Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 277, Issue 9, Pages 7386-7395Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M110350200
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Funding
- EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH &HUMAN DEVELOPMENT [R01HD036291] Funding Source: NIH RePORTER
- NATIONAL CANCER INSTITUTE [T32CA009537] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [R37DK035008, R01DK035008] Funding Source: NIH RePORTER
- HHS [P30 HH 2815-08] Funding Source: Medline
- NCI NIH HHS [CA 09537] Funding Source: Medline
- NICHD NIH HHS [R01 HD 36291] Funding Source: Medline
- NIDDK NIH HHS [DK 35008] Funding Source: Medline
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We observed that the human CD40 ligand (CD40L) gene W-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 W-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 W-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 NTF-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 W-flanking region acts as a T cell-specific classical transcriptional enhancer by a NF-kappaB p50-dependent mechanism.
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