Regulated gene expression in reconstituted chromatin and synthetic nuclei

Abstract

This chapter discusses the regulation of gene expression in reconstituted chromatin and synthetic nuclei. The initial opening of chromatin structure and reprogramming of gene expression during development or disease may depend on the ability to bind protein activators to DNA sites assembled within nucleosomes. DNA replication during the cell cycle presents such an occasion where promoters and enhancers are accessible to transacting factors during concomitant chromatin assembly. Incubation of cloned DNA in Xenopus egg extracts, consisting of both soluble and vesicular components, leads to a stepwise assembly of chromatin structures that bind nuclear membrane vesicles prior to a single round of semi-conservative DNA synthesis within synthetic nuclei. The fractionated Xenopus egg extracts is used as an in vitro system to assemble reconstituted nuclei in two stages: chromatin formation followed by nuclear membrane encapsulation. This stagewise approach allows the analysis of the transcriptional regulation of β-globin gene loci in chromatin or in synthetic nuclei before and after DNA replication. The interplay of activators, repressors, architectural effectors, and ubiquitous factors in the induction or repression of gene expression can be addressed to develop a more thorough structural and mechanistic view of gene switching and regulation.