fascin gene-specific product of similar length was amplified in parallel reactions, using an intron 1-specific primer (hFasINT-1) in conjunction with the same exon 3-specific primer hFasORF-2. In all human genomic samples (Caucasian, Chinese, Japanese), the fascin-related retroposon was detected in addition to the gene (Fig. 1b). Even with the DNA of chimpanzee and orangutan, the {Psi}fascin-specific PCR product was apparent, as confirmed by direct sequencing (not shown). However, in gorilla, a PCR product of expected size was obtained under less stringent PCR conditions only, which still yielded {Psi}fascin-specific products in control reactions (not shown). During our studies, genomic sequences of unordered contigs of BAC clones were added to the databases, encompassing the human fascin gene and {Psi}fascin (GenBank accession nos. AC006483, AC027139) that map to chromosomes 7p22 (fascin) and 15q12 ({Psi}fascin), respectively. The location on different chromosomes hints, again, at a transposition event. Nevertheless, we checked for promoter activity of the 5'-flanking regions of both loci.

To assess promoter activity of the genomic region upstream flanking the fascin gene, a corresponding fragment of ~3.1 kb in length, including the major part of the subcloned 5'-UTR was cloned upstream of a luciferase reporter gene (phF3.1). For initial analysis of promoter activity, fascin-negative human monocytes and fascin-positive human monocyte-derived DC (Fig. 1c) were used. We had to establish a convenient DC transfection method, as to our knowledge, human DC have not been used for promoter studies before, probably in part due to low efficiency of conventional transfection methods (29). We used the biolistic gene transfection method (28) for this purpose and tested activity of phF3.1 initially in monocytes isolated from PBMC, and in mature DC generated from the same batches of monocytes by cultivation with proinflammatory cytokines. The fascin gene promoter proved to be silent in monocytes, but exerted robust activity in mature DC (Fig. 1d), reflecting the specificity of the endogenous gene. Furthermore, the promoter proved to be active in the neuroblastoma cell line SHSY-5Y, which expresses moderate levels of fascin (not shown), while a 3.1-kb fragment 5'-flanking {Psi}fascin, that included part of the putative 5'-UTR, showed no activity (Fig. 1e). As the latter result rendered a transcriptional activity of {Psi}fascin unlikely, we concentrated on analysis of the regulatory elements of the fascin gene.

The human fascin promoter isolated reflects the specificity of fascin expression in vivo and contains enhancing elements active in mature DC

To define the extension of the human fascin promoter and to detect regulatory regions within the promoter, we generated serial 5' deletion constructs of phF3.1. Luciferase activity of the reporter constructs was first analyzed in mature DC and in the human fascin-negative (data not shown) monocytic cell line THP-1. As depicted in Fig. 2a, deletion construct phF0.21 was the shortest construct displaying considerable reporter activity in both cell types tested, thus comprising a functional core promoter. In DC, increasing the length of reporter constructs up to 1.6 kb (phF1.6.) resulted in a stepwise increase of reporter activity up to 2.7-fold compared with core promoter activity. A further increase of fragment length (phF1.9, phF3.1) resulted in moderate reduction of activity compared with the 1.6-kb promoter fragment. In contrast, in THP-1, promoter constructs containing up to 0.94 kb of the genomic sequence displayed similar activity as the basal promoter, and inclusion of an additional 297 bp even resulted in a significant drop of reporter activity to ~30–40% of core promoter activity. Although reporter construct phF1.6 exerted some higher promoter activity (~80% of the basal promoter), all of the longer constructs displayed markedly reduced reporter activity (~30–40% of core promoter activity). Thus, the promoter region distal to the core promoter contains positive regulatory elements enhancing expression in DC and, presumably, negative regulatory elements suppressing core promoter activity in THP-1.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 2. Activity of the 3.1-kb fascin gene promoter (phF3.1) and derived 5'-deletion constructs in a set of fascin-positive and -negative cell types. Cells were transiently transfected with luciferase reporter constructs and reporter activity was detected by dual luciferase assays. Serial 5'-deletion constructs of phF3.1 were tested in fascin-positive mature monocyte-derived DC (a), in the fascin-negative monocytic cell line THP-1 (a), and in the fascin-positive neuroblastoma cell line SHSY-5Y (b). The approximate length of the promoter fragments is indicated by the names of the constructs. c, The 3.1-kb full-length promoter (phF3.1), an intermediate fragment of 1.6-kb length (phF1.6), and the 0.21-kb core promoter (phF0.21) were tested for promoter activity in CD83+ mature DC and CD83- immature DC, which had been separated immunomagnetically from the same culture. d, For analysis of cell type specificity fascin-positive (mature DC, SHSY-5Y) and -negative (MO, HaCaT) cell types were transfected phF3.1 and analyzed for reporter activity. Parallel transfections with reporter construct pGL3-EF1{alpha}1, driving expression of luciferase by the ubiquitously expressed EF1{alpha}1 promoter, allowed for comparison of promoter activity between cell types. Reporter activities were normalized in all cases by cotransfection with pRL-CMV. Reporter activities represent mean ± SD of two (DC, MO) or three (SHSY-5Y, HaCaT) experiments performed in triplicate or duplicate (SHSY-5Y) and are given relative to the activity of the shortest functional fascin promoter construct (phF0.21) (a–c) or of pGL3-EF1{alpha}1 (d). pGL3-Basic (Basic) served as negative control. *, Not tested.

