Engelman J A, Zhang X L, Lisanti M P
Department of Molecular Pharmacology and The Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
FEBS Lett. 1999 Apr 9;448(2-3):221-30. doi: 10.1016/s0014-5793(99)00365-8.
The CA microsatellite repeat marker, D7S522, is located at the center of a approximately 1000 kb smallest common deleted region that is lost in many forms of human cancer. It has been proposed that a putative tumor suppressor gene lies in close proximity to D7S522, within this smallest common deleted region. However, the genes located in proximity to D7S522 have remained elusive. Recently, we identified five independent BAC clones (approximately 100-200 kb) containing D7S522 and the human genes encoding caveolins 1 and 2. Here, we present the detailed organization of the caveolin locus and its relationship to D7S522, as deduced using a shot-gun sequencing approach. We derived two adjacent contigs for a total coverage of approximately 250 kb. Analysis of these contigs reveals that D7S522 is located approximately 67 kb upstream of the caveolin-2 gene and that the caveolin-2 gene is located approximately 19 kb upstream of the caveolin-1 gene, providing for the first time a detailed genetic map of this region. Further sequence analysis reveals many interesting features of the caveolin genes; these include the intron-exon boundaries and several previously unrecognized CA repeats that lie within or in close proximity to the caveolin genes. The first and second exons of both caveolin genes are embedded within CpG islands. These results suggest that regulation of caveolin gene expression may be controlled, in part, by methylation of these CpG regions. In support of this notion, we show here that the CGs in the 5' promoter region of the caveolin-1 gene are functionally methylated in two human breast cancer cell lines (MCF7 and T-47D) that fail to express the caveolin-1 protein. In contrast, the same CGs in cultured normal human mammary epithelial cells (NHMECs) are non-methylated and these cells express high levels of the caveolin-1 protein. Comparison of the human locus with the same locus in the pufferfish Fugu rubripes reveals that the overall organization of the caveolin-1/-2 locus is conserved from pufferfish to man. In conclusion, our current studies provide a systematic basis for diagnostically evaluating the potential deletion, mutation, or methylation of the caveolin genes in a variety of human tumors.
CA微卫星重复标记D7S522位于一个约1000 kb的最小常见缺失区域的中心，该区域在多种人类癌症中缺失。有人提出，一个假定的肿瘤抑制基因位于这个最小常见缺失区域内，靠近D7S522。然而，位于D7S522附近的基因一直难以确定。最近，我们鉴定出五个独立的BAC克隆（约100 - 200 kb），它们包含D7S522以及编码小窝蛋白1和2的人类基因。在此，我们展示了小窝蛋白基因座的详细结构及其与D7S522的关系，这是通过鸟枪法测序方法推导出来的。我们得到了两个相邻的重叠群，总覆盖范围约为250 kb。对这些重叠群的分析表明，D7S522位于小窝蛋白-2基因上游约67 kb处，小窝蛋白-2基因位于小窝蛋白-1基因上游约19 kb处，首次提供了该区域的详细遗传图谱。进一步的序列分析揭示了小窝蛋白基因的许多有趣特征；这些特征包括内含子-外显子边界以及位于小窝蛋白基因内部或附近的几个先前未被识别的CA重复序列。两个小窝蛋白基因的第一和第二外显子都嵌入在CpG岛中。这些结果表明，小窝蛋白基因表达的调控可能部分受这些CpG区域甲基化的控制。为支持这一观点，我们在此表明，在两个不表达小窝蛋白-1蛋白的人乳腺癌细胞系（MCF7和T - 47D）中，小窝蛋白-1基因5'启动子区域的CGs发生了功能性甲基化。相反，在培养的正常人乳腺上皮细胞（NHMECs）中，相同的CGs未甲基化，并且这些细胞表达高水平的小窝蛋白-1蛋白。将人类基因座与河豚红鳍东方鲀中的相同基因座进行比较，发现从小窝蛋白-1/-2基因座的整体结构从河豚到人类是保守的。总之，我们目前的研究为诊断评估多种人类肿瘤中小窝蛋白基因的潜在缺失、突变或甲基化提供了系统依据。
