Understanding of chromatin infrastructure that allows transcription initiation

这个项目是为了了解染色质的基础结构，它允许激活剂在转录的第一阶段结合。将合成的176bp的左旋超螺旋DNA(T20)连接到HSV tk启动子或hsp70B启动子上，产生两个报告基因。已发现T20可激活tk启动子约100倍。第三个报告含有小鼠PGK-1启动子，其螺旋轴略微向左弯曲。这些记者中的每一个都被瞬时引入COS-7核。结果表明，细胞核中T20侧翼的tk启动子和PGK-1启动子上均有核小体形成。初步数据还表明，将T20引入hsp70B启动子的上游，在一定程度上缓解了“启动子-近端停顿”现象。这种影响可能是由转录起始点下游区域形成的染色质结构引起的。然后，我们建立了一个HeLa细胞系，该细胞系在其基因组的异染色质区域含有T20-tk启动子的单一拷贝。令人惊讶的是，发现启动子在该基因座上是活跃的。最后，对177个人类启动子的三维结构进行了理论建模。结果表明，在大多数情况下，含有TATA盒的区域重叠着一个左旋或右旋的超螺旋DNA结构，位于转录起始点下游附近的区域几乎是直的。所有这些结果有力地表明，模仿左手超螺旋的弯曲DNA通过活跃地形成核小体而在转录中发挥作用。另一方面，据推测，模仿右旋超螺旋结构的弯曲DNA可能通过抑制核小体的形成来帮助转录。

This project has been carried out to understand chromatin infrastructure that allows activator binding at the first stage of transcription. A synthetic left-handed superhelical bent DNA of 176 bp (T20) was ligated to HSV tk promoter or human hsp70B promoter to generate two reporters. The T20 had been found to activate the tk promoter by about 100-fold. The third reporter contained mouse pgk-1 promoter whose helical axis is slightly curved toward left. Each of these reporters was transiently introduced into COS-7 nuclei. It was revealed that nucleosomes were formed on both the T20-flanked tk promoter and the pgk-1 promoter in the nuclei. A preliminary data also suggested that the phenomenon called "promoter-proximal pausing" was relieved to some extent when the T20 was introduced into upstream of the hsp70B promoter. This effect was presumably caused by the chromatin structure formed in the downstream region of the transcription initiation site. Then, we have established a HeLa cell line that contains a single copy of the T20-tk promoter construct in heterochromatin region of its genome. Surprisingly, it was found that the promoter was active in the locus. Finally, three-dimensional architectures of 177 human promoters were theoretically modeled. The result showed that in most cases the region containing the TATA box overlaps a left- or right-handed superhelical bent DNA structure and the region locating in the downstream vicinity of the transcription initiation site is almost straight.All of these results strongly indicate that bent DNAs mimicking left-handed superhelix function in transcription by actively forming nucleosomes. On the other hand, it is speculated that bent DNAs mimicking right-handed superhelical structure may help transcription by inhibiting nucleosome formation.