In vivo protein-DNA interactions at a human DNA replication origin
In vivo protein-DNA interactions at a human DNA replication origin
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DOI：
10.1073/pnas.93.4.1498
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发表时间：
1996-02-20
影响因子：
11.1
通讯作者：
Falaschi, A
中科院分区：
文献类型：
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作者：
Dimitrova, DS;Giacca, M;Falaschi, A
在体内研究了含有人类DNA复制起点的区域的蛋白质-DNA相互作用，该区域位于lamin B2基因的3‘端，并部分重叠了另一基因的启动子，位于下游。DNase I处理从指数生长和非增殖的HL-60细胞中分离的细胞核表明，该区域具有改变的、高度可及的染色质结构，通过基于连接介导的聚合酶链式反应的体内足迹技术，对围绕起点的600bp区域的蛋白质-DNA相互作用进行了高分辨率分析。在下游基因启动子对应的区域检测到与已知转录因子(碱性-螺旋-环-螺旋家族成员、核呼吸因子1、转录因子Sp1和上游结合因子)结合位点同源的序列上的足迹，当细胞转换到非增殖状态时，观察到这些足迹的强度降低，这与基因组区域转录活性的降低平行。除了这些保护外，与复制起始点密切对应的是一个显著的足迹，它只在一条链上延伸超过70个核苷酸，而在非增殖的HL-60细胞中没有这种足迹，这表明这种特定的蛋白质-DNA相互作用可能参与了起源激活过程。
Protein-DNA interactions were studied in vivo at the region containing a human DNA replication origin, located at the 3' end of the lamin B2 gene and partially overlapping the promoter of another gene, located downstream, DNase I treatment of nuclei isolated from both exponentially growing and nonproliferating HL-60 cells showed that this region has an altered, highly accessible, chromatin structure, High-resolution analysis of protein-DNA interactions in a 600-bp area encompassing the origin was carried out by the in vivo footprinting technique based on the ligation-mediated polymerase chain reaction, In growing HL-60 cells, footprints at sequences homologous to binding sites for known transcription factors (members of the basic-helix-loop-helix family, nuclear respiratory factor 1, transcription factor Sp1, and upstream binding factor) were detected in the region corresponding to the promoter of the downstream gene, Upon conversion of cells to a nonproliferative state, a reduction in the intensity of these footprints was observed that paralleled the diminished transcriptional activity of the genomic area. In addition to these protections, in close correspondence to the replication initiation site, a prominent footprint was detected that extended over 70 nucleotides on one strand only, This footprint was absent from nonproliferating HL-60 cells, indicating that this specific protein-DNA interaction might be involved in the process of origin activation.