 
Next, we tested whether the distal promoter elements enhance activity in DC per se or in a maturation-dependent fashion. Therefore, we immunomagnetically separated fully mature CD83-positive DC from immature CD83-negative DC of the same culture using anti-CD83 mAb HB15e. Activity was tested in both cell fractions in parallel, using the core promoter construct phF0.21, reporter construct phF1.6, that had exerted the highest reporter activity in DC before (see Fig. 2a), and the full-length reporter construct phF3.1. The core promoter-containing reporter construct (phF0.21) showed considerable activity above negative control (Basic) in both DC subpopulations (Fig. 2c). However, the longer reporter constructs (phF1.6, phF3.1) exerted a 5.5-fold higher reporter activity in CD83+ mature DC only. In CD83-negative DC, activity of these constructs was not markedly enhanced compared with the core promoter construct, indicating tight regulation of the distal enhancing promoter elements.

We also used the complete set of 5'-deletion reporter constructs with the neuronal cell line SHSY-5Y in luciferase assays (Fig. 2b). Deletion construct phF0.21 retained core promoter activity in SHSY-5Y, as demonstrated for DC and THP-1 before. In contrast to DC, reporter activity was not increased by the upstream located enhancer region. Rather, because phF0.66 and all of the longer reporter constructs displayed diminished promoter activity of ~60% of the core promoter, a moderately active repressor element might be located in the vicinity (-831 to -548) distal of the core promoter. Taken together, distinct regulatory elements contribute to promoter activity in different cell types.

Moreover, we directly compared activity of the full-length promoter (phF3.1) in different cell types. For this purpose, we generated a firefly luciferase construct, driven by the promoter of the human elongation factor 1{alpha}1 (EF1{alpha}1) gene. EF1{alpha}1 is a housekeeping gene, which is expressed in all cell types at similar levels and the promoter is, therefore, ideally suited to compare expression levels between cell types (26). Fascin promoter and EF1{alpha}1 promoter-driven firefly constructs, respectively, were cotransfected in parallel reactions with CMV promoter-driven renilla luciferase reporter constructs, allowing for normalization of fascin promoter activity against EF1{alpha}1 promoter activity.

The full-length fascin promoter was most active in DC (Fig. 2d), exerting ~50% higher activity than the EF1{alpha}1 promoter. In primary monocytes that resemble progenitor cells of DC, the fascin promoter was inactive, as expected. Considerable activity of ~25% of the EF1{alpha}1 promoter was denoted in the neuroblastoma cell line SHSY-5Y, which expresses moderate levels of fascin. Negligible activity (~8%) was detected in the fascin-negative (not shown) keratinocyte cell line HaCaT.

To confirm these results on the single cell level and to underscore that the minimal values obtained with HaCaT keratinocytes represent background activity, we used as a second reporter gene EGFP. Cells were transfected with expression constructs and synthesized EGFP was detected intracellularly by fluorescence microscopy. Photographs were taken at optimal time points for each cell type (24–48 h following transfection), based on visual control (Fig. 3). The fascin promoter mediated EGFP expression only in fascin-positive cells tested, namely DC (Fig. 3a) and the neuronal cell line SHSY-5Y (Fig. 3c), but not in the fascin-negative cell lines HaCaT and THP-1 (Fig. 3, e and g), while CMV promoter-driven EGFP expression constructs conferred strong EGFP expression in all cell types examined (Fig. 3, b, d, f, and h). The possibility that lack of EGFP-positive cells was due to low transfection efficiency was excluded by cotransfection of a renilla luciferase reporter construct. A promoterless EGFP expression construct was inactive in all cell types (not shown). In cultures of immature, quantitatively CD83-negative DC, no EGFP-positive cells were detected following transfection with the fascin expression construct (data not shown).



View larger version (66K):
[in this window]
[in a new window]
  		FIGURE 3. Qualitative assessment of cell type-specific activity of the fascin promoter. Fascin-positive mature DC (a and b) and SHSY-5Y (c and d) and fascin-negative HaCaT (e and f) and THP-1 (g and h) were transfected in parallel with EGFP expression constructs driven by the fascin promoter (a, c, e, and g), and the CMV promoter (b, d, f, and h), respectively. A promoterless EGFP expression construct served as a negative control (not shown). Experiments were performed twice in duplicate. Fluorescence images were taken at optimal time points. A renilla luciferase reporter construct was cotransfected and luciferase assays indicated efficient transfection of all samples (not shown).

 
Regulatory elements of the fascin promoter

Upstream of the fascin coding sequence a consensus TATA box (TATAAAA) is separated by 27 bp from the first homologue base in {Psi}fascin and may represent the transcription start site. This distance is well within the range of 25–30 bp between the TATA box as a target site for the RNA polymerase II transcription machinery and the transcription start site of many genes. The proximal fascin gene-flanking region between 160 bp 5' to the TATA box and the gene displays several structural features of a prototypic promoter. High GC content of 82%, as compared with only 50–60% in the more distal genomic regions, indicates a CpG island characteristic of gene promoters. Correspondingly, 28% of all CpG dinucleotides located within 3 kb of the cloned gene-flanking sequence are situated in this proximal region.

