Heritable capture of heterochromatin dynamics in Saccharomyces cerevisiae

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.DOI: http://dx.doi.org/10.7554/eLife.05007.001

Nutritional Control of Chronological Aging and Heterochromatin inSaccharomyces cerevisiae

“…Quite surprisingly, increasing glucose levels caused a marked destabilization of silencing in a unique spatio-temporal pattern (Figure 8). All previously studied interventions that destabilized silencing in the CRASH assay resulted in loss-of-silencing events relatively early in colony growth and continued throughout the growth of the colony, forming visible green sectors of sizes proportional to the timing of the events (Dodson and Rine 2015;Liu et al 2016). Here, however, loss-of-silencing events, particularly in 6 and 12% glucose, occurred late in colony development; preventing their outgrowth into a visible green sector.…”

“…The Sir2/ 3/4 complex is necessary for silencing HML, and loss of Sir function results in constitutive transcription of the a1 and a2 genes contained within (Rusche et al 2003). We employed a recently developed method from our laboratory that marks cells that have experienced transient losses of silencing at the HML locus (Dodson and Rine 2015) (Figure 6). In this method, known as the cre-reported altered states of heterochromatin (CRASH) assay (Liu et al 2016), the coding sequence of a2 at HML has been replaced with the coding sequence of cre, and cre is expressed only if Sir-based silencing at HML is disrupted.…”

Impact of Homologous Recombination on Silent Chromatin inSaccharomyces cerevisiae

“…Conceivably, the timing of either the double-strand break or the recombination event itself could influence whether or not the HML::cre donor locus lost silencing. For example, the sirtuin deacetylase Hst3 is a cell cycle-regulated protein important for maintaining silencing stability at HML (Dodson and Rine 2015) and is degraded after S phase but before anaphase (Delgoshaie et al 2014).…”

“…To monitor silencing stability, we employed an assay previously developed by our lab called the Cre-Reported Altered States of Heterochromatin, or CRASH, assay (Dodson and Rine 2015). In this assay, the cre recombinase gene replaces the native α2 sequence at HML.…”

“…However, traditional assays of silencing loss that rely on mating phenotypes have limited ability to reveal whether a donor locus becomes expressed as a result of a mating-type switch. In this study, we used a recently developed assay capable of detecting even transient disruptions of silencing (Dodson and Rine 2015) to investigate whether changes to silent chromatin dynamics at the HML locus result from its participation in homologous recombination. NatMX, lys2, ChrVIII:13192-13237::pseudo-MAT, leu2∆::pGAL10:HO, HMLα1-INC:α2∆::cre, hmr∆::K.l.LEU2, ura3∆::pGPD:loxP:yEmRFP;tCYC1:KanMX:loxP:yEGFP:tADH1, can1-100, his3-11 JRY10818 mat∆::NatMX, lys2, ChrVIII:13192-13237::pseudo-MAT-INC, leu2∆::pGAL10:HO, HMLα1-INC:α2∆::cre, hmr∆::K.l.LEU2, ura3∆::pGPD:loxP:yEmRFP;tCYC1:KanMX:loxP:yEGFP:tADH1, can1-100, his3-11 JRY10819 mat∆::NatMX, lys2, ChrVIII:13192-13237::K.l.URA3-HO, ChrIX:428440::K.l.URA3-HO-INC, leu2∆::pGAL10:HO, HMLα1∆HO:α2∆::cre, hmr∆::K.l.LEU2, ura3∆::pGPD:loxP:yEmRFP;tCYC1:KanMX:loxP:yEGFP:tADH, can1-100, his3-11 JRY10820 mat∆::NatMX, lys2, swi2∆::swi2∆10R, ChrVIII:13192-13237::pseudo-MAT, leu2∆::pGAL10:HO, HMLα1-INC:α2∆::cre, hmr∆::K.l.LEU2, ura3∆::pGPD:loxP:yEmRFP;tCