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

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. © 2009 American Association of Blood Banks.