Identification of a rosette-enriched chromatin fraction from mouse fibroblast nuclei

Abstract

We have characterized two components of DNA isolated from mouse L-M cell nuclei. These components, designated as HMW (high molecular weight) and VHMW (very high molecular weight) DNA, were characterized by rate zonal sedimentation, agarose gel electrophoresis, and for protein content. Our electron micrographs revealed that HMW-DNA contained mainly linear molecules with few single rosette structures, while the VHMW-DNA was enriched in rosettes, many of which were significantly larger and linked together in multimeric structural forms. The VHMW-DNA component was also enriched for residual protein, which we believe represents the core of the rosette. The characteristics of this residual protein are consistent with reported findings of the most tightly bound proteins. The rosette conformation does not appear to be an artifact of microscopy or of an aggregate nature for several reasons: (i) rosettes are preferentially found in the VHMW-DNA component; (ii) further manipulation or purification of the DNA disrupts the rosette structure and produces linear fragments; (iii) the amount of proteinaceous material at the core of the rosette is diminished when the DNA is further purified; and (iv) treatment of intact nuclei with a novel bisamine reagent putatively crosslinks DNA in vivo and minimizes the disruption of rosettes by shear. We believe this separation of chromatin is critical to establish the architectural forms of euchromatin and heterochromatin of interphase DNA in the eucaryotic system. Once established, fractionated chromatin can be used to identify specifically expressed or repressed genes with linear form DNA and rosette form DNA. We discuss rosettes as derivatives of chromosomal domains that retain structural features because of residual peptide elements.