Due to the high degree of conservation of fascin in evolution and the congruency of fascin expression profiles in mouse and man (7, 10, 14, 16), regulatory transcription factors and their binding sites might be conserved as well. We screened for longer continuous stretches of homology hinting at important sites and identified three blocks of ~70% sequence identity each, all located within the proximal half of the putative fascin promoter. Stretch A of ~50 bp in length contains a conserved consensus NF-{kappa}B binding site. In adjacent region B with a size of ~170 bp in length, a number of potential transcription factor binding sites is clustered in three separated composite sequence motifs (motif I: Oct-1, NF-1, Lmo2-containing complex, USF, MzF1, IK, NF-{kappa}B; motif II: IK-2, Nrf-2, Nkx-2.5; motif III: IK-2, IK, NF-{kappa}B). Region C of ~100 bp in length contains the TATA box in its center, and harbors a conserved GC box, overlapping with a potential AHR-ARNT binding motif and an adjacent composite binding motif for both CREB and AP-1, overlapping in part with a putative TCF11 binding sequence.

For a more detailed analysis of the relevant promoter elements, we concentrated on the core promoter region harboring box C (Fig. 4a). To evaluate the actual contribution of the several conserved potential cis-acting elements located within the core promoter region, a series of fascin promoter constructs with small internal deletions in the core promoter region were tested in both DC and SHSY-5Y. As shown in Fig. 4b, deletion of most of the 5'-UTR had no effect on reporter activity (Fig. 4, b and c, lanes I and II). In contrast, deletion of the composite cAMP responsive element (CRE)/AP-1 binding site located between the GC box and TATA sequence resulted in considerable loss of promoter activity down to 53% in DC and 25% in SHSY-5Y (Fig. 4, b and c, lane III). Interestingly, partial deletion of the TATA box in addition did not decrease reporter activity in SHSY-5Y any further (Fig. 4, b and c, lane IV). In contrast, deletion of both the functional GC box and the composite CRE/AP-1 site had a deleterious effect, as reporter activity was reduced to ~6% of the wild-type promoter in both cell types, which is still significantly higher than the promoterless negative control (Fig. 4, b and c, lane V). Only in the case of the additional deletion of the TATA box reporter activity was abolished completely (Fig. 4, b and c, lane VI).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 4. Identification of fascin core promoter elements. a, Sequence of the fascin core promoter. A region conserved between human and mouse (GenBank accession no. U90355) fascin promoter is boxed. Conserved transcription factor recognition sites within this region are labeled and named. An AccI restriction site used for subcloning is boxed. The putative transcriptional start point is denoted by an arrow. The encoded fascin amino acid sequence is given in single letter code above the corresponding nucleotide sequence. b, The full-length fascin promoter construct (phF3.1) was modified by small internal deletions within the core promoter region (II-VI). The scale above the overview indicates the distances relative to the putative start of transcription (arrow). Relevant restriction sites are indicated and named: A, AccI; Aa, Aat II; Ap, ApaI; Sa, SauI; Sc, SacII. Potential transcription factor binding sites are boxed and named. Sequences deleted are indicated by a dashed line. C, Activity of putative promoter elements in the core promoter region was analyzed in mature DC and cell line SHSY-5Y by comparison of reporter activity of phF3.1 (I) with the reporter activity of the internal deletion constructs displayed (II-VI). Cotransfection of pRL-CMV allowed for normalization of reporter activities in dual luciferase assay. Reporter activities represent mean ± SD of two (DC) or three (SHSY-5Y) experiments performed in triplicate and are given relative to the activity of the parental fascin promoter (I). pGL3-Basic served as negative control (not shown). *, Not tested.

 
The exons, introns, and the 3'-UTR of the fascin gene do not contain enhancer elements of relevance for gene expression in DC

Regulation of gene expression is frequently influenced by cis-acting enhancer/suppressor elements located within introns, in the 3'-UTR, or even in exons of a gene. The proximal 1.02 kb of intron 1 of the fascin gene is characterized by an unusually high GC content of 75% and a lack of repetitive elements. Moreover, the corresponding sequence in the mouse fascin gene shares similar characteristics, suggesting evolutionary conservation due to sequence-inherent functional properties.

Thus, we generated a set of fascin promoter reporter constructs, containing 3–5 kb fragments of the fascin gene, cloned downstream of the luciferase polyadenylation (PA) signal in sense orientation (Fig. 5a). However, when tested in fascin-positive mature DC and SHSY-5Y, none of the constructs exerted significant changes in reporter activity compared with the parental promoter construct in either cell type (Fig. 5a).



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 5. Test for cis-regulatory elements in the fascin gene. a, Parts of the human fascin gene locus were cloned downstream of the fascin promoter luciferase expression cassette of construct phF3.1. Results of luciferase assays are depicted next to the corresponding constructs. b, The heterologous SV40 PA signal of construct phF3.1 was replaced by the complete fascin 3'-UTR ± the SV40 PA signal or a small region encompassing the fascin PA signal ± the SV40 PA signal. Constructs were tested for activity in mature DC and SHSY-5Y. Reporter activities were normalized by cotransfection with pRL-CMV and represent mean ± SD of two (DC) or three (SHSY-5Y) experiments performed in triplicate. Reporter activities are given relative to the activity of parental construct phF3.1. pGL3-Basic served as negative control (not shown). *, Not tested.

