Understanding of chromatin infrastructure that allows transcription initiation

点击翻译按钮获取中文摘要

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.