Nucleic Acids Research, 1989, Vol. 17, No. 11 4339-4352
© 1989
MOLECULAR BIOLOGY |
Mutations in two regions upstream of the A
globin gene canonical promoter affect gene expression
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, College of Medicine 231 Bethesda Avenue, Cincinnati, OH 45267/0524, USA
*To whom correspondence should be addressed
Received February 8, 1989. Revised April 17, 1989. Accepted April 17, 1989.
Two regions upstream of the human fetal (A
) globin gene, which interact with protein factors from K562 and HeLa nuclear extracts, have functional significance in gene expression. One binding site (site I) is at a position –290 to –267 bp upstream of the transcription initiation site, the other (site II) is at –182 to –168 bp. Site II includes the octamer sequence (ATGCAAAT) found in an immunoglobulin enhancer and the histone H2b gene promoter. A point mutation (T
C) at –175, within the octamer sequence, is characteristic of a naturally occurring HPFH (hereditary persistence of fetal hemoglobin), and decreases factor binding to an oligonucleotide containing the octamer motif. Expression assays using a A globin promoter-CAT (chloramphenicol acetyl transferase) fusion gene show that the point mutation at –175 increases expression in erythroid, but not non-erythroid cells when compared to a wild-type construct. This correlates with the actual effect of the HPFH mutation in humans. This higher expression may result from a mechanism more complex than reduced binding of a negative regulator. A site I clustered-base substitution gives -CAT activity well below wild-type, suggesting that this factor is a positive regulator.
+Present address: The Molecular Diagnostics Center, Good Samaritan Hospital, 3217 Clifton Avenue, Cincinnati, OH 45220, USA
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