 
Finally, we checked whether the 3'-UTR of mRNA is involved in the regulation of fascin gene expression. The 3'-UTRs of mRNAs are well-known targets in gene regulation. A class of tightly regulated RNA binding proteins interact with specific sequences within the 3'-UTR of certain genes, thereby influencing stability and half-life of the corresponding mRNA and, in consequence, protein expression. We replaced the vector-derived SV40 late PA signal adjacent to the reporter gene luciferase by a fragment of the fascin gene encompassing the 1.5 kb 3'-UTR, putative PA signal and immediate downstream flanking genomic sequence. Reporter activity of this construct was reduced by 70% in mature DC and by 62% in SHSY-5Y (Fig. 5b). To determine whether repression was due to inefficient usage of the fascin PA site, the strong SV40 PA signal was inserted immediately downstream. In SHSY-5Y reporter activity of this construct was diminished by 25% only, confirming low strength of the fascin PA site. Because reporter activity was not restored completely, the possibility of moderate repressing potential of the fascin 3'-UTR was not ruled out. Deletion of the major part of the fascin 3'-UTR did not result in increased reporter activity in SHSY-5Y. However, when the strong SV40 PA signal was inserted downstream of the remaining fascin PA signal, reporter activity of the derived construct exceeded the activity of both the construct solely encompassing the fascin PA signal and even the construct solely encompassing the SV40 PA signal (Fig. 5b). Taken together, the fascin 3'-UTR contains a weaker PA site and, if any, moderately inhibitory regulatory elements.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The high expression levels of the actin-bundling protein fascin and expression among hemopoietic immune cells in mature DC only prompted us to isolate the human fascin gene and to characterize its regulatory elements. Although both functional states of DC, immature and mature, are of crucial importance for the immune system, little is known about regulation of gene expression during DC maturation. The importance of NF-{kappa}B family members, PU.1 and Ikaros for the ontogeny and maturation of DC has been demonstrated. However, regulatory mechanisms on the gene level have scarcely been studied in DC, which is in part due to low transfection efficiency (29). The present study sheds light on this process exemplified by regulation of the fascin gene. Moreover, our data suggest that the fascin promoter is suited for transcriptional targeting of human DC, offering intriguing new possibilities in immunotherapy, applicable to fields as different as treatment of cancer, autoimmunity, and allergy or vaccination against microbial infections.

In addition to the fascin gene, we identified a second conserved genomic locus with all hallmarks of a fascin-derived processed retroposon, including target site duplications and lack of introns. Its upstream end is extended by 10 bp compared with the fascin cDNA sequences published. The integration event occurred at least before speciation of anthropoids, because the locus is conserved not only in man but as well in the chimpanzee, gorilla, and orangutan. A functional role of the retroposon cannot be excluded, but transcription of the locus is rendered unlikely, as our cDNA library screenings and database searches revealed no corresponding cDNAs and the 5'-flanking genomic region did not mediate reporter gene activity in the fascin-positive cell line SHSY-5Y.

The mouse and human fascin gene display identical organization, except for sequence variation of the intron 4 splice donor site. Promoter activity of a subcloned 3.1-kb promoter fragment correlated with the endogenous fascin expression state, being silent in fascin-negative monocytes, but highly active in fascin-positive monocyte-derived mature DC. By progressive 5'-deletion analysis, we identified a core promoter as defined by its functional ability to confer basal reporter expression. Its nucleotide sequence is highly homologous to the corresponding murine fascin gene flanking region and contains several conserved potential transcription factor binding sites and a consensus TATA box. Internal deletion of a composite CRE/AP-1 site located in the core promoter region resulted in significant reduction of reporter activity of the full-length promoter, which was almost abolished in the case of additional deletion of the upstream flanking GC box. The GC box is recognized by members of the Sp/XKLF family (Sp1, Sp3, Sp4, TIEG2, and BTEB1). Expression of Sp1 family members in DC is controversial, because Granelli-Piperno and coworkers (30) reported absence of Sp1 and Sp3 in DC, while Bakri et al. (31) recently demonstrated constitutive nuclear presence of either factor in DC. Moreover, Prieschl and coworkers (32) detected binding of Sp1 to the human TNF-{alpha} promoter in a murine DC-like cell line. The GC box of the fascin promoter overlaps with a potential AHR-ARNT heterodimer binding site. AHR-ARNT is activated in response to halogenated aromatic hydrocarbon compounds like dioxin and binds to xenobiotic response elements of target genes that encode for detoxifying enzymes (33). Expression of these factors in DC has not yet been investigated.

The downstream neighboring composite CRE/AP-1 site can be occupied by (hetero)dimers of the numerous distinct CREB family proteins, dimeric AP-1 family members, and heterodimers formed by members of either family. In different DC models, activation of the p38 signal transduction pathway has been shown to result in phosphorylation of both the prototypic AP-1 family member c-Jun (34) and CRE binding transcription factors CREB and ATF-2 (35). Recently, Mann and coworkers (36) have demonstrated stimulus-dependent phosphorylation and subsequent binding of several AP-1 members to the promoter of the human IL-6 gene in a murine DC-like cell line. In the human fascin gene promoter, the composite CRE/AP-1 binding site overlaps in part with a Nrf-1 binding site. Nrf-1/AP-1 heterodimers bind in response to cell stress to antioxidative responsive elements of target genes (37). Prieschl and coworkers (32) have demonstrated stimulus-dependent binding of Nrf-1 to the human TNF-{alpha} promoter in a murine DC-like cell line.

