DNA in Transcriptionally Silent Chromatin Assumes a Distinct Topology That Is Sensitive to Cell Cycle Progression

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Abstract
Transcriptionally silent regions of the Saccharomyces cerevisiae genome, the silent mating type loci and telomeres, represent the yeast equivalent of metazoan heterochromatin. To gain insight into the nature of silenced chromatin structure, we have examined the topology of DNA spanning the HML silent mating type locus by determining the superhelical density of mini-circles excised from HML (HML circles) by site-specific recombination. We observed that HML circles excised in a wild-type (SIR+) strain were more negatively supercoiled upon deproteinization than were the same circles excised in a sir- strain, in which silencing was abolished, even when HML alleles in which neither circle was transcriptionally competent were used. cis-acting sites flanking HML, called silencers, are required in the chromosome for establishment and inheritance of silencing. HML circles excised without silencers from cells arrested at any point in the cell cycle retained SIR-dependent differences in superhelical density. However, progression through the cell cycle converted SIR+ HML circles to a form resembling that of circles from sir- cells. This decay was not observed with circles carrying a silencer. These results establish that (i) DNA in transcriptionally silenced chromatin assumes a distinct topology reflecting a distinct organization of silenced versus active chromatin; (ii) the altered chromatin structure in silenced regions likely results from changes in packaging of individual nucleosomes, rather than changes in nucleosome density; and (iii) cell cycle progression disrupts the silenced chromatin structure, a process that is counteracted by silencers.