Genomic organization and promoter analysis of human KCNN3 gene

KCNN3 is a member of the gene family, KCNN1-4, encoding the small and inter mediate conductance calcium-activated potassium channels. Long CAG-repeat a lleles of this gene have been found to be over-represented in patients with schizophrenia in a number of population-based association studies, and thi s gene maps to human chromosome 1q21, a region recently implicated in schiz ophrenia by linkage. To set the stage for a further functional evaluation o f KCNN3, we defined the nature of the genomic locus in the size, structure, and sequence of its introns and exons and the function of potential upstre am regulatory regions. We isolated P1-derived artificial chromosome (PAC) c lones from a genomic library and identified an overlapping available bacter ial artificial chromosome (BAC) clone. Cosmids subcloned from the PAC and B AC clones were then sequenced and merged with the sequence in the public da tabase. The KCNN3 gene spans over 163.1 kb and is composed of eight exons a nd seven introns. All of the exon-intron junctions conform closely to conse nsus splice sites. The proximal 2.5kb of the 5 ' -flanking sequence was obt ained and analyzed for potential transcription factor binding sites. In the proximal 2.5kb upstream region, potential sites for the Ikaros factor (IK2 ), homeodomain factor Nkx-2.5/Csx (NKX25), nuclear factor of activated T-ce lls (NFAT), upstream stimulating factor (USF), c-AMP responsive element bin ding protein (CREB), POU factor Brn2 (BRN-2), myeloid zinc finger protein ( MZF1), vitellogenin binding protein (VBP), HNF3 forkhead homologue 2 (HFH2) , and transcription initiation were identified, as well as several potentia l AP-1 and AP-4 sites. Finally, a 2261-bp fragment of this upstream region was cloned into a promoterless pGL3-luciferase vector, where it produced or ientation-dependent expression of the reporter gene in transiently transfec ted PC12 cells, cells which natively express functional KCNN3 channels, sug gesting that this cloned fragment includes competent promoter elements of t his gene.