The functional response of upstream DNA to dynamic supercoiling in vivo

Abstract

Because RNA polymerase is a powerful motor, transmission of transcription-generated forces might directly alter DNA structure, chromatin or gene activity in mammalian cells. Here we show that transcription-generated supercoils streaming dynamically from active promoters have considerable consequences for DNA structure and function in cells. Using a tamoxifen-activatable Cre recombinase to excise a test segment of chromatin positioned between divergently transcribed metallothionein-IIa promoters, we found the degree of dynamic supercoiling to increase as transcription intensified, and it was very sensitive to the specific arrangement of promoters and cis elements. Using psoralen as an in vivo probe confirmed that, during transcription, sufficient supercoiling is produced to enable transitions to conformations other than B-DNA in elements such as the human MYC far upstream element (FUSE), which in turn recruit structure-sensitive regulatory proteins, such as FUSE Binding Protein (FBP) and FBP-Interacting Repressor (FIR). These results indicate that mechanical stresses, constrained by architectural features of DNA and chromatin, may broadly contribute to gene regulation.

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Figure 1: Overview of the approaches used.
Figure 2: Topological analysis of chromatin circles excised between of the divergent metal-inducible promoters.
Figure 3: Inhibition of transcription reveals dynamic supercoiling.
Figure 4: Topoisomerases dissipate transcription-induced DNA supercoiling.
Figure 5: FUSE is a supercoil-dependent sensor of transcription.
Figure 6: FUSE melting absorbs transcription-induced dynamic DNA supercoiling.
Figure 7: Ongoing transcription authorizes binding of FBP and FIR to interpromoter region of FUSE-containing episomes.
Figure 8: The FUSE modulation of MTIIa promoter activity is supercoil-dependent.

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Acknowledgements

Our research is supported by the Intramural Research Program of the US National Institutes of Health, National Cancer Institute, Center for Cancer Research. We thank B. Lewis, H.-J. Chung and A. Mikaelyan for critical comments.

Author information

F.K. and D.L. designed research; F.K., S.S. and Z.E.-F. performed research; F.K. and D.L. analyzed data and wrote the paper.

Correspondence to David Levens.

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Kouzine, F., Sanford, S., Elisha-Feil, Z. et al. The functional response of upstream DNA to dynamic supercoiling in vivo. Nat Struct Mol Biol 15, 146–154 (2008). https://doi.org/10.1038/nsmb.1372

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