Sun G, Tomita H, Shakkottai V G, Gargus J J
Department of Physiology and Biophysics, University of California, Irvine, 92697-4560, USA.
J Hum Genet. 2001;46(8):463-70. doi: 10.1007/s100380170046.
KCNN3 is a member of the gene family, KCNN1-4, encoding the small and intermediate conductance calcium-activated potassium channels. Long CAG-repeat alleles of this gene have been found to be over-represented in patients with schizophrenia in a number of population-based association studies, and this gene maps to human chromosome 1q21, a region recently implicated in schizophrenia by linkage. To set the stage for a further functional evaluation of 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 upstream regulatory regions. We isolated P1-derived artificial chromosome (PAC) clones from a genomic library and identified an overlapping available bacterial artificial chromosome (BAC) clone. Cosmids subcloned from the PAC and BAC clones were then sequenced and merged with the sequence in the public database. The KCNN3 gene spans over 163.1 kb and is composed of eight exons and seven introns. All of the exon-intron junctions conform closely to consensus splice sites. The proximal 2.5 kb of the 5'-flanking sequence was obtained and analyzed for potential transcription factor binding sites. In the proximal 2.5 kb upstream region, potential sites for the Ikaros factor (IK2), homeodomain factor Nkx-2.5/Csx (NKX25), nuclear factor of activated T-cells (NFAT), upstream stimulating factor (USF), c-AMP responsive element binding 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 potential 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 orientation-dependent expression of the reporter gene in transiently transfected PC12 cells, cells which natively express functional KCNN3 channels, suggesting that this cloned fragment includes competent promoter elements of this gene.
KCNN3是KCNN1 - 4基因家族的成员之一，该家族编码小电导和中电导钙激活钾通道。在多项基于人群的关联研究中发现，该基因的长CAG重复等位基因在精神分裂症患者中过度表达，并且该基因定位于人类染色体1q21，这是一个最近通过连锁分析与精神分裂症相关的区域。为了为进一步对KCNN3进行功能评估奠定基础，我们确定了其基因组位点的性质，包括其内含子和外显子的大小、结构和序列以及潜在上游调控区域的功能。我们从基因组文库中分离出P1衍生人工染色体（PAC）克隆，并鉴定出一个重叠的可用细菌人工染色体（BAC）克隆。然后对从PAC和BAC克隆亚克隆的黏粒进行测序，并与公共数据库中的序列合并。KCNN3基因跨度超过163.1 kb，由八个外显子和七个内含子组成。所有外显子 - 内含子连接均与共有剪接位点紧密相符。获得了5'侧翼序列的近端2.5 kb，并分析了潜在的转录因子结合位点。在近端2.5 kb上游区域，鉴定出了Ikaros因子（IK2）、同源结构域因子Nkx - 2.5/Csx（NKX25）、活化T细胞核因子（NFAT）、上游刺激因子（USF）、c - AMP反应元件结合蛋白（CREB）、POU因子Brn2（BRN - 2）、髓系锌指蛋白（MZF1）、卵黄生成素结合蛋白（VBP）、HNF3叉头同源物2（HFH2）以及转录起始的潜在位点，以及几个潜在的AP - 1和AP - 4位点。最后，将该上游区域的一个2261 bp片段克隆到无启动子的pGL3 - 荧光素酶载体中，在瞬时转染的PC12细胞（天然表达功能性KCNN3通道的细胞）中，它产生了报告基因的方向依赖性表达，这表明该克隆片段包含该基因的有效启动子元件。
