Experimental analysis of hFACT action during Pol II transcription in vitro.

FACT (facilitates chromatin transcription) is a histone chaperone that facilitates transcription through chromatin and promotes histone recovery during transcription. Here, we describe a highly purified experimental system that recapitulates many important properties of transcribed chromatin and the key aspects of hFACT action during this process in vitro. We present the protocols describing how to prepare different forms of nucleosomes, including intact nucleosome, covalently conjugated nucleosome, nucleosome missing one of the two H2A/2B dimers (hexasome) and tetrasome (a nucleosome missing both H2A/2B dimers). These complexes allow analysis of various aspects of FACT's function. These approaches and other methods described below can also be applied to the study of other chromatin remodelers and chromatin-targeted factors.