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Single-copy transduction and expression of human gamma-globin in K562
erythroleukemia cells using recombinant adeno-associated virus vectors: the
effect of mutations in NF-E2 and GATA-1 binding motifs within the
hypersensitivity site 2 enhancer [published erratum appears in Blood 1995
Feb 1;85(3):862]
JL Miller, CE Walsh, PA Ney, RJ Samulski and AW Nienhuis
Clinical Hematology Branch, National Heart, Lung and Blood Institute,
National Institutes of Health, Bethesda, MD 20892.
The use of recombinant adeno-associated virus (rAAV) vectors provides a new
strategy to investigate the role of specific regulatory elements and
trans-acting factors in globin gene expression. We linked hypersensitivity
site 2 (HS2) from the locus control region (LCR) to a A gamma-globin gene
(A gamma*) mutationally marked to allow its transcript to be distinguished
from endogenous gamma-globin mRNA. The vector also contains the
phosphotransferase gene that confers resistance to neomycin (NeoR). HS2
region mutations within the NF-E2 motifs prevented NF-E2 binding while
preserving AP-1 binding. Another set in the GATA-1 motif prevented binding
of the factor. Several NeoR K562 clones containing a single unrearranged
RAAV genome with the A gamma* gene linked to the native HS2 core fragment
(WT), mutant NF-E2 HS2 (mut-NFE2), mutant GATA-1 HS2 (mut-GATA1), or no HS
[(-)HS] were identified. In uninduced K562 cells, mut-NFE2 clones expressed
A gamma* mRNA at the same level as the WT clones, compared with a lack of A
gamma* signal in the (-)HS2 clones. However, hemin induction of mut- NFE2
clones did not result in an increase in the A gamma* signal above the level
seen in uninduced cells. Mut-GATA1 clones expressed the A gamma* mRNA at
the same level as WT clones in both uninduced and induced cells. Thus,
GATA-1 binding to this site does not appear to be required for the
enhancing function of HS2 in this context. This single- copy rAAV
transduction model is useful for evaluating the effects of specific
mutations in regulatory elements on the transcription of linked genes.
Volume 82,
Issue 6,
pp. 1900-1906,
09/15/1993
Copyright © 1993 by The American Society of Hematology
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