Functional Analysis of the ProximalCCR5Promoter

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Abstract
Two promoters for the CCR5 gene, termed Pu and Pd, corresponding to the upstream and downstream initiation sites, respectively, have been described. We show here that the proximal promoter, Pd, is used two- to fivefold more frequently than Pu in primary activated T cells and in the transformed T cell line PM1. Because of its importance in CCR5 transcription we characterized the transcriptional activity of this promoter. Pd contains a pair of consensus TATA elements (nt −19 and −31) and several potential regulatory elements and transcription factor-binding sites, including those for STAT, NF-κB, AP-1, NF-AT, and CD28RE. Using a transfected reporter vector, we found the promoter to be highly active and cell type specific. By 5′ deletion analysis, the minimal CCR5 promoter was localized to a 225-nucleotide region (nt −189 to +36). This region contained the two TATA elements, a CD28RE consensus sequence, an AP-1-binding site, and two STAT-binding sites. The 1.9-kb intron appeared to have a negative influence on reporter gene activity, suggesting the presence of a negative element in this region. In addition, an upstream negative element was detected in the region nt −988 to −588. Mutagenesis of the TATA elements, of the NF-κB-, and AP-1-, and STAT-binding sites, and of the CD28RE indicated the importance of each of these in transcription. Finally, the NF-κB/Rel family member, p65(RelA), was a potent activator of the CCR5 promoter.