Abstract
Islet cell autoantigen 69-kDa (ICA69), protein product of the human ICA1 gene, is one target of the immune processes defining the pathogenesis of Type 1 diabetes. We have characterized the genomic structure and functional promoters within the 5'-regulatory region of ICA1. 5'-RNA ligase-mediated rapid amplification of cDNA ends evaluation of ICA1 transcripts expressed in human islets, testis, heart, and cultured neuroblastoma cells reveals that three 5'-untranslated region exons are variably expressed from the ICA1 gene in a tissue-specific manner. Surrounding the transcription initiation sites are motifs characteristic of non-TATA, non-CAAT, GC-rich promoters, including consensus Sp1/GC boxes, an initiator element, cAMP-responsive element-binding protein (CREB) sites, and clusters of other putative transcription factor sites within a genomic CpG island. Luciferase reporter constructs demonstrate that the first two ICA1 exon promoters reciprocally stimulate luciferase expression within islet- (RIN 1046-38 cells) and brain-derived (NMB7) cells in culture; the exon A promoter exhibits greater activity in islet cells, whereas the exon B promoter more efficiently activates transcription in neuronal cells. Mutation of a CREB site within the ICA1 exon B promoter significantly enhances transcriptional activity in both cell lines. Our basic understanding of expression from the functional core promoter elements of ICA1 is an important advance that will not only add to our knowledge of the ICA69 autoantigen but will also facilitate a rational approach to discover the function of ICA69 and to identify relevant ICA1 promoter polymorphisms and their potential associations with disease.
Highlights
Islet cell autoantigen 69 kDa (ICA69) is identified with a group of Type 1 diabetes-related islet autoantigens considered
ICA69 is encoded on human chromosome 7p22 by the ICA1 gene [7], which is composed of 14 coding exons and three 5Ј-untranslated region (UTR)1 sequences, each of which splices with exon 1 in a mutually exclusive manner [4]
Genomic Organization of the ICA1 5Ј-UTR Exons—We identified a single ICA1-containing Centre d’Etude du Polymorphisme Humain (CEPH) Mega-YAC clone (CEPH813G2) in a PCR-based YAC library screen
Summary
Cell Lines—Two adherent cell lines were maintained in culture in order to provide RNA for transcript analysis and for testing promoter activity of cloned ICA1 5Ј-flanking sequences in a firefly luciferase reporter assay. GC-rich regions of ICA1 sequence from human genomic, YAC, and BAC DNA samples were amplified using reagents from the Advantage GC Genomic PCR kit (Clontech, Palo Alto, CA), whereas the Advantage GC cDNA Enzyme (Clontech) facilitated amplification of GC-rich plasmid inserts and cDNA templates generated for 5Ј-RACE analysis. The Advantage GC cDNA enzyme was used in all nested RLM-RACE PCRs. Cloning of ICA1 Sequences into pGL3 Basic—To clone segments of the ICA1 5Ј-flanking region and UTR exons, a 1028-bp genomic segment spanning the entire region was amplified from CITB-503D2 DNA via PCR, followed by T/A ligation of the product into the pCR2.1 vector (amplification primers, 5Ј-TAGGAAGCAGCTATGCCAACACT-3Ј and 5Ј-CAGAGAAGGCAGCTCCTACCA-3Ј). A core similarity of Ն0.900 from the MatInspector analysis was used as a cut-off for consideration of potential query sequence matches with known transcription factor recognition sequences
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