Wang X, Tolstonog G, Shoeman R L, Traub P
Max-Planck-Institut für Zellbiologie, Ladenburg, Germany.
DNA Cell Biol. 1996 Mar;15(3):209-25. doi: 10.1089/dna.1996.15.209.
Mouse vimentin intermediate filaments (IFs) reconstituted in vitro were analyzed for their capacity to select certain DNA sequences from a mixture of about 500-bp-long fragments of total mouse genomic DNA. The fragments preferentially bound by the IFs and enriched by several cycles of affinity binding and polymerase chain reaction (PCR) amplification were cloned and sequenced. In general, they were G-rich and highly repetitive in that they often contained Gn, (GT)n, and (GA)n repeat elements. Other, more complex repeat sequences were identified as well. Apart from the capacity to adopt a Z-DNA and triple helix configuration under superhelical tension, many fragments were potentially able to form cruciform structures and contained consensus binding sites for various transcription factors. All of these sequence elements are known to occur in introns and 5'/3'-flanking regions of genes and to play roles in DNA transcription, recombination and replication. A FASTA search of the EMBL data bank indeed revealed that sequences homologous to the mouse repetitive DNA fragments are commonly associated with gene-regulatory elements. Unexpectedly, vimentin IFs also bound a large number of apparently overlapping, AT-rich DNA fragments that could be aligned into a composite sequence highly homologous to the 234-bp consensus centromere repeat sequence of gamma-satellite DNA. Previous experiments have shown a high affinity of vimentin for G-rich, repetitive telomere DNA sequences, superhelical DNA, and core histones. Taken together, these data support the hypothesis that, after penetration of the double nuclear membrane via an as yet unidentified mechanism, vimentin IFs cooperatively fix repetitive DNA sequence elements in a differentiation-specific manner in the nuclear periphery subjacent to the nuclear lamina and thus participate in the organization of chromatin and in the control of transcription, replication, and recombination processes. This includes aspects of global regulation of gene expression such as the position effects associated with translocation of genes to heterochromatic centromere and telomere regions of the chromosomes.
对体外重构的小鼠波形蛋白中间丝（IFs）进行分析，以研究其从约500个碱基对长的小鼠基因组DNA总片段混合物中选择特定DNA序列的能力。通过几个亲和结合和聚合酶链反应（PCR）扩增循环，优先被IFs结合并富集的片段被克隆并测序。一般来说，它们富含G且高度重复，因为它们经常包含Gn、（GT）n和（GA）n重复元件。还鉴定出了其他更复杂的重复序列。除了在超螺旋张力下形成Z-DNA和三螺旋结构的能力外，许多片段还可能形成十字形结构，并包含各种转录因子的共有结合位点。所有这些序列元件都已知存在于基因的内含子和5'/3'侧翼区域，并在DNA转录、重组和复制中发挥作用。对EMBL数据库的FASTA搜索确实显示，与小鼠重复DNA片段同源的序列通常与基因调控元件相关。出乎意料的是，波形蛋白IFs还结合了大量明显重叠的富含AT的DNA片段，这些片段可以排列成与γ-卫星DNA的234个碱基对共有着丝粒重复序列高度同源的复合序列。先前的实验表明波形蛋白对富含G的重复端粒DNA序列、超螺旋DNA和核心组蛋白具有高亲和力。综上所述，这些数据支持这样一种假说，即波形蛋白IFs通过一种尚未确定的机制穿透双层核膜后，以分化特异性方式在核纤层下方的核周边协同固定重复DNA序列元件，从而参与染色质的组织以及转录、复制和重组过程的控制。这包括基因表达的全局调控方面，例如与基因易位到染色体异染色质着丝粒和端粒区域相关的位置效应。
