Markose E R, Stein J L, Stein G S, Lian J B
Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655.
Proc Natl Acad Sci U S A. 1990 Mar;87(5):1701-5. doi: 10.1073/pnas.87.5.1701.
By the combined use of DNase I footprinting, electrophoretic mobility-shift assay, and methylation interference analysis, we have identified a series of sequence-specific protein-DNA interactions in the 5' flanking region of the rat osteocalcin gene. Stimulation of osteocalcin gene expression by 1,25-dihydroxyvitamin D3, a physiologic mediator of this bone-specific gene in vitro and in vivo, is associated with modifications in the binding of ROS 17/2.8 cell nuclear factors to two promoter segments that up-regulate transcription. One segment located between -462 and -437 exhibits a vitamin D-dependent increase in sequence-specific binding of nuclear factors. This element (CTGGGTGAATGAGGACATTACTGACC), identified at single nucleotide resolution, contains a region of hyphenated dyad symmetry and shares sequence homology with consensus steroid-responsive elements and with the sequence that has been identified as the vitamin D receptor binding site in the human osteocalcin gene. We have also observed that vitamin D stimulation of osteocalcin gene expression results in a 5-fold increase in protein binding to the region of the osteocalcin box, a 24-nucleotide segment in the proximal promoter with a CCAAT motif as the central core. Our results demonstrate protein-DNA interactions in a vitamin D-responsive element and in a second sequence, the osteocalcin box, both of which are involved in the physiologic regulation of the osteocalcin gene in response to 1,25-dihydroxyvitamin D3.
通过联合使用DNA酶I足迹法、电泳迁移率变动分析和甲基化干扰分析，我们在大鼠骨钙素基因的5'侧翼区域鉴定出了一系列序列特异性的蛋白质-DNA相互作用。1,25-二羟基维生素D3是该骨特异性基因在体外和体内的生理介质，它对骨钙素基因表达的刺激作用与ROS 17/2.8细胞核因子与两个上调转录的启动子片段的结合改变有关。位于-462至-437之间的一个片段显示出核因子序列特异性结合的维生素D依赖性增加。这个在单核苷酸分辨率下鉴定出的元件（CTGGGTGAATGAGGACATTACTGACC）包含一个间断的二元对称区域，与共有类固醇反应元件以及已被鉴定为人骨钙素基因中维生素D受体结合位点的序列具有序列同源性。我们还观察到，维生素D对骨钙素基因表达的刺激导致与骨钙素盒区域的蛋白质结合增加了5倍，骨钙素盒是近端启动子中一个24个核苷酸的片段，以CCAAT基序为核心。我们的结果证明了在维生素D反应元件和第二个序列即骨钙素盒中的蛋白质-DNA相互作用，这两者都参与了骨钙素基因对1,25-二羟基维生素D3的生理调节。
