The human fascin gene promoter is highly active in mature dendritic cells due to a stage-specific enhancer

Authors:
Bros M, Ross XL, Pautz A, Reske-Kunz AB and Ross R
In:
Source: J Immunol
Publication Date: (2003)
Issue: 171: 1825-1834
Cells used in publication:
Dendritic cell (NHDC), human
Species: human
Tissue Origin: blood
Platform:
Nucleofectorâ„¢ I/II/2b
Experiment
Fascin, an evolutionarily highly conserved cytoskeletal protein is involved in cell motility and cell structure. It is expressed in dendritic cells (DC), neuronal and glial cells, and capillary endothelial cells. The authors report the isolation of the fascin promoter and the characterization of its regulatory elements. To reveal the specifity of the promoter, fascin-positive mature DC and SHSY-5Y and fascin-negative HaCaT and THP-1 were transfected with an EGFP expression construct driven by the fascin promoter. In this experiment nucleofection was used for DC transfection. Control transfections were performed with an EGFP construct driven by CMV promoter and a promoterless EGFP vector. The fascin promoter mediated EGFP expression only in fascin positive cells tested, namely DC and the neuronal cell line SHSY-5Y, but not in fascin-negative cell lines (HaCaT, THP-1). Other experiments showed a markedly increased promoter activity with maturation of DC. The authors demonstrated that the human fascin promoter allows for transcriptional targeting of mature DC and represents a promising tool for immunotherapy.
Abstract
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.