A T Cell-specific Enhancer of the Human CD40 Ligand Gene

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-B/Rel ciselement, 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-B/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-B/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-B1 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-B 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-B p50-dependent mechanism.

A T-cell-specific CD154 transcriptional enhancer located just upstream of the promoter

“…24,25 In vitro, both the hCD154 transcriptional promoter and 3 0 enhancer element are relatively weak, despite the abundant and rapid generation of CD154 mRNA following CD4 T-cell activation. 5 This led us to hypothesize that other CD154 cis-transcriptional regulatory elements contribute to CD154 expression.…”

“…The 345 bp fragment augmented CD154 promoter-driven transcription fivefold over the promoter alone in either orientation (Figure 3a), compared to threefold using the previously published 3 0 enhancer. 5 Unlike the 3 0 enhancer, 5 the novel 5 0 enhancer did not act heterologously with the IL-2 promoter (Figure 3b). Nevertheless, by acting at a distance from the promoter and by enhancing promoter activity in either orientation, this upstream 345 bp fragment surrounding HSS 2 fulfilled the definition of a transcriptional enhancer element in vitro.…”

“…Nuclear preparations and DNase I hypersensitive site mapping Nuclei from established transformed cell lines or primary human cells were prepared by NP-40 lysis as described by Siebenlist et al, 38 using 0.1-0.3% NP-40 followed by either a sucrose gradient 5 or gentle (980 g) centrifugation and subsequent wash in buffer not containing NP-40 after treatment to obtain pure nuclei. Nuclei were gently (by vortex and stirring) resuspended in nuclear isolation buffer (60 mM KCl, 15 mM NaCl, 5 mM MgCl 2 , 0.1 mM EGTA, 15 mM Tris-HCl (pH 7.0), 0.5 mM dithiothreitol, 0.1 mM AEBSF (4-(2-aminoethyl)-benzenesulfonylfluoride), 0.3 M sucrose) containing 5% glycerol, and aliquoted into 0.5 ml samples each containing 5-10 Â 10 6 nuclei.…”

“…3 We previously identified and characterized the human CD154 (hCD154) transcriptional promoter 4 and a 3 0 enhancer element. 5 The promoter is activation dependent and requires the cyclosporin-sensitive nuclear factor of activated T cells (NFAT) transcription factor for expression.…”

“…Both the promoter and enhancer display relatively weak transcriptional activity in reporter gene assays in primary human T cells. The observation that the CD154 promoter has weak transcriptional activity as compared with other cytokine genes (for example, interleukin (IL)-2), but has similarly abundant mRNA transcripts, 2,5 led us to search for other cis-acting elements that could account for its transcriptional regulation.…”