The functional response of upstream DNA to dynamic supercoiling in vivo.
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.
Kouzine
Fedor
F
Laboratory of Pathology, National Cancer Institute, 10 Center Drive, Building 10, Room 2N106, Bethesda, Maryland 20892-1500, USA.
Sanford
Suzanne
S
Elisha-Feil
Zichrini
Z
Levens
David
D
D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D026942:DNA Topoisomerases / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry* / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D005786:Gene Expression Regulation / Q000502:physiology*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*
D002843:Chromatin; D004278:DNA, Superhelical; C577426:FARP2 protein, human; D020662:Guanine Nucleotide Exchange Factors; C105789:KHSRP protein, human; D016601:RNA-Binding Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D015534:Trans-Activators; D026942:DNA Topoisomerases