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

(1)我们在EPD（真核启动子数据库）中对所有1871个人类和196个小鼠RNA聚合酶II启动子进行了分类，并研究了仅包含TATA盒或启动子（Inr）序列的启动子的平均灵活性。我们发现，TATA盒和Inr序列有一个共同的异常的机械性能：它们是由独特的灵活和刚性序列，相比启动子区的其他部分。此外，还发现TATA盒或Inr序列上游的DNA区域比每个元件下游的区域更刚性。我们还计算了不含典型启动子元件的人类启动子的平均柔性概况，并发现TATA或Inr对应区域位于转录起始位点周围的几个核苷酸中。(2)We建立了六个HeLa细胞系，其携带具有T2O侧翼的tk启动子的报告基因，以及它们的从报告基因座缺失T2O的对照细胞系。T2O是模拟负超螺旋的一部分的180 bp的合成弯曲DNA片段。发现T2O在瞬时和稳定的测定系统中激活转录。在后一种系统中，我们不仅使用了上述稳定的转化体，而且还使用了携带报道基因的附加体。染色质分析表明，T2O是旋转设置在希斯顿核心和核小体没有定位或周围的T2O的位置。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.