Shehin-Johnson S E, Palmer K C, Hines R N
Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201-1998, USA.
Drug Metab Dispos. 1996 Aug;24(8):891-8.
Previous studies demonstrated that the flavin-containing monooxygenases (FMO) are expressed in a tissue-specific manner. To begin an elucidation of mechanisms regulating this expression pattern, genomic clones for rabbit FMO2 were isolated and characterized. Two clones were isolated from a lambda EMBL3 genomic library and shown to span approximately 30 kilobase pairs but to contain only about 800 base pairs of coding information. Primer extension analysis was used to map the transcription start site, extending the previously published cDNA sequence by 17 base pairs. The FMO2 promoter does not utilize a classical TATA box, nor are HTF islands present (DNA domains rich in cleavable sites for 5-methyl-cytidine/guanosine-sensitive restriction enzymes). Rather, homology with promoters controlled by initiator elements is observed. Previous studies demonstrated FMO2 expression in rabbit pulmonary Clara and type II cells [Overby, Nishio, Lawton, Plopper, and Philpot: Exp. Lung Res. 18, 131, (1992)]. In the present study, a highly enriched Clara/type II cell population was prepared, and the FMO2 gene was analyzed for DNase I-hypersensitive and methylated regions. These data were then contrasted with those obtained from a similar analysis of the nonexpressed, hepatocyte FMO2 gene. No difference in the methylation status was observed. However, Clara/type II cell-specific DNase I-hypersensitive sites are located within the promoter region of the FMO2 gene. Thus, tissue-specific transcription factors likely are more prominent than methylation in regulating FMO2 expression. Consistent with this observation, both polyomavirus enhancer activator 3 (E26 transformation specific) and thyroid transcription factor 1 consensus sequences are present within the tissue-specific DNase I-hypersensitive domain.
先前的研究表明，含黄素单加氧酶（FMO）以组织特异性方式表达。为了开始阐明调节这种表达模式的机制，分离并鉴定了兔FMO2的基因组克隆。从λEMBL3基因组文库中分离出两个克隆，显示其跨度约为30千碱基对，但仅包含约800个碱基对的编码信息。引物延伸分析用于定位转录起始位点，将先前发表的cDNA序列延长了17个碱基对。FMO2启动子不使用经典的TATA盒，也不存在HTF岛（富含对5-甲基胞嘧啶/鸟苷敏感的限制性内切酶可切割位点的DNA结构域）。相反，观察到与由起始元件控制的启动子具有同源性。先前的研究表明FMO2在兔肺克拉拉细胞和II型细胞中表达[Overby，Nishio，Lawton，Plopper和Philpot：《实验肺研究》18，131，（1992）]。在本研究中，制备了高度富集的克拉拉/II型细胞群体，并分析了FMO2基因的DNA酶I超敏和甲基化区域。然后将这些数据与从非表达的肝细胞FMO2基因的类似分析中获得的数据进行对比。未观察到甲基化状态的差异。然而，克拉拉/II型细胞特异性DNA酶I超敏位点位于FMO2基因的启动子区域内。因此，在调节FMO2表达方面，组织特异性转录因子可能比甲基化更为突出。与此观察结果一致，多瘤病毒增强子激活剂3（E26转化特异性）和甲状腺转录因子1共有序列均存在于组织特异性DNA酶I超敏结构域内。
