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. 2006 Oct;26(19):7056–7067. doi: 10.1128/MCB.01033-06

FIG. 7.

FIG. 7.

Cell type-specific enhancer activity of the −77 kb region. (A) Sequence of the −77 and +9.5 kb regions analyzed in the transient-transfection assay. Conserved GATA motifs within the −77 and +9.5 kb fragments are highlighted. (B) G1E and MEL cells were transiently transfected with reporter plasmids derived from the pGL3 luciferase vector containing the Gata2 1S promoter cloned upstream of luciferase (1SLuc): the GATA motifs were intact in the (−77)1SLuc, (−3.9)1SLuc, and (+9.5)1SLuc plasmids, whereas the GATA motifs were mutated in the (−77mt1)1SLuc, (−77mt2)1SLuc, (−77mt3)1SLuc, and (+9.5mt)1SLuc plasmids. Mutations: −77mt1, TTATCA to TATGAA; −77mt2, GGATAC to ACGCGT; −77mt3, CTATCTATCA to TAGCTAGCGT; +9.5mt, CTATCC to ACGCGT, AGATAA to GACTTC, and GTATCT to GCTAGC. The plots depict luciferase activities of the cell lysates normalized by the protein concentrations of the lysates. The activity of the 1SLuc construct was designated 1.0 (mean ± standard error, 3 to 12 independent experiments). In each experiment, transfections were performed in triplicate. RLU, relative luciferase units. (C) Summary of enhancer activities of the GATA switch sites. +, enhancer activity; +++, strong enhancer activity (∼30-fold); −, no enhancer activity. Parentheses indicate results from our previously published study (35).