Deletion of the TATA box in addition to a deletion of the GC box and the CRE/AP-1 site abolished promoter activity completely. Deletion of the consensus TATA box in addition to deletion of the CRE/AP-1 site alone had, if any, only a marginal effect. Activity may be compensated in part by sequence motifs acting as initiator element(s), frequently found in TATA-less gene promoters in the vicinity of the start of transcription. Moreover, deletion of the CRE/AP-1 site already affects promoter activity severely.

The 5'-UTR of the fascin gene is most probably devoid of functional activity, because deletion of its major part, encompassing a conserved SRY and AP-2 binding site, had no effect on promoter activity.

Though internal deletions within the core promoter region can completely abrogate activity, there are regulatory elements within the distal part of the promoter as well and, importantly, these elements mediate the specificity of the fascin promoter. Mature DC are the only cells tested using enhancer elements within the distal part of the fascin promoter. Reporter constructs with increasing length of the distal part induced stepwise enhancement of reporter activity, suggesting contribution of several neighboring enhancers of low strength each, as demonstrated, e.g., for the promoter of the murine MARCKS gene (38). The distal enhancer elements are involved in regulation of differential fascin expression in DC, as indicated by parallel transfections of CD83+ mature DC and CD83- immature DC. The full-length promoter mediated a markedly stronger expression in CD83+ mature DC (Fig. 2c). A slight increase in reporter activity in CD83- DC using the full-length promoter vs the core promoter may be due to maturing CD83-, fascin+ DC, as fascin expression is induced earlier during maturation than CD83 expression (14). Reporter activity of the full-length promoter construct in mature DC even exceeded activity of the 1.2-kb EF1{alpha}1 gene promoter construct that is well-known not only for its ubiquitous expression but also for its high activity (39).

The neuroblastoma cell line SHSY-5Y showed the highest reporter activity with the core promoter construct. Activity was moderately but reproducibly reduced with constructs longer than 0.37 kb, hinting at a repressor element distal from this region. Activity of the full-length promoter reporter construct (Figs. 2d and 3c) reflects the intermediate expression level of the endogenous fascin gene in SHSY-5Y (data not shown), suggesting that the relevant regulatory elements used in SHSY-5Y are included in the 3.1-kb promoter region. Fascin expression in neurons may be quite complex, as fascin is most prominently located in the growth cone filopodia. Thus, fascin expression may be stage-specific in neurons.

THP-1, an example of a fascin-negative cell line, showed basal activity with the core promoter construct. Reduced activity of longer reporter constructs in THP-1 hint at repressor elements in the distal promoter region. All fascin-negative cells tested, including THP-1, freshly isolated monocytes and HaCaT keratinocytes showed only background activity with the 3.1-kb fascin promoter luciferase and EGFP reporter constructs, respectively (Figs. 2d and 3). However, large scale screenings of relevant cell types and extensive ex vivo studies are necessary to further confirm promoter specificity. Moreover, it would be interesting to analyze whether the promoter is functional when integrated in chromatin.

Regulatory elements of the 5'-flanking promoter region are often complemented by elements located further downstream, e.g., in introns or exons. Examples are the human {alpha}- and {beta}-actin genes, which both contain an enhancer in the first intron (40, 41). Interestingly, we observed preferential enrichment of repetitive elements in the same region of large intron 1 in mouse and man, though most integration events occurred after mammalian radiation, as indicated (data not shown) by mainly species-specific short-interspersed nucleotide elements and different numbers and distribution of elements common to all mammals like ancestrally arisen mammalian-wide interspersed repeats and long-interspersed nucleotide elements. However, neither the region of intron 1 enriched in repetitive elements nor the region devoid of such elements or any other part including all exons or introns revealed activity of cis-regulatory elements (Fig. 5a).

Another level of gene regulation we investigated was mRNA stability. The half-life of certain mRNAs is influenced by proteins binding to specific sequences located in the 3'-UTR, either increasing or decreasing mRNA half-life and, thus, the time frame for translation. Half-life of {beta}-actin mRNA e.g. is down-regulated during myogenesis by binding of a differentially regulated protein to a site located within the {beta}-actin mRNA 3'-UTR (42). However, we did not identify regulatory elements in the fascin 3'-UTR.

Taken together, regulatory elements appear to be restricted to the promoter sequence of the fascin gene. However, the presence of elements located outside of the genomic region examined, either upstream or downstream, cannot be excluded. Yet, the fact that the promoter activity reflects expression levels of the endogenous gene in the cells analyzed suggests that the most important elements are probably included.

We recently demonstrated transcriptional targeting of DC in mice using the murine fascin promoter (17). Expression plasmids driving expression of the reporter gene EGFP under control of the murine promoter, which were adsorbed to microscopic gold particles, were administered to mouse skin biolistically using a gene gun. Only a few cells expressed the transgene EGFP, which were identified as LC by use of a mAb against langerin, a LC-specific surface marker (43). Interestingly, the EGFP-positive cells were not located in the epidermis but, following the route of activated emigrating LC, in the dermis and in the draining lymph nodes. This is in line with the notion that transgene expression requires maturation of LCs, as LCs mature during migration. It is in perfect agreement with our previous results that LC induce fascin expression during maturation (7). In humans, further studies of both endogenous fascin expression and promoter specificity are required to answer the question of whether the fascin promoter is a tool to restrict gene expression exclusively to DC. This study shows that the fascin promoter is strong and allows for transcriptional targeting of mature DC. Depending on the route of DNA delivery (e.g., by transfection of unseparated blood cells, bone marrow cells, or epidermal cells with fascin promoter-driven expression constructs) it may be possible to restrict transgene expression exclusively to DC.

