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

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(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.