Identification of GATA elements on the CCR5 promoter region. (A) Map and sequence of the CCR5 promoter region. Location and sequences of three GATA elements (GATA#1, GATA#2, and GATA#3) are shown. The sequence shown for GATA#3 was also used as an oligonucleotide probe (F5) for gel-mobility shift assays (see B). Nuleotides shown in upward arrows indicate mutated nucleotides in plasmids pGL-CCR5▵GATA#1, pGL-CCR5▵GATA#2, pGL-CCR5▵GATA#3, and pGL-CCR5▵GATA#1+2+3. Brackets indicate GATA elements. (B) Identification of GATA#3 as a binding site for GATA-1 by gel mobility shift assays. A radiolabeled oligonucleotide F5 corresponding to FP-5 (a DNase-protected area in footprinting assays4) was incubated with nuclear extracts from PM1 cells. Lanes 1 and 9 represent probe alone. Where indicated, a 50- or 500-fold molar excess of oligonucleotide indicated above the figure was added as a competitor. F5-m has mutation on the GATA element as in plasmid pGL-CCR5▵GATA#3. For GATA or AP1 consensus oligonucleotide, see Moriuchi et al.4 Gel shift disruption experiments (lanes 9 through 14) were performed by adding either control Ab or monoclonal antibodies to either GATA-1, GATA-2, or GATA-3 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), as indicated above the figure. The arrow indicates the GATA-1 complex. NS, nonspecific bands; FP, free probe. The experiments were repeated twice with similar results.