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.
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.


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.


CONCLUSIONS
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.
Denomme
Gregory A
GA
Research & Development, Canadian Blood Services, 67 College Street, Toronto, Ontario, Canada. greg.denomme@blood.ca
Wang
Duncheng
D
Matheson
Kimberly A
KA
Titolo
Danny
D
D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*
D011993:Recombinant Fusion Proteins; C117760:RhD fusion protein, human