Heinrichs A A, Banerjee C, Bortell R, Owen T A, Stein J L, Stein G S, Lian J B
Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655.
J Cell Biochem. 1993 Nov;53(3):240-50. doi: 10.1002/jcb.240530309.
The rat osteocalcin gene encodes a 6-kD osteoblast-specific protein that is expressed post-proliferatively. The developmental and steroid hormone responsive expression of the osteocalcin gene is transcriptionally regulated by a promoter with multiple basal and enhancer elements that exhibit activity controlled by a series of physiological mediators (e.g., 1.25(OH)2D3, glucocorticoids). In this study, we established the contribution of the rat osteocalcin (OC) box domain (-99 to -76), a proximal basal element with a CCAAT motif as a central core, to transcriptional activity of the rat osteocalcin gene with in vivo co-transfection assays. By this same assay, however, the highly homologous (22 of 24 nt) human OC box element was unable to compete for transcription factor binding with the rat OC promoter. In vitro protein/DNA interaction studies confirm the presence of two protein binding sites in the OC box region, one of which overlaps the CCAAT motif and, at least in part, accounts for species-specific expression. Competition analysis established that the single nucleotide substitution of adenine for thymine, which converts the core motif of the rat OC box (CCAAT) to the core motif of the human OC box (CCAAA), accounts for observed species differences in transcription factor interactions. The CCAAT-specific protein/DNA interactions are heat stable and insensitive to phosphatase treatment. At second protein/DNA interaction located upstream of the CCAAT motif includes two steroid-like half-elements. These interactions are heat labile and sensitive to phosphatase treatment in contrast to the CCAAT-specific interactions. The human OC promoter contains only a single steroid-like half-element, while two steroid half-elements with an 11 nucleotide spacer are present in the rat OC promoter. These observed variations in sequence organization and transactivation factor binding in analogous proximal basal regulatory regions of the OC gene promoter may provide a basis for species-restricted variations in responsiveness to physiological mediators of OC gene expression at the transcriptional level.
大鼠骨钙素基因编码一种6kD的成骨细胞特异性蛋白，该蛋白在增殖后期表达。骨钙素基因的发育及类固醇激素反应性表达受到一个具有多个基础元件和增强子元件启动子的转录调控，这些元件的活性受一系列生理介质（如1,25(OH)₂D₃、糖皮质激素）控制。在本研究中，我们通过体内共转染实验确定了大鼠骨钙素（OC）盒结构域（-99至-76）（一个以CCAAT基序为核心的近端基础元件）对大鼠骨钙素基因转录活性的贡献。然而，通过同样的实验，高度同源（24个核苷酸中有22个相同）的人OC盒元件无法与大鼠OC启动子竞争转录因子结合。体外蛋白质/DNA相互作用研究证实OC盒区域存在两个蛋白质结合位点，其中一个与CCAAT基序重叠，至少部分解释了物种特异性表达。竞争分析表明，腺嘌呤对胸腺嘧啶的单核苷酸取代将大鼠OC盒的核心基序（CCAAT）转变为人OC盒的核心基序（CCAAA），这解释了观察到的转录因子相互作用中的物种差异。CCAAT特异性蛋白质/DNA相互作用对热稳定且对磷酸酶处理不敏感。位于CCAAT基序上游的第二个蛋白质/DNA相互作用包含两个类类固醇半元件。与CCAAT特异性相互作用相反，这些相互作用对热不稳定且对磷酸酶处理敏感。人OC启动子仅包含一个类类固醇半元件，而大鼠OC启动子中存在两个间隔11个核苷酸的类固醇半元件。在OC基因启动子类似的近端基础调控区域中观察到的序列组织和反式激活因子结合的这些差异，可能为OC基因表达在转录水平上对生理介质反应性的物种限制差异提供基础。
