RECONSTITUTION OF HYPERACETYLATED, DNASE I-SENSITIVE CHROMATIN CHARACTERIZED BY HIGH CONFORMATIONAL FLEXIBILITY OF NUCLEOSOMAL DNA

Increased acetylation at specific N-terminal lysines of core histones is a hallmark of active chromatin in vivo. Set the structural conseque nces of acetylation leading to increased gene activity are only poorly defined, We employed a new approach to characterize the effects of hi stone acetylation: A Drosophila embryo-derived cell-free system for ch romatin reconstitution under physiological conditions was programmed w ith exogenous histones to assemble hyperacetylated or matching control chromatin of high complexity. Hyperacetylated chromatin resembled unm odified chromatin at similar nucleosome density with respect to its se nsitivity toward microccal nuclease, its nucleosomal repeat length, an d the incorporation of the linker histone H1. In contrast, DNA in acet ylated chromatin showed an increased sensitivity toward DNase I and a surprisingly high degree of conformational flexibility upon temperatur e shift pointing to profound alterations of DNA/histone interactions, This successful reconstitution of accessible and flexible chromatin ou tside of a nucleus paves the way for a thorough analysis of the causal relationship between histone acetylation and gene function.