Abstract
We observed that the human CD40 ligand (CD40L) gene 5'-flanking region conferred weak promoter activity in activated CD4 T cells, suggesting that additional regions are required for optimal CD40L gene transcription. We therefore examined a 3'-flanking segment of the CD40L gene, which contained a putative NF-kappaB/Rel cis-element, for its ability to enhance CD40L promoter function. This segment augmented CD40L promoter activity in an orientation-independent manner in CD4 T-lineage cells but not in human B cell or monocyte cell lines. Mapping of CD4 T-lineage cell nuclei identified a DNase I-hypersensitive site in the flanking region near the NF-kappaB/Rel sequence, suggesting a transcriptional regulatory role. This was further supported by truncation analysis and site-directed mutagenesis, which indicated that the CD40L 3'-flanking NF-kappaB/Rel cis-element was critical for enhancer function. Electrophoretic mobility shift assays showed that the cis-element preferentially bound the p50 form of the NF-kappaB1 gene contained in human T cell nuclear protein extracts. This binding also appeared to occur in vivo in CD4 T cells based on chromatin immunoprecipitation assays using NF-kappaB p50-specific antiserum. Together, these results suggest that the CD40L gene 3'-flanking region acts as a T cell-specific classical transcriptional enhancer by a NF-kappaB p50-dependent mechanism.
Highlights
CD40 ligand (CD40L),1 known as CD154, is a member of the tumor necrosis factor (TNF) ligand superfamily that is transiently expressed on the cell surface of activated CD4positive T cells [1]
The relatively low promoter activity of the 1.3-kb 5Јflanking CD40L gene segment does not appear to be due to strong negative regulatory elements, since we have previously shown that truncation of the 5Ј region of this segment does not increase promoter activity [22]
A 1284-bp Segment of the CD40L 3Ј-Flanking Region Acts as an Enhancer of CD40L Promoter Activity in CD4 T Cells— Because the 3Ј-flanking region of many genes expressed by T lymphocytes, such as the genes encoding CD2 and IL-4 [49], contain transcriptional enhancers, we investigated whether the 3Ј-flanking region of the CD40L gene might contribute to its transcriptional activation in CD4 T-lineage cells
Summary
This adjacent region behaves as a classic enhancer (i.e. it increases the activity of the core IL-2 promoter and does so in an orientation-independent manner) [17] This enhancer contains most of the cis-activating elements known to be required for optimal IL-2 gene transcription, including binding sites for NF-B/Rel, NFAT, and leucine zipper proteins [11, 12]. A transcriptional enhancer active in mononuclear phagocyte cells has been identified in the 3Ј-flanking region of the human TNF gene [18] This region includes a NF-B/Rel binding site that may contribute to increased TNF promoter activity [19]. A putative NF-B/Rel cis-element, GGGATTTCCA, has been identified in the 5Ј-flanking region of the CD40L gene based on sequence analysis [25] and has recently been reported to enhance promoter activity, possibly by binding NF-B p65 [26]. We show that the human CD40L gene contains a T cell-specific, NF-B/Rel protein-dependent, classical enhancer in its 3Ј-flanking region that may be important in the regulation of CD40L transcription
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