Biophysical Characterization of a Recombinant Chromatin-Liposome Aggregates

Abstract

Self-assembled aggregates of recombinant chromatin and liposomes were characterized by synchrotron small angle x-ray spectroscopy (SAXS) and optical microscopy. Recombinant nucleosome core particles (NCP) and 12-mer nucleosome arrays, where half of the negative DNA charge is neutralized by histone octamers, were used as a chromatin model. Liposomes of different charge and charge density were prepared from the either cationic DOTAP or anionic DOPG lipid molecules mixed with zwitterionic DOPC at different molar percentages.Two distinct phases were identified in aggregates with low and high charge density cationic liposomes. The former appeared as translucent precipitate, probably due to higher water retention, compared to latter appearing compact and opaque, and exhibiting birefringence. Systematic SAXS measurements revealed chromatin-lipid bilayer complexes at 3-10% DOTAP, with lamellar distance around 160 A for NCP, and 200 A for arrays. At 10-100% DOTAP, proteins were displaced from DNA by cationic liposomes forming DNA lamellar phase. Confocal microscopy with triple fluorescent labeling displayed homogeneous distribution of DNA, proteins and lipids in both phases at the resolution scale determined by the diffraction limit.Array adopted compacted 30-nm fiber and NCP formed columnar-hexagonal phase in the aggregates with anionic liposomes at 2-50 [Mg2+] revealed by SAXS. These Mg-induced effects are known to occur in the absence of anionic liposomes. Variations of charge density and lipid concentration were found not to influence the dimensions of NCP columnar phase. Lipid molecules may prevent resolubilization of condensed chromatin by neutralizing excess of Mg ions.Chromatin aggregates with liposomes of different charge may serve as a model of the eukaryotic nucleus where chromatin is surrounded by the nuclear envelope. The current results may demonstrate why cationic lipids destabilizing for chromatin are not prevalent in the nucleus, and how anionic membranes may influence the chromatin aggregation state.