The RRM domain of MINT, a novel Msx2 binding protein, recognizes and regulates the rat osteocalcin promoter
The RRM domain of MINT, a novel Msx2 binding protein, recognizes and regulates the rat osteocalcin promoter
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DOI：
10.1021/bi990967j
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发表时间：
1999-08-17
期刊：
影响因子：
2.9
通讯作者：
Towler, DA
中科院分区：
文献类型：
--
作者：
Newberry, EP;Latifi, T;Towler, DA
MSX2是一种同源结构域转录抑制因子，在颅面部骨骼和神经发育过程中发挥组织特异性作用。为了确定参与MSX2转录抑制的协同调节分子，我们应用Farwest表达克隆策略来确定编码与MSX2结合的蛋白的转录本。用含MSX2核心抑制区的放射性标记GST-MSX2融合蛋白筛选小鼠脑组织中的lambda gt11表达文库。分离到一个编码与放射性标记的MSX2结合的新蛋白片段的cDNA。对T7表位标记的重组蛋白片段进行Farwest分析证实了同源蛋白结合活性，体外相互作用需要体内转录抑制所必需的MSX2残基。在生化分析的基础上，这种新的蛋白质被命名为MINI，这是MSX2相互作用核靶蛋白的首字母缩写。最初的克隆是12.6kb转录本的一部分，在睾丸中高水平表达，在颅骨成骨细胞和脑中低水平表达。对从小鼠睾丸文库中分离到的多个克隆进行了测序，构建了11kb的mint基因重叠群。从Kozak环境良好的启动子Met开始，预测了一个由3576个氨基酸组成的大的新生多肽，在与MSX2相互作用结构域残基2070-2394相邻的开放阅读框中。蛋白质序列分析表明，MINT具有3个N-末端RNA识别基序(RRMS)和4个核定位信号。对分离细胞提取物的Western印迹分析表明，类似110 kDa(N-端)和250 kDa(C-端)的成熟薄荷蛋白片段聚集在染色质和核基质部分，与MSX2和拓扑异构酶II共分离。在凝胶位移分析中，薄荷RRM结构域选择性地结合T-和G-丰富的DNA序列；这包括在大鼠骨钙素(OC)启动子近端存在的一个大的G/T丰富的反向重复元件，重叠了三个支持OC在成骨细胞中表达的同源基因。在MC3T3E1成骨细胞分化过程中，Mint和OC的mRNAs相互调控。与其作为核转录因子的作用一致，mint(1-812)的瞬时表达抑制了成纤维细胞生长因子/forsklin激活的OC启动子，对CMV启动子的活性没有明显的调节作用，但显著上调了HSV胸苷激酶启动子的表达。总之，这些数据表明，新的核蛋白薄荷结合了同源蛋白MSX2，并在颅面发育过程中协同调节OC。MSX2和MINT都针对OC近端启动子的信息密集、成骨细胞特异性调控区域，即核苷酸-141至-111。N端的mint RRM结构域代表了这种新的脊椎动物核基质蛋白的真实dsDNA结合模块。作为一种支架蛋白，mini通过在核基质中组织转录复合体，潜在地发挥着积极和消极的调节作用。
Msx2 is a homeodomain transcriptional repressor that exerts tissue-specific actions during craniofacial skeletal and neural development. To identify coregulatory molecules that participate in transcriptional repression by Msx2, we applied a Farwestern expression cloning strategy to identify transcripts encoding proteins that bind Msx2. A lambda gt11 expression library from mouse brain was screened with radiolabeled GST-Msx2 fusion protein encompassing the core suppressor domain of Msx2. A cDNA was isolated that encodes a novel protein fragment that binds radiolabeled Msx2. Homeoprotein binding activity was confirmed by Farwestern analysis of the T7-epitope-tagged recombinant protein fragment, and interactions in vitro require Msx2 residues necessary for transcriptional suppression in vivo. On the basis of biochemical analyses, this novel protein was named MINI, an acronym for Msx2-interacting nuclear target protein. The original clone is part of a 12.6 kb transcript expressed at high levels in testis and at lower levels in calvarial osteoblasts and brain. Multiple clones isolated from a mouse testis library were sequenced to construct a MINT cDNA contig of 11 kb. Starting from an initiator Met in good Kozak context, a large nascent polypeptide of 3576 amino acids is predicted, in contiguous open reading frame with the Msx2 interaction domain residues 2070-2394. Protein sequence analysis reveals that MINT has three N-terminal RNA recognition motifs (RRMs) and four nuclear localization signals. Western blot analysis of fractionated cell extracts reveals that mature similar to 110 kDa (N-terminal) and similar to 250 kDa (C-terminal) MINT protein fragments accumulate in chromatin and nuclear matrix fractions, cosegregating with Msx2 and topoisomerase II. In gel shift assays, the MINT RRM domain selectively binds T- and G-rich DNA sequences; this includes a large G/T-rich inverted repeat element present in the proximal rat osteocalcin (OC) promoter, overlapping three cognates that support OC expression in osteoblasts. MINT and OC mRNAs are reciprocally regulated during differentiation of MC3T3E1 calvarial osteoblasts. Consistent with its proposed role as a nuclear transcriptional factor, transient expression of MINT(1-812) suppresses the FGF/forskolin-activated OC promoter, does not significantly regulate CMV promoter activity, but markedly upregulates the HSV thymidine kinase promoter in MC3T3E1 cells. In tote, these data indicate that the novel nuclear protein MINT binds the homeoprotein Msx2 and coregulates OC during craniofacial development. Msx2 and MINT both target an information-dense, osteoblast-specific regulatory region of the OC proximal promoter, nucleotides -141 to -111. The N-terminal MINT RRM domain represents an authentic dsDNA binding module for this novel vertebrate nuclear matrix protein. Acting as a scaffold protein, MINI potentially exerts both positive and negative regulatory actions by organizing transcriptional complexes in the nuclear matrix.