CELL-FREE SYSTEM FOR ASSEMBLY OF TRANSCRIPTIONALLY REPRESSED CHROMATIN FROM DROSOPHILA EMBRYOS

We describe a cell-free system, derived from preblastoderm Drosophila embryos, for the efficient assembly of cloned DNA into chromatin. The chromatin assembly system utilizes endogenous core histones and assembly factors and yields long arrays of regularly spaced nucleosomes with a repeat length of 180 bp. The assembly system is also capable of complementary-strand DNA synthesis accompanied by rapid nucleosome formation when the starting template is single-stranded circular DNA. Chromatin assembled with the preblastoderm embryo extract is naturally deficient in histone H1, but exogenous H1 can be incorporated during nucleosome assembly in vitro. Regular spacing of nucleosomes with or without histone H1 is sufficient to maximally repress transcription from hsp70 and fushi tarazu gene promoters. The Drosophila assembly system should be particularly useful for in vitro studies of chromatin assembly during DNA synthesis and for elucidating the action of transcription factors in the context of native chromatin.