A GC Box Element Is Critical for Transcriptional Regulation of the K-Cl Cotransporter Isoform KCC3a in Hematopoetic Cells.
Authors: Crable Scott , Joiner Clinton H. , Gallagher Patrick G. 
Published: April 2020

Abstract
The KCl Cotransporter (KCC) is a key component of the volume regulation system of human reticulocytes, and its excessive activity in sickle cells contributes to cellular dehydration and therefore to sickling pathology. Three of the four KCC genes, are expressed in erythroid cells (Crable et al. Exp. Hematol.2005;33:624). Although the relative contribution of the three KCC isoforms to KCC fluxes and volume regulation in red cells remains unknown, KCC3 appears to be the dominant transcript in late erythroid cells. Heterogenity of the 5′ ends of the KCC3 mRNA transcripts has been described by Mount and colleagues (Mercado et al. Am J Physiol289:F1246, 2005), including two untranslated exons (ex1D and 1C) 1.2 kb upstream from ex1A of KCC3a, which contains the originally described translation initiation site. We used RT-PCR, 5′ RACE (rapid amplification of cDNA ends), and primer extension analyses to study the 5′ ends of KCC3 cDNA transcripts in erythroid cells. The large exon 1 of KCC3 was identified in fetal liver and bone marrow RNA as a 1718bp region (containing exons 1A, 1C, and 1D described by Mercado et al) that undergoes complex patterns of alternate splicing to generate 10 different transcripts. Six major splicing isoforms are expressed in hematopoietic cell RNA. One isoform incorporates the first 167bp of the exon (exon 1D) as 5′ untranslated sequence which splices to the last 208bp of exon 1, including additional 5′ untranslated sequence and an alternative in-frame initiator methionine. The translated protein exhibits a 59 amino acid N-terminal truncation of KCC3a lacking several potential phosphorylation sites (called KCC3a-Short by Mercado et al). This transcript was the most abundant isoform in hematopoietic cell RNA. A second major isoform contains 629bp of the exon immediately 3′ of exon 1D as 5′ untranslated sequence (exon 1C), then splices out the next 735bp to join the last 243bp of the exon, including additional 5′ untranslated sequence and the alternate initiator methionine of KCC3a-Short. A third novel transcript includes the entire exon, utilizing the first initiator methionine of full-length KCC3a. Two other novel transcripts were found, both of which code for KCC3a-Short. These transcripts were also identified in EST databases. We examined the genomic region around exon 1 for promoter activity using luciferase promoter constructs expressed in erythroid K562 cells. Promoter activity was minimal with constructs which spanned from exon 1D to the beginning of exon 1A, but increased substantially in constructs that included this region plus 900 bp 5′ sequence. Further deletion analysis shows that a minimal promoter containing less than 125 base pairs yields full promoter activity. A mutation of a GC box within this region reduces activity over 15 fold, implicating SP-1 as a trans-regulatory factor. Thus, KCC3 exhibits a complex pattern of alternative splicing in erythroid cells, producing several novel transcripts, some of which encode an N-terminal truncation of KCC3a. Identifying the factors modulating transcriptional and translational control of KCC3 expression and the functional behavior of this truncated protein in erythroid cells is important to understanding volume regulation in reticulocytes and its abnormalities in sickle cells.