DC are powerful tools in immunotherapy and in vitro-generated DC are currently being used in numerous clinical trials. DC are ideal targets for intervention as 1) they are the principal inducers of novel T cell responses (1) and 2) they are decisive for the type of the resulting immune response by changing their repertoire of immunostimulatory molecules in response to external stimuli (44). DC-specific promoters would allow for expression of regulatory molecules exclusively in DC to modulate the immune response. Moreover, DNA vaccination, i.e., transfection of expression plasmids to induce immune responses against the encoded Ag, is a rapidly expanding, powerful new field of vaccination. We have shown that transcriptional targeting of DC by biolistic DNA vaccination using the murine fascin promoter induces strong Ag-specific responses of IFN-{gamma}-producing CD8+ effector T cells. The humoral immune response was markedly decreased compared with parallel immunizations using the ubiquitously expressed CMV promoter, which is probably due to the reduced levels of free protein of the intracellular model Ags (EGFP, {beta}-galactosidase) used (17). The Ag-specific Ab response was dominated by IgG2a Ab in contrast to CMV promoter-driven biolistic vaccinations, and the cytokine profile of CD4+ T cells in draining lymph nodes following Ag stimulation in vitro indicated a Th1 response.5 For many human disease states, such type 1 immune responses are thought to be beneficial. Until now, only murine CD11c and dectin-2 gene promoters have been ascribed DC-restricted activity. For the CD11c gene promoter DC specificity has been demonstrated in transgenic mice only (45). Despite of the high degree of sequence homology between the human and murine CD11c promoters, the human CD11c gene displays a strikingly different expression pattern: lymphoid DC lack CD11c (46), but a subpopulation of CD8+ T cells in intestinal epithelium (47) and thymic eosinophils (48) express CD11c. Therefore, the potential of the CD11c promoter to drive DC-specific transgene expression in humans remains uncertain. Recently, the murine dectin-2 promoter was shown to exert LC-restricted reporter gene expression in transgenic mice, as well as after biolistic transfection of expression constructs into mouse skin (49). Ag-specific cellular immune responses were elicited (50). However, the promoter was more active in DC lines of an immature than a mature state. As immature DC induce inefficient T cell stimulation or even T cell tolerance (18), use of the dectin-2 promoter may be limited. The fascin promoter, which is induced during maturation, may be favorable for many applications. An additional advantage is its high activity in DC, while Morita et al. (50) observed similar expression levels of the dectin-2 promoter and the relatively weak SV40 promoter. Thus, we think that a vaccination approach based on the human fascin promoter offers significant opportunities, especially when cellular immune responses are curative.


   Footnotes
 
1 This work was supported by Stiftung Rheinland-Pfalz für Innovation (8312-386261/280) and by the Deutsche Forschungsgemeinschaft (Ro2208/2-1). Back

2 Current address: Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany. Back

3 Address correspondence and reprint requests to Dr. Ralf Ross, Johannes Gutenberg Universität Mainz, Obere Zahlbacher Strasse 63, D-55131 Mainz, Germany. E-mail address: ross{at}mail.uni-mainz.de Back

4 Abbreviations used in this paper: DC, dendritic cell; LC, Langerhans cell; rh, recombinant human; ORF, open reading frame; UTR, untranslated region; EGFP, enhanced green fluorescent protein; psi, pounds per square inch; EF1{alpha}1, elongation factor 1{alpha}1; CRE, cAMP responsive element; PA, polyadenylation. Back

5 S. Sudowe, I. Ludwig-Portugall, E. Montermann, R. Ross, and A. B. Reske-Kunz. Transcriptional targeting of dendritic cells in gene gun-mediated DNA immunization favors the induction of type 1 immune responses. Submitted for publication. Back

