Understanding of chromatin infrastructure that allows transcription initiation

这个项目已经进行，以了解染色质的基础设施，允许激活剂结合在转录的第一阶段。将合成的176 bp的左手超螺旋弯曲DNA（T20）连接到HSV tk启动子或人hsp 70 B启动子以产生两个报告基因。已经发现T20激活tk启动子约100倍。第三个报告基因含有小鼠pgk-1启动子，其螺旋轴略微向左弯曲。将这些报告基因中的每一种瞬时引入COS-7细胞核。结果表明，在细胞核中，T20侧翼的tk启动子和pgk-1启动子上都形成了核小体。初步研究结果表明，在hsp 70 B启动子上游引入T20后，启动子近端停顿现象得到一定程度的缓解。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HeLa细胞系，其基因组的异染色质区域中含有T20-tk启动子构建体的单拷贝。令人惊讶的是，发现启动子在基因座中是有活性的。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，这些结果强烈地表明，模仿左旋超螺旋的弯曲DNA通过积极地形成核小体而在转录中发挥作用。另一方面，据推测，模仿右手超螺旋结构的弯曲DNA可能通过抑制核小体形成来帮助转录。

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.