Purification of Yeast Native Reagents for the Analysis of Chromatin Function-I: Nucleosomes for Reconstitution and Manipulation of Histone Marks.

Purification of native biological material provides powerful tools for the functional analysis of enzymes and proteins in chromatin. In particular, histone proteins harbor numerous post-translational modifications, which may differ between species, tissues, and growth conditions and are lacking on recombinant histones. Moreover, the physiological substrate of most enzymes that modify histones is chromatin and the majority of these enzymes need to be part of a multiprotein assembly to be able to act on chromatin. For the yeast Saccharomyces cerevisiae different chromatin purification protocols are available but often result in poor yields or rely on genetic manipulation. We present a simple purification protocol that can yield up to 150 μg of pure native chromatin per liter of yeast culture. The purified material can be obtained from mutant cells lacking specific histone modifications and can be used in in vitro chromatin assembly for biochemical studies. Based on the extremely high degree of conservation throughout eukaryotes, this modifiable native chromatin can be used in studies with factors from other organisms including humans.