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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(s) -
Denomme Gregory A.,
Wang Duncheng,
Matheson Kimberly A.,
Titolo Danny
Publication year - 2009
Publication title -
transfusion
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.045
H-Index - 132
eISSN - 1537-2995
pISSN - 0041-1132
DOI - 10.1111/j.1537-2995.2009.02162.x
Subject(s) - promoter , transcription factor , transcription (linguistics) , biology , gene , electrophoretic mobility shift assay , microbiology and biotechnology , genetics , binding site , regulatory sequence , cis regulatory module , enhancer , gene expression , linguistics , philosophy
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.