Received for publication February 19, 2003. Accepted for publication June 13, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bancherau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245.[Medline]
  2. Ross, R., J. Schwing, K. Kumpf, A. Endlich, A. B. Reske-Kunz. 1997. Isolation of differentially expressed genes in epidermal Langerhans cells. Adv. Exp. Med. Biol. 417:481.[Medline]
  3. Ross, R., X.-L. Ross, T. Längin, A. B. Reske-Kunz. 1999. Maturation of epidermal Langerhans cells: increased expression of {beta}- and {gamma}-actin isoforms as a basis of specialized cell functions. Exp. Dermatol. 8:487.[Medline]
  4. Ross, R., A. Endlich, K. Kumpf, J. Schwing, A. B. Reske-Kunz. 1998. Maturation of epidermal Langerhans cells in vitro is accompanied by downregulation of 4F2 (CD98) as determined by differential display. J. Invest. Dermatol. 110:57.[Medline]
  5. Ross, R., X.-L. Ross, H. Ghadially, T. Lahr, J. Schwing, J. Knop, A. B. Reske-Kunz. 1999. Mouse Langerhans cells differentially express an activated T cell-attracting CC chemokine. J. Invest. Dermatol. 113:991.[Medline]
  6. Schaniel, C., E. Pardali, F. Sallusto, M. Speletas, C. Ruedl, T. Shimizu, T. Seidl, J. Andersson, F. Melchers, A. G. Rolink, P. Sideras. 1998. Activated murine B lymphocytes and dendritic cells produce a novel CC chemokine which acts selectively on activated T cells. J. Exp. Med. 188:451.[Abstract/Free Full Text]
  7. Ross, R., X.-L. Ross, J. Schwing, T. Längin, A. B. Reske-Kunz. 1998. The actin-bundling protein fascin is involved in the formation of dendritic processes in maturing epidermal Langerhans cells. J. Immunol. 160:3776.[Abstract/Free Full Text]
  8. Edwards, R. A., J. Bryan. 1995. Fascins, a family of actin bundling proteins. Cell Motil. Cytoskeleton 32:1.[Medline]
  9. Mosialos, G., M. Birkenbach, S. Ayehunie, F. Matsumura, G. S. Pinkus, E. Kieff, E. Langhoff. 1996. Circulating human dendritic cells differentially express high levels of a 55-kD actin-bundling protein. Am. J. Pathol. 148:593.[Abstract]
  10. Mosialos, G., S. Yamashiro, R. W. Baughman, P. Matsudaira, L. Vara, F. Matsumura, E. Kieff, M. Birkenbach. 1994. Epstein-Barr virus infection induces expression in B lymphocytes of a novel gene encoding an evolutionarily conserved 55-kilodalton actin-bundling protein. J. Virol. 68:7320.[Abstract/Free Full Text]
  11. Pinkus, G. S., J. L. Pinkus, E. Langhoff, F. Matsumura, S. Yamashiro, G. Mosialos, J. W. Said. 1997. Fascin, a sensitive new marker for Reed-Sternberg cells of Hodgkin’s disease: evidence for a dendritic or B cell derivation?. Am. J. Pathol. 150:543.[Abstract]
  12. Adams, J. C., M. A. Schwartz. 2000. Stimulation of fascin spikes by thrombospondin-1 is mediated by the GTPases Rac and Cdc42. J. Cell Biol. 150:807.[Abstract/Free Full Text]
  13. Yamashiro, S., Y. Yamakita, S. Ono, F. Matsumura. 1998. Fascin, an actin-bundling protein, induces membrane protrusions and increases cell motility of epithelial cells. Mol. Biol. Cell 9:993.[Abstract/Free Full Text]
  14. Ross, R., H. Jonuleit, M. Bros, X.-L. Ross, S. Yamashiro, F. Matsumura, A. H. Enk, J. Knop, A. B. Reske-Kunz. 2000. Expression of the actin-bundling protein fascin in cultured human dendritic cells correlates with dendritic morphology and cell differentiation. J. Invest. Dermatol. 115:658.[Medline]
  15. Al-Alwan, M. M., G. Rowden, T. D. Lee, K. A. West. 2001. The dendritic cell cytoskeleton is critical for the formation of the immunological synapse. J. Immunol. 166:1452.[Abstract/Free Full Text]
  16. Al-Alwan, M. M., G. Rowden, T. D. Lee, K. A. West. 2001. Fascin is involved in the antigen presentation activity of mature dendritic cells. J. Immunol. 166:338.[Abstract/Free Full Text]
  17. Ross, R., S. Sudowe, J. Beisner, X.-L. Ross, I. Ludwig-Portugall, J. Steitz, T. Tüting, J. Knop, and A. B. Reske-Kunz. 2003. Transcriptional targeting of dendritic cells for gene therapy using the promoter of the cytoskeletal protein fascin. Gene Therapy. In press.
  18. Jonuleit, H., E. Schmitt, G. Schuler, J. Knop, A. H. Enk. 2000. Induction of interleukin 10-producing, nonproliferating CD4+ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J. Exp. Med. 192:1213.[Abstract/Free Full Text]
  19. Yamashiro-Matsumura, S., F. Matsumura. 1986. Intracellular localization of the 55-kD actin-bundling protein in cultured cells: spatial relationships with actin, {alpha}-actinin, tropomyosin, and fimbrin. J. Biol. Chem. 103:631.
  20. Ross, R., R. Kleinz, A. B. Reske-Kunz. 1995. A method for rapid generation of competitive standard molecules for RT-PCR avoiding the problem of competitor/probe cross-reactions. PCR Methods Appl. 4:371.[Medline]
  21. Waiyawuth, W., L. Zhang, C. Rittner, P. M. Schneider. 1998. Genetic analysis of the short tandem repeat system D12S391 in the German and three Asian populations. Forensic Sci. Int. 94:25.[Medline]
  22. Schneider, P. M., K. Witzel-Schlömp, C. Steinhauer, B. Stradmann-Bellinghausen, C. Rittner. 2001. Rapid detection of the ERV-K(C4) retroviral insertion reveals further structural polymorphism of the complement C4 genes in old world primates. Exp. Clin. Immunogenet. 18:130.[Medline]
