ASSEMBLY OF MMTV PROMOTER MINICHROMOSOMES WITH POSITIONED NUCLEOSOMESPRECLUDES NF1 ACCESS BUT NOT RESTRICTION ENZYME CLEAVAGE

To generate long arrays of nucleosomes within a topologically defined chromatin domain we have assembled minichromosomes on negatively super coiled plasmid DNA with extracts from Drosophila preblastoderm embryos . These minichromosomes are dynamic substrates for energy-dependent nu cleosome remodeling machines that facilitate the binding of various tr anscription factors but do not exhibit nucleosome positioning, In cont rast, if such minichromosomes include the mouse mammary tumour virus ( MMTV) promoter we find it wrapped around a nucleosome with similar tra nslational and rotational position as in vivo. This structure preclude d binding of NF1 to its cognate site at -75/-65 at salt concentrations between 60 and 120 mM, even in the presence of ATP, which rendered th e NF1 site accessible to the restriction enzyme HinfI. However, insert ion of 30 bp just upstream of the NF1 site, which moves the site to th e linker DNA, allowed ATP-dependent binding of NF1 to a fraction of th e minichromosomes, even in the presence of stoichiometric amounts of h istone H1, The minichromosomes assembled in the Drosophila embryo extr act reproduce important features of the native MMTV promoter chromatin and reveal differences in the ability of transcription factors and re striction enzymes to access their binding sites in positioned nucleoso mes.