Biochemical analysis of chromatin containing recombinant Drosophila core histones.
To investigate the effects of histone modifications upon chromatin structure and function, we studied the assembly and properties of chromatin that contains unmodified recombinant core histones. To this end, we synthesized the Drosophila core histones in Escherichia coli. The purified histones were lacking covalent modifications as well as their N-terminal initiating methionine residues. The recombinant histones were efficiently assembled into periodic nucleosome arrays in a completely purified recombinant system with Drosophila ATP-utilizing chromatin assembly and remodeling factor (ACF), Drosophila nucleosome assembly protein-1, plasmid DNA, and ATP. With the Gal4-VP16 activator and a crude transcription extract, we found that the transcriptional properties of ACF-assembled chromatin containing unmodified histones were similar to those of chromatin containing native histones. We then examined ACF-catalyzed chromatin remodeling with completely purified factors and chromatin consisting of unmodified histones. In these experiments, we observed promoter-specific disruption of the regularity of nucleosome arrays upon binding of Gal4-VP16 as well as nucleosome positioning by R3 Lac repressor and subsequent nucleosome remobilization upon isopropyl-beta-D-thiogalactopyranoside-induced dissociation of R3 from the template. Thus, chromatin assembly and remodeling by ACF can occur in the absence of histone modifications.
Levenstein
Mark E
ME
Section of Molecular Biology, University of California, San Diego, La Jolla, California 92093-0347, USA.
Kadonaga
James T
JT
D000818:Animals; D015139:Blotting, Southern; D002843:Chromatin / Q000737:chemistry*; D004247:DNA / Q000378:metabolism; D004278:DNA, Superhelical; D003850:Deoxyribonuclease I / Q000378:metabolism; D019281:Dimerization; D004330:Drosophila; D004926:Escherichia coli / Q000378:metabolism; D006657:Histones / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic
D002843:Chromatin; D004278:DNA, Superhelical; D006657:Histones; D011994:Recombinant Proteins; D004247:DNA; D003850:Deoxyribonuclease I