The functional response of upstream DNA to dynamic supercoiling in vivo
The functional response of upstream DNA to dynamic supercoiling in vivo
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DOI：
10.1038/nsmb.1372
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发表时间：
2008-02-01
影响因子：
16.8
通讯作者：
Levens, David
中科院分区：
文献类型：
--
作者：
Kouzine, Fedor;Sanford, Suzanne;Levens, David
由于RNA聚合酶是一个强大的马达，转录产生的力量的传递可能会直接改变哺乳动物细胞的DNA结构、染色质或基因活性。在这里，我们表明，从活性启动子动态产生的转录产生的超级线圈对细胞中的DNA结构和功能有相当大的影响。利用三苯氧胺可激活的Cre重组酶切割位于不同转录的金属硫蛋白-IIa启动子之间的染色质测试片段，我们发现动态超卷曲的程度随着转录的加强而增加，并且它对启动子和顺式元件的特定排列非常敏感。使用补骨脂素作为体内探针证实，在转录过程中，产生了足够的超螺旋，使人类MYC远上游元件(FUSE)等元件能够转变为B-DNA以外的构象，进而招募结构敏感的调节蛋白，如融合结合蛋白(FBP)和FBP相互作用抑制因子(FIR)。这些结果表明，机械应力，受DNA和染色质结构特征的限制，可能对基因调控有广泛的贡献。
Because RNA polymerase is a powerful motor, transmission of transcription-generated forces might directly alter DNA structure, chromatin or gene activity in mammalian cells. Here we show that transcription-generated supercoils streaming dynamically from active promoters have considerable consequences for DNA structure and function in cells. Using a tamoxifen-activatable Cre recombinase to excise a test segment of chromatin positioned between divergently transcribed metallothionein-IIa promoters, we found the degree of dynamic supercoiling to increase as transcription intensified, and it was very sensitive to the specific arrangement of promoters and cis elements. Using psoralen as an in vivo probe confirmed that, during transcription, sufficient supercoiling is produced to enable transitions to conformations other than B-DNA in elements such as the human MYC far upstream element (FUSE), which in turn recruit structure-sensitive regulatory proteins, such as FUSE Binding Protein (FBP) and FBP-Interacting Repressor (FIR). These results indicate that mechanical stresses, constrained by architectural features of DNA and chromatin, may broadly contribute to gene regulation.