  23. Ioannou, P. A., C. T. Amemiya, J. Garnes, P. M. Kroisel, H. Shizuya, C. Chen, M. A. Batzer, P. J. de Jong. 1994. A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nat. Genet. 6:84.[Medline]
  24. Ross, R., X.-L. Ross, B. Rueger, T. Längin, A. B. Reske-Kunz. 1999. Nonradioactive detection of differentially expressed genes using complex RNA or DNA hybridization probes. BioTechniques 26:150.[Medline]
  25. Kline, J. N., G. M. Hunninghake, B. He, M. M. Monick, G. W. Hunninghake. 1998. Synergistic activation of the human cytomegalovirus major immediate early promoter by prostaglandin E2 and cytokines. Exp. Lung Res. 24:3.[Medline]
  26. Kim, D. W., T. Uetsuki, Y. Kaziro, N. Yamaguchi, S. Sugano. 1990. Use of the human elongation factor 1{alpha} promoter as a versatile and efficient expression system. Gene 91:217.[Medline]
  27. Mizushima, S., S. Nagata. 1990. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 18:5322.[Free Full Text]
  28. Kalkbrenner, F., E. Dippel, B. Wittig, G. Schultz. 1996. Specificity of interaction between receptor and G protein: use of antisense techniques to relate G-protein subunits to function. Biochim. Biophys. Acta 1314:125.[Medline]
  29. Arthur, J. F., L. H. Butterfield, M. D. Roth, L. A. Bui, S. M. Kiertscher, R. Lau, S. Dubinett, J. Glaspy, W. H. McBride, J. S. Economou. 1997. A comparison of gene transfer methods in human dendritic cells. Cancer Gene Ther. 4:17.[Medline]
  30. Granelli-Piperno, A., M. Pope, K. Inaba, R. M. Steinman. 1995. Coexpression of NF-{kappa}B/Rel and Sp1 transcription factors in human immunodeficiency virus 1-induced, dendritic cell-T-cell syncytia. Proc. Natl. Acad. Sci. USA 92:10944.[Abstract/Free Full Text]
  31. Bakri, Y., C. Schiffer, V. Zennou, P. Charneau, E. Kahn, A. Benjouad, J. C. Gluckman, B. Canque. 2001. The maturation of dendritic cells results in postintegration inhibition of HIV-1 replication. J. Immunol. 166:3780.[Abstract/Free Full Text]
  32. Prieschl, E. E., V. Novotny, R. Csonga, D. Jaksche, A. Elbe-Burger, W. Thumb, M. Auer, G. Stingl, T. Baumruker. 1998. A novel splice variant of the transcription factor Nrf1 interacts with the TNF{alpha} promoter and stimulates transcription. Nucleic Acids Res. 26:2291.[Abstract/Free Full Text]
  33. Rowlands, J. C., J. A. Gustafsson. 1997. Aryl hydrocarbon receptor-mediated signal transduction. Crit. Rev. Toxicol. 27:109.[Medline]
  34. Häcker, H., H. Mischak, T. Miethke, S. Liptay, R. Schmid, T. Sparwasser, K. Heeg, G. B. Lipford, H. Wagner. 1998. CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation. EMBO J. 17:6230.[Medline]
  35. Ardeshna, K. M., A. R. Pizzey, S. Devereux, A. Khwaja. 2000. The PI3 kinase, p38 SAP kinase, and NF-{kappa}B signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells. Blood 96:1039.[Abstract/Free Full Text]
  36. Mann, J., F. Oakley, P. W. Johnson, D. A. Mann. 2002. CD40 induces interleukin-6 gene transcription in dendritic cells: regulation by TRAF2, AP-1, NF-{kappa}B, and CBF1. J. Biol. Chem. 277:17125.[Abstract/Free Full Text]
  37. Venugopal, R., A. K. Jaiswal. 1996. Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene. Proc. Natl. Acad. Sci. USA 93:14960.[Abstract/Free Full Text]
  38. Wang, L., X. Liu, R. H. Lenox. 2002. Transcriptional regulation of mouse MARCKS promoter in immortalized hippocampal cells. Biochem. Biophys. Res. Commun. 292:969.[Medline]
  39. Salmon, P., V. Kindler, O. Ducrey, B. Chapuis, R. H. Zubler, D. Trono. 2000. High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors. Blood 96:3392.[Abstract/Free Full Text]
  40. Ng, S. Y., P. Gunning, S. H. Liu, J. Leavitt, L. Kedes. 1989. Regulation of the human {beta}-actin promoter by upstream and intron domains. Nucleic Acids Res. 17:601.[Abstract/Free Full Text]
  41. Nakano, Y., T. Nishihara, S. Sasayama, T. Miwa, S. Kamada, T. Kakunaga. 1991. Transcriptional regulatory elements in the 5' upstream and first intron regions of the human smooth muscle (aortic type) {alpha}-actin-encoding gene. Gene 99:285.[Medline]
  42. DePonti-Zilli, L., A. Seiler-Tuyns, B. M. Paterson. 1988. A 40-base pair sequence in the 3' end of the {beta}-actin gene regulates {beta}-actin mRNA transcription during myogenesis. Proc. Natl. Acad. Sci. USA 85:1389.[Abstract/Free Full Text]
  43. Valladeau, J., V. Clair-Moninot, C. Dezutter-Dambuyant, J. J. Pin, A. Kissenpfennig, M. G. Mattei, S. Ait-Yahia, E. E. Bates, B. Malissen, F. Koch, et al 2002. Identification of mouse langerin/CD207 in Langerhans cells and some dendritic cells of lymphoid tissues. J. Immunol. 168:782.[Abstract/Free Full Text]
  44. Kalinski, P., C. M. Hilkens, E. A. Wierenga, M. L. Kapsenberg. 1999. T-cell priming by type-1 and type-2 polarized dendritic cells: the concept of a third signal. Immunol. Today 20:561.[Medline]
  45. Riedinger, M., K. Karjalainen, T. Brocker. 1997. Targeted expression of MHC class II genes to dendritic cells in vivo. Immunol. Lett. 57:155.[Medline]