Proteomic analysis of nuclear factors binding to an intronic enhancer in the myelin proteolipid protein gene
Proteomic analysis of nuclear factors binding to an intronic enhancer in the myelin proteolipid protein gene
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DOI：
10.1111/j.1471-4159.2008.05288.x
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发表时间：
2008-06-01
影响因子：
4.7
通讯作者：
Wight, Patricia A.
中科院分区：
文献类型：
--
作者：
Dobretsova, Anna;Johnson, Jennifer W.;Wight, Patricia A.
髓鞘蛋白脂质蛋白基因（Plp 1）编码CNS髓鞘中发现的最丰富的蛋白质，占总蛋白质的近一半。其在少突胶质细胞中的表达受发育调节-在CNS发育的活跃髓鞘形成期达到峰值。以前，我们已经确定了一种新的增强子（指定ASE）的内含子1 DNA，似乎是重要的，在介导激增的Plp 1基因活性在活跃的髓鞘形成期间。有证据表明ASE参与了一种称为增强体的特殊多蛋白/DNA复合物的形成。目前的研究描述了一种优化的，五步，DNA亲和层析纯化程序，以纯化小鼠脑细胞核蛋白结合85 bp ASE序列，具体。电泳迁移率变动分析表明，在整个纯化过程中保留了特定的DNA结合活性，导致伴随的核蛋白复合物的富集。通过质谱分析鉴定了所声称的调节因子，包括20多种序列特异性DNA结合蛋白。补充的蛋白质印迹分析，以确定这些序列特异性因子中存在于少突胶质细胞，其在全脑的发育和区域表达，表明Pur α和Pur β排名最高的候选因子的组成部分的多蛋白复合物上形成的ASE。
The myelin proteolipid protein gene (Plp1) encodes the most abundant protein found in CNS myelin, accounting for nearly one-half of the total protein. Its expression in oligodendrocytes is developmentally regulated - peaking during the active myelination period of CNS development. Previously, we have identified a novel enhancer (designated ASE) in intron 1 DNA that appears to be important in mediating the surge of Plp1 gene activity during the active myelination period. Evidence suggests that the ASE participates in the formation of a specialized multi-protein/DNA complex called an enhanceosome. The current study describes an optimized, five-step, DNA affinity chromatography purification procedure to purify nuclear proteins from mouse brain that bind to the 85-bp ASE sequence, specifically. Electrophoretic mobility shift assay analysis demonstrated that specific DNA-binding activity was retained throughout the purification procedure, resulting in concomitant enrichment of nucleoprotein complexes. Identification of the purported regulatory factors was achieved through mass spectrometry analysis and included over 20 sequence-specific DNA-binding proteins. Supplementary western blot analyses to determine which of these sequence-specific factors are present in oligodendrocytes, and their developmental and regional expression in whole brain, suggest that Pur alpha and Pur beta rank highest among the candidate factors as constituents of the multi-protein complex formed on the ASE.