Histone release during transcription: NAP1 forms a complex with H2A and H2B and facilitates a topologically dependent release of H3 and H4 from the nucleosome.
Transcription through a multinucleosomal template was studied to determine why histones are released to the nascent RNA. It was first determined in competition experiments between DNA and RNA that histones H2A and H2B have a 20-fold preference for binding RNA over DNA; a preference was not seen for histones H3 and H4. Histones H3 and H4 would preferentially bind RNA, provided they were in an octameric complex with H2A and H2B. In transcription studies with T7 RNA polymerase, H3 and H4 were transferred to the nascent RNA, provided the template was linear. If the DNA was topologically restrained, which is a condition that more closely maintains transcription-induced stresses, H3 and H4 would not release. Histones H3 and H4 would be released from this template when H2A and H2B were present, a release that was enhanced by the presence of nucleosome assembly protein-1 (NAP1). Since a small quantity of H2A and H2B is sufficient to facilitate this transfer, it is proposed that H2A and H2B function to repeatedly shuttle H3 and H4 from the template DNA to the RNA. Cross-linked histones (dimethylsuberimidate-cross-linked octamer) were reconstituted into nucleosomes and found to be transferred to the RNA at the same frequency as un-cross-linked histones, an indication that such large complexes can be released during transcription. Transcription was carried out in the presence of Escherichia coli topoisomerase I so that positive coils would accumulate on the DNA. Histones H3 and H4 would again not be transferred from this DNA, unless H2A and H2B were present. In this instance, however, when NAP1 was present, the shuttling of H3 and H4 to the RNA caused a significant depletion of H2A and H2B from the positively coiled DNA. These results are discussed with regard to current models for transcription through nucleosomes.
Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.
Vladislav
V
Levchenko
Vaughn
V
Jackson
D000818:Animals; D018797:Cell Cycle Proteins; D002461:Cell Line, Transformed; D002645:Chickens; D004264:DNA Topoisomerases, Type I / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D009687:Nuclear Proteins; D056490:Nucleosome Assembly Protein 1; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism*; D010443:Peptide Chain Termination, Translational / Q000235:genetics; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D056490:Nucleosome Assembly Protein 1; D009707:Nucleosomes; D011506:Proteins; D016601:RNA-Binding Proteins; D004264:DNA Topoisomerases, Type I