Abstract
Heterochromatin exerts a heritable form of eukaryotic gene repression and contributes to chromosome segregation fidelity and genome stability. However, to date there has been no quantitative evaluation of the stability of heterochromatic gene repression. We designed a genetic strategy to capture transient losses of gene silencing in Saccharomyces as permanent, heritable changes in genotype and phenotype. This approach revealed rare transcription within heterochromatin that occurred in approximately 1/1000 cell divisions. In concordance with multiple lines of evidence suggesting these events were rare and transient, single-molecule RNA FISH showed that transcription was limited. The ability to monitor fluctuations in heterochromatic repression uncovered previously unappreciated roles for Sir1, a silencing establishment factor, in the maintenance and/or inheritance of silencing. In addition, we identified the sirtuin Hst3 and its histone target as contributors to the stability of the silenced state. These approaches revealed dynamics of a heterochromatin function that have been heretofore inaccessible.
Highlights
Heterochromatin is a heritable, condensed chromatin structure that silences the expression of most genes within or near it
Heterochromatic gene repression provides a means for genetically identical cells to differentiate into stable, distinct cell types
It should be noted that the recombination event would be essentially irreversible in that the excised DNA lacked an origin of replication and would thereby be lost upon cell division
Summary
Heterochromatin is a heritable, condensed chromatin structure that silences the expression of most genes within or near it. Phenomena such as clonal inheritance of inactivated X-chromosomes in female mammals, as well as position-effect variegation in Drosophila and yeast, demonstrate the remarkable ability of cells to propagate heterochromatic repression through mitosis. In Saccharomyces cerevisiae, heterochromatin forms at the silent mating-type loci, HML and HMR, through the recruitment and subsequent spreading of Sir proteins (Grunstein and Gasser, 2013). DNA elements known as the E and I silencers flank each locus and nucleate complexes of Sir, Sir and Sir. Sir complexes deacetylate histones and bind nucleosomes throughout the region, thereby rendering HML and HMR transcriptionally silenced and largely inaccessible to DNA-interacting proteins. Since each locus contains either a or α mating-type information, as does the MAT locus, heterochromatic repression of HML and HMR ensures that the MAT genotype is the only determinant of whether haploids mate as a or α cells
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