The human fascin gene promoter is highly active in mature dendritic cells due to a stage-specific enhancer.
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.
Bros
Matthias
M
Clinical Research Unit Allergology, Department of Dermatology, Johannes Gutenberg University, Mainz, Germany.
Ross
Xiao-Lan
XL
Pautz
Andrea
A
Reske-Kunz
Angelika B
AB
Ross
Ralf
R
D020413:3' Untranslated Regions; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002454:Cell Differentiation / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D002478:Cells, Cultured; D017124:Conserved Sequence; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005091:Exons; D005786:Gene Expression Regulation; D020868:Gene Silencing; D005819:Genetic Markers; D015894:Genome, Human; D006801:Humans; D007438:Introns; D008840:Microfilament Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011544:Pseudogenes; D012045:Regulatory Sequences, Nucleic Acid; D018626:Retroelements; D014407:Tumor Cells, Cultured
D020413:3' Untranslated Regions; D002352:Carrier Proteins; D005819:Genetic Markers; D008840:Microfilament Proteins; D018626:Retroelements; C020324:fascin