Histone Release during Transcription: Displacement of the Two H2A−H2B Dimers in the Nucleosome Is Dependent on Different Levels of Transcription-Induced Positive Stress

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Abstract
Both indirect (transcription-induced stress) and direct effects of polymerase elongation on histone−DNA interactions were studied on closed circular DNA that was either moderately or positively coiled. The templates were reconstituted with 3H-labeled H2A, H2B, H3, and H4 to form nucleosomes, and transcription was done with T7 RNA polymerase in the presence of a negatively coiled competitor DNA (reconstituted with unlabeled H3 and H4). The first of the two labeled H2A−H2B dimers readily displaced from the highly positively coiled template to the competitor even in the absence of transcription, while the indirect effect of transcription-induced stress was required for the moderately coiled template. The second labeled H2A−H2B dimer required transcription-induced stress for both moderately and highly positively coiled DNA. The displacement of the labeled H3−H4 tetramer also occurred, provided it was associated with an H2A−H2B dimer and a moderately positively coiled DNA. This displacement occurred independent of transcription-induced stress and is likely due to the direct effect of polymerase disruption of histone−DNA interactions. The inclusion of the histone chaperone, NAP1, greatly enhanced the release of both of the two H2A−H2B dimers. These observations are consistent with in vivo observations which indicate that during transcription H2A and H2B are significantly more mobile than H3 and H4 and indicate that transcription-induced positive stress is a likely cause for this selective movement.