A study on the role of bent DNA in constructing chromatin infrastructure for transcription initiation

(1)我们对EPD(真核生物启动子数据库)中的1871个人RNA聚合酶II启动子和196个小鼠RNA聚合酶II启动子进行了分类，并研究了仅含有TATA盒或启动子(INR)序列的启动子的平均柔韧性。我们发现，TATA盒和INR序列有一个共同的异常力学性质：与启动子区域的其他部分相比，它们由不同的柔性和刚性序列组成。此外，还发现TATA盒或INR序列上游的DNA区域比每个元件的下游区域更刚性。我们还计算了不包含典型启动子元件的人类启动子的平均柔性谱，发现TATA或INR对应的区域位于转录起始点周围的几个核苷酸中。(2)我们建立了六个携带T2O侧翼tk启动子的报告基因的HeLa细胞系及其从报告基因座上删除T2O的对照细胞株。T2O是一段合成的弯曲DNA片段，长180个碱基对，模仿部分负极超级线圈。在暂态和稳定的检测系统中，T2O都能激活转录。在后一种系统中，我们不仅使用了上述稳定的转化子，还使用了携带报告基因的异构体。染色质分析表明，T2O以旋转方式定位在组蛋白核心上，核小体没有平移地定位在T2O的内部或周围。T2O可能通过帮助核小体滑动来激活转录。

(1) We classified all of the 1871 human and 196 mouse RNA polymerase II promoters in the EPD (eukaryotic promoter database) and investigated average flexibility profiles of the promoters containing either a TATA box or an initiator (Inr) sequence only. We found that TATA boxes and Inr sequences have a common anomalous mechanical property : they are comprised of distinctively flexible and rigid sequences, compared to the other parts of the promoter region. Additionally, it was also found that DNA region upstream of TATA box or Inr sequence is more rigid than region downstream of each element. We also calculated the average flexibility profiles of the human promoters that do not contain canonical promoter elements and found that the TATA- or Inr-corresponding region lies in the several nucleotides around the transcription initiation site.(2)We established six HeLa cell lines that carry a reporter with a T2O-flanked tk promoter and their control cell lines that delete T2O from the reporter locus. The T2O is a synthetic bent DNA segment of 180 bp that mimics a part of negative supercoils. The T2O was found to activate transcription in both transient and stable assay systems. In the latter system, we used not only the stable transformants described above but also episomes carrying the reporter. Chromatin analyses indicated that the T2O were rotationally set on histon cores and that nucleosomes did not position in or around the T2O translationally. The T2O may activate transcription by helping nucleosome sliding.