Maintenance of the chromatin states and histone modification patterns during transcription is essential for proper gene regulation and cell survival. Histone octamer survives moderate transcription, but is evicted during intense transcription in vivo by RNA polymerase II (Pol II). Previously we have shown that nucleosomes can survive transcription by single Pol II complexes in vitro. To study the mechanism of histone displacement from DNA, the encounter between multiple complexes of RNA polymerase and a nucleosome was analyzed in vitro. Multiple transcribing Pol II complexes can efficiently overcome the high nucleosomal barrier and displace the entire histone octamer, matching the observations in vivo. DNA-bound histone hexamer left behind the first complex of transcribing enzyme is evicted by the next Pol II complex. Thus transcription by single Pol II complexes allows survival of the original H3/H4 histones, while multiple, closely spaced complexes of transcribing Pol II can induce displacement of all core histones along the gene.

Enhancers are regulatory DNA elements that can activate their genomic targets over a large distance. The mechanism of enhancer action over large distance is unknown. Activation of the glnAp2 promoter by NtrC-dependent enhancer in Escherichia coli was analyzed by using a purified system supporting multiple-round transcription in vitro . The data suggest that DNA supercoiling is an essential requirement for enhancer action over a large distance (2,500 bp) but not over a short distance (110 bp). DNA supercoiling facilitates functional enhancer–promoter communication over a large distance, probably by bringing the enhancer and promoter into close proximity.