Structure-function analysis of Msx2-mediated transcriptional suppression.
Structure-function analysis of Msx2-mediated transcriptional suppression.
复制标题
DOI：
10.1021/bi971008x
复制
发表时间：
1997-08
期刊：
影响因子：
2.9
通讯作者：
Elizabeth P. Newberry;T. Latifi;John T. Battaile;D. Towler
中科院分区：
文献类型：
--
作者：
Elizabeth P. Newberry;T. Latifi;John T. Battaile;D. Towler
骨钙素(OC)是一种钙结合蛋白，在成熟的成骨细胞矿化过程中表达。OC基因已被确定为MSX2转录抑制的靶点，MSX2是一种同源结构域转录因子，控制发育中的头盖骨的骨化。我们已经启动了MSX2的系统结构-功能分析，以MC3T3-E1颅骨成骨细胞中OC启动子抑制(荧光素酶报告)为实验对象。对MSX2变异体进行表位(“FLAG”)标记，通过Western印迹分析监测MSX2蛋白的表达。对N-末端和C-末端截短分子的功能分析表明，MSX2残基97-208为核心抑制域。内部缺失分析表明，抑制功能取决于同源结构域上游的残基132-148编码的结构特征，并与同源结构域N端延伸重叠--但不依赖于三个同源结构域螺旋中的残基。增强DNA结合活性的突变并不成比例地增强MSX2抑制功能；此外，完全缺乏DNA结合活性的MSX2点突变MSX2(T147A)在抑制OC启动子的能力方面与野生型MSX2没有区别，这表明MSX2抑制功能不需要与DNA直接相互作用。这表明MSX2通过蛋白质-蛋白质与基本转录机制的组件相互作用来抑制转录，无论是单独的还是与辅助调节因子协同作用的。使用相互作用“远西方”印迹分析，我们系统地测试了MSX2与已知的介导转录激活的基本转录机制组件(TBP、TFIIB和TFIIF)之间的蛋白质-蛋白质相互作用。MSX2结合TFIIF(RAP74、RAP30)的两个组分，但不结合TFIIB或TBP。MSX2(55-208)包含核心抑制域残基并与TFIIF结合；在此背景下，缺失核心抑制区功能所需的17个氨基酸残基132-148会取消与TFIIF组分的相互作用。RAP74在MC3T3-E1细胞中的共表达可部分逆转(50%)MSX2对OC启动子活性的抑制，而TFIIB或RAP30的共表达不起作用。因此，MSX2的核心抑制域参与了与RAP74的重要功能相互作用，该作用调节了颅骨成骨细胞中OC启动子的活性。
Osteocalcin (OC) is a calcium binding protein expressed in mature osteoblasts undergoing mineralization. The OC gene has been identified as a target for transcriptional suppression by Msx2, a homeodomain transcription factor that controls ossification in calvarial bone of the developing skull. We have initiated systematic structure-function analyses of Msx2, using OC promoter suppression (luciferase reporter) in MC3T3-E1 calvarial osteoblasts as an assay. Msx2 variants were epitope ("FLAG")-tagged for monitoring Msx2 protein expression by Western blot analysis. Functional analyses of N- and C-terminally truncated molecules identify Msx2 residues 97-208 as the core suppressor domain. Internal deletion analyses indicate that suppressor function is dependent upon structural features encoded by residues 132-148--upstream of the homeodomain and overlapping the homeodomain N-terminal extension--but not upon residues in the three homeodomain helices. Mutations that enhance DNA binding activity do not proportionally enhance Msx2 suppressor function; moreover, a Msx2 point mutant Msx2(T147A) that completely lacks DNA binding activity is indistinguishable from wild-type Msx2 in its ability to suppress the OC promoter, demonstrating that direct interaction with DNA is not required for Msx2 suppressor function. This suggests that Msx2 suppresses transcription via protein-protein interactions with components of the basal transcriptional machinery, either alone or in concert with co-regulators. Using interaction "Far Western" blotting assays, we systematically tested for protein-protein interactions between Msx2 and components of the basal transcriptional machinery known to mediate transcriptional activation (TBP, TFIIB, and TFIIF). Msx2 binds both components of TFIIF (RAP74, RAP30), but not TFIIB or TBP. Msx2(55-208) encompasses core suppressor domain residues and binds TFIIF; in this context, deletion of the seventeen amino acid residues 132-148 that are required for core suppressor function abrogates interactions with TFIIF components. Co-expression of RAP74 in MC3T3-E1 cells partially reverses (>50%) suppression of OC promoter activity by Msx2, while co-expression of TFIIB or RAP30 has no effect. Thus the core suppressor domain of Msx2 participates in functionally important interactions with RAP74 that regulate OC promoter activity in calvarial osteoblasts.