A T cell-specific enhancer of the human CD40 ligand gene.
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.
Schubert
Lisa A
LA
Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195, USA.
Cron
Randy Q
RQ
Cleary
Aileen M
AM
Brunner
Michael
M
Song
An
A
Lu
Li-Sheng
LS
Jullien
Pascale
P
Krensky
Alan M
AM
Lewis
David B
DB
D001402:B-Lymphocytes; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D015704:CD4 Antigens / Q000096:biosynthesis; D023201:CD40 Ligand / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004742:Enhancer Elements, Genetic; D017930:Genes, Reporter; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism; D019169:Jurkat Cells; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020569:Proto-Oncogene Proteins c-rel / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000378:metabolism*; D014158:Transcription, Genetic
D015704:CD4 Antigens; D018076:DNA, Complementary; D007376:Interleukin-2; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D020569:Proto-Oncogene Proteins c-rel; D012333:RNA, Messenger; D023201:CD40 Ligand; D004247:DNA; D003850:Deoxyribonuclease I