Characterization of the functional promoter for CCR3 in human T cells.

(A) Map of the upstream region of the human CCR3 gene. Black boxes and angle dashed lines represent exons and splicing patterns, respectively. Exon 1 contains the main 5′ UTR part and exon 2 contains the ATG coding region. Grey arrows represent primers subsequently used for PCR in Figure 2A. H and K, respectively, indicate the position ofHindIII and KpnI sites. The 1, 2, and 3 located with gray lines indicate the regions analyzed by ChIP and DNaseI HS experiments. (B) Identification of the T-cell minimal promoter region for CCR3. Construct maps and data for relative luciferase expression in Jurkat human T cells (filled bars) and 721.221 human B cells (open bars). PCR fragments of variable sizes of the CCR3 5′-flanking region were generated and cloned in the Firefly luciferase pGL3 Basic vector. Exon 1 (gray box) and Luciferase (open box) are represented on the maps. Plasmids were transiently transfected by electroporation with a control plasmid pRL-TK (Renillaluciferase) into Jurkat and 721.221 cells. Relative luciferase activities of the cell lysates are representative of at least 5 independent experiments performed with different clones for each construct. Errors bars indicate SDs. (-) indicates not tested. (C) Identification of an NRE located in the flanking intronic region. An intronic PCR fragment (+134 to +237) was cloned either in sense (pGL3 C NRE S) or antisense orientation (pGL3 C NRE AS) into theFirefly luciferase vector pGL3 control containing the SV40 promoter and enhancer. Relative luciferase activities were determined as described in (B).