STUDIES ON THE ISOLATED TRANSCRIPTIONALLY ACTIVE AND INACTIVE CHROMATIN FRACTIONS FROM RAT-LIVER NUCLEI

Using mild sonication, nucleoplasmic, nucleolar, and subnucleolar P-3 and S-3 chromatin fractions are isolated from rat liver nuclei. These fractions differ widely (over 80-fold) from each other in transcriptio nal activity as measured by the chromatin bound engaged RNA polymerase s. Chemical analyses indicate that the active chromatin, e.g. P-3 and nucleolar fractions, are rich in RNA and protein as compared to the in active chromatin, e.g. nucleoplasmic, and S-3 fractions. However, the DNA base content are all the same, showing 40% GC and 60% AT, includin g P-3 which is enriched in rDNA. Polyacrylamide gel electrophoresis of the 0.25 N HCl extracted proteins shows that all five histones are pr esent in active chromatin. Additionally, the gel reveals two protein b ands, one ahead of histone H2B and another ahead of histone H4, that a re diminished or missing from the inactive chromatin. On the other han d, there is a fast moving protein band ahead of H4 in the inactive chr omatin that is almost absent in the active chromatin. Transcriptional tests using E. coli RNA polymerase and several synthetic DNA templates of known base content and sequence indicate that the 0.25 N HCl solub le protein extracts from active chromatin contain activator proteins w hich are capable of countering the histone suppressors present in the extracts in a DNA base and sequence specific manner. The data show tha t although the histone suppressors are able to strongly inhibit the te mplate function of poly[d(A-T)], the protein activators are able to ov ercome the suppressor activity and stimulate RNA synthesis several-fol d when poly(dA).poly(dT) or poly(dT) is used.