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The Human Potassium-Chloride Cotransporter 1 Gene Promoter Is Regulated by AP-2 Binding Proteins and Contains a Functional Downstream Promoter Element

The Human Potassium-Chloride Cotransporter 1 Gene Promoter Is Regulated by AP-2 Binding Proteins and Contains a Functional Downstream Promoter Element

Blood 100(11): Abstract No. 434

K-Cl cotransport is a major pathway for the coupled, electroneutral release of K+ and Cl- from nonexcitable cells. The majority of K-Cl cotransport in the erythrocyte is attributed to KCC1, a widely expressed member of a family of cation-Cl cotransporters. K-Cl cotransport is elevated in sickle cell erythrocytes and the KCC1 gene has been proposed as a candidate modifier gene contributing to the clinical course and response to therapy in sickle cell disease. To provide insight into the regulation of KCC1, we have identified and characterized the KCC1 gene promoter. Screening a human genomic DNA BAC library yielded aapprx90kb genomic clone containing the entire KCC1 gene. A 14kb XbaI fragment containing exons 1-3 and the 5' flanking sequence was isolated from the BAC clone. No TATA or CCAAT-sequences were found in the 5' flanking DNA; consensus sequences for the DNA-binding proteins GATA-1, AP-2, and CGCC-binding proteins were found. Primer extension identified a single transcription initiation site 27bp 5' from the end of the previously reported cDNA; this site encodes an initiation recognition element (InR). To identify sequences essential for KCC1 gene expression, plasmids containing 5' flanking sequences linked to a luciferase reporter gene were transiently transfected into K562 cells. A 368bp KCC1 gene fragment from -367 to +1 directed high levels of luciferase expression, 138.75+11.8 fold over background. Deletional analysis demonstrated that a minimal promoter fragment from -107 to +1also directed activity in erythroid cells. DNAseI footprinting of the core promoter region identified a protected region from -21 to -11 that encodes a potential AP-2 binding site. To determine if AP-2 proteins bind these sequences in vitro, we performed gel mobility shift assays. A major complex was identified using a KCC1 AP-2 probe that migrated at the same mobility as a control AP-2 probe. This complex was competed away by an excess of unlabeled KCC1 or control AP-2 probe. AP-2 antibodies inhibited formation of the KCC1 AP-2 complex. Mutation of this AP-2 site in the 368bp KCC1 promoter/reporter plasmid reduced luciferase activity by more than half in transfection assays. When co-transfected with AP-2alpha, beta, gamma, or delta cDNA expression plasmids, the KCC1 promoter/luciferase plasmid was transactivated in AP-2 deficient HepG2 cells in a dose dependent manner. Additional transfections demonstrated that addition of 63-bp of sequence 3' to the transcription initiation site significantly increased reporter gene activity, over 10 fold. This region contains the sequence GGGCGTG at position +34 to +40, matching the consensus sequence and location for a downstream promoter element (DPE). Both mutation of the KCC1 DPE consensus sequence and variation in the spacing from the InR to the DPE decreased reporter gene activity. In a minimal 60bp core promoter fragment, mutation of the InR, the DPE, or both completely abolished promoter function. Recent work has demonstrated that DPEs mediate transcription initiation and are critical regulatory elements in core promoter function. The presence of an InR and a DPE in a TATA-less promoter, a combination found primarily in Drosophila gene promoters, is beginning to be recognized as an important component of mammalian core promoters. Thus the human KCC1 gene promoter, which is regulated by AP-2 binding proteins, is one of the first mammalian promoters identified that contains this combination of core promoter elements.

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