Chromatin assembly in Xenopus extracts

This chapter describes chromatin assembly in Xenopus extracts. Chromatin is a periodic structure made up of repeating, regularly spaced subunits called “nucleosomes.” To determine the precise molecular details of the way chromatin regulates transcription, mimicking the in vivo transcriptional activation process in vitro within a chromatin context is essential. Numerous studies employing cell-free protein extracts have reproduced in vivo transcription factor–nucleosome interactions in vitro. Protein extracts prepared from Xenopus laevis ovaries have been particularly useful in assembling cloned genes into chromatin in vitro for transcription analysis. The advantage of using Xenopus toads is that oocytes provide a rich source of nuclear proteins, including chromatin assembly components and transcription factors. The chapter presents various protocols that describe the preparation method of an efficient extract that can assemble the large amounts of chromatin necessary for biochemical analysis. The chapter describes various methods for the isolation of key nucleosome assembly components from the oocyte extract.