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.
Key wordsTranscription Histone chaperone Histone cross-linking Chromatin assembly
This work was supported by the National Institutes of Health RO1 grant GM58650 to V.M.S. and by RAS Presidium Program “Basic research for the development of biomedical technologies.”