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.
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.
Clinical Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
J L
JL
Miller
C E
CE
Walsh
P A
PA
Ney
R J
RJ
Samulski
A W
AW
Nienhuis
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D015870:Gene Expression; D015321:Gene Rearrangement; D005822:Genetic Vectors; D005914:Globins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D016297:Mutagenesis, Site-Directed; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D011995:Recombination, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D016335:Zinc Fingers / Q000235:genetics / Q000502:physiology*
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; D012333:RNA, Messenger; D014157:Transcription Factors; D005914:Globins