Merriman H L, van Wijnen A J, Hiebert S, Bidwell J P, Fey E, Lian J, Stein J, Stein G S
Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655, USA.
Biochemistry. 1995 Oct 10;34(40):13125-32. doi: 10.1021/bi00040a025.
The nuclear matrix protein, NMP-2, was originally identified as an osteoblast-specific DNA-binding complex localized exclusively to the nuclear matrix. NMP-2 was shown to recognize two binding sites, site A (nt-605 to -599) and site B (nt -441 to -435), in the rat bone-specific osteocalcin gene promoter. This study shows that the NMP-2 binding sites A and B as well as a third NMP-2 binding site (nt -135 to -130) constitute a consensus sequence, ATGCTGGT, and represent an AML-1 recognition motif. AML-1 is a member of the AML transcription factor family which is associated with acute myelogenous leukemia and binds to the sequence TGCTGGT via its DNA-binding runt domain. Electrophoretic mobility shift assays reveal that a component of NMP-2 is a member of the AML/PEBP2/runt domain transcription factor family based on cross-competition with AML-1 consensus oligonucleotide. Limited immunoreactivity of NMP-2 with a polyclonal N-terminal AML-1 antibody and inability of the AML-1 partner protein CBF-beta to form complexes with NMP-2 indicate that NMP-2 is not identical to AML-1 but represents a variant AML/PEBP2/runt domain protein. Western and Northern blots reveal the presence of multiple AML-related proteins and AML-1 transcripts in several osseous cell lines. Furthermore, our results indicate that AML family members may selectively partition between nuclear matrix and nonmatrix compartments. Because proteins that contain a runt domain are implicated in tissue-specific transcriptional regulation, our results support the concept that the nuclear matrix mediates osteoblast-specific expression of the osteocalcin gene.
核基质蛋白NMP-2最初被鉴定为一种仅定位于核基质的成骨细胞特异性DNA结合复合物。研究表明，NMP-2可识别大鼠骨特异性骨钙素基因启动子中的两个结合位点，即位点A（核苷酸-605至-599）和位点B（核苷酸-441至-435）。本研究显示，NMP-2结合位点A和B以及第三个NMP-2结合位点（核苷酸-135至-130）构成共有序列ATGCTGGT，代表AML-1识别基序。AML-1是AML转录因子家族的成员，与急性髓性白血病相关，并通过其DNA结合矮 runt 结构域与序列TGCTGGT结合。电泳迁移率变动分析表明，基于与AML-1共有寡核苷酸的交叉竞争，NMP-2的一个组分是AML/PEBP2/runt结构域转录因子家族的成员。NMP-2与多克隆N端AML-1抗体的免疫反应性有限，且AML-1伴侣蛋白CBF-β无法与NMP-2形成复合物，这表明NMP-2与AML-1不同，但代表一种变异的AML/PEBP2/runt结构域蛋白。蛋白质免疫印迹法和Northern印迹法显示，在几种骨细胞系中存在多种AML相关蛋白和AML-1转录本。此外，我们的结果表明，AML家族成员可能在核基质和非基质区室之间选择性分配。由于含有矮 runt 结构域的蛋白质与组织特异性转录调控有关，我们的结果支持核基质介导骨钙素基因成骨细胞特异性表达这一概念。
