DNA superhelicity enhances the assembly of transcriptionally active chromatin in vitro

Abstract

Using an in vitro chromatin assembly system, we analyzed the influence of DNA superhelicity on the development of transcriptionally active minichromosomes. Plasmid DNA molecules containing either a Xenopus borealis 5S RNA gene or an X. laevis methionine tRNA gene were utilized as templates for the assembly of chromatin. Both plasmids were processed into active minichromosomes if introduced as supercoiled molecules into the extract (S-150). The degree of superhelicity is a determining factor in the assembly of active chromatin. Molecules containing varying superhelical densities were processed into minichromosomes with different transcriptional activities. The absence of supercoils leads to the assembly of chromatin with substantially lower transcriptional activity. Assembled minichromosomes are stable enough to be isolated by sucrose gradient centrifugation while retaining their transcriptional phenotype. The formation of nucleosomes with a periodic spacing occurred with the same efficiency and to the same degree regardless of the initial DNA topology. Hence, a determining factor in the development of transcriptionally active chromatin may be the initial superhelicity of the DNA molecule to which activator (trans-acting factors) or repressor (histones) proteins bind. Once the chromatin assembly process has begun, the transcriptional activity of the resulting minichromosome may already have been determined.