A Method for the In Vitro Reconstitution of a Defined “30 nm” Chromatin Fibre Containing Stoichiometric Amounts of the Linker Histone

Abstract

An understanding of the role of higher-order chromatin structure in the regulation of cellular processes such as transcription will require knowledge of the structure of the "30 nm" chromatin fibre and its folding and unfolding pathways. We report an in vitro chromatin reconstitution system, which uses arrays of 12 and 19 copies of a 200 bp repeat of the Widom 601 DNA sequence. Since this DNA sequence binds the histone octamer with much higher affinity than mixed sequence DNA, we have used competitor DNA in the reconstitutions to control the loading of both the histone octamer and linker histone onto the 601 DNA arrays. Using this method we have obtained nucleosome arrays that have one histone octamer and one H5 bound per 200 bp repeat, and hence have the stoichiometric composition of native chromatin. To obtain highly compact 30 nm chromatin fibres, we have investigated a number of folding buffer conditions including varying NaCl or MgCl(2) concentrations. Sedimentation velocity analysis shows that the reconstituted nucleosome arrays have the same folding properties as native chromatin and form highly compact structures in high NaCl concentrations or 1mM MgCl(2). Negative stain and electron cryo-microscopy of the folded arrays show a homogeneous population of folded particles with a uniform diameter of 34 nm. The data presented provide good evidence that the reconstitution method we have developed produces, for the first time, a defined population of folded 30 nm fibres suitable for detailed structural investigation.