Understanding of chromatin infrastructure that allows transcription initiation

该项目旨在了解在转录的第一阶段允许激活子结合的染色质基础结构。将合成的176 bp （T20）的左旋超螺旋弯曲DNA连接到HSV tk启动子或人hsp70B启动子上，生成两个报告子。T20已被发现激活tk启动子约100倍。第三个报告基因包含小鼠pgk-1启动子，其螺旋轴略微向左弯曲。这些报告基因中的每一个都被短暂地引入COS-7核中。结果表明，核小体在细胞核中t20侧的tk启动子和pgk-1启动子上都有形成。初步数据还表明，将T20引入hsp70B启动子上游，一定程度上缓解了“启动子-近端暂停”现象。这种效应可能是由转录起始位点下游区域形成的染色质结构引起的。然后，我们建立了一个在其基因组异染色质区域含有单个T20-tk启动子结构的HeLa细胞系。令人惊讶的是，发现启动子在基因座上是活跃的。最后，对177个人体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，包含TATA盒的区域与左旋或右旋超螺旋弯曲DNA结构重叠，位于转录起始位点下游附近的区域几乎是直的。所有这些结果强烈表明，弯曲dna通过主动形成核小体来模仿左旋超螺旋的转录功能。另一方面，据推测，弯曲dna模仿右手超螺旋结构可能有助于转录抑制核小体的形成。

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.