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  • Title: The proximal cis-regulatory region of the RHD/RHCE promoter is 105 bp and contains a 55-bp core devoid of known binding motifs but necessary for transcription.
    Author: Denomme GA, Wang D, Matheson KA, Titolo D.
    Journal: Transfusion; 2009 Jul; 49(7):1361-9. PubMed ID: 19374729.
    Abstract:
    BACKGROUND: The RhD and RhCE polypeptides are erythroid-specific members of the RH gene family. Little is known about the promoter cis-regulatory proximal region responsible for transcription. STUDY DESIGN AND METHODS: The 1246-bp 5'-flanking regions of the RHD and RHCE promoter were amplified and ligated to a luciferase reporter vector and erythroid-specific transcription was evaluated in K562, HEL, U937, and HeLa cell lines. Deletion and substitution promoter-reporter constructs were generated to define the minimal cis-regulatory region. RESULTS: Deletion analysis in K562 cells revealed that the cis-regulatory region extended to a position between -78 and -120 relative to the site of initiation of transcription. Electrophoretic mobility shift assays confirmed binding motifs for Sp1, GATA-1, and E2F transcription factors. The use of 15-bp substitution mutagenesis showed that the minimal region required for transcription extended to -105 bp, and 6-bp substitution mutants from -35 to -90 identified a region necessary for transcription yet devoid of known cis-regulatory binding motifs. CONCLUSION: The proximal RHD/RHCE promoter regions contain cis-regulatory binding motifs and an internal sequence-dependent region that together regulate transcription. The results suggest that this region may be relevant in the weak expression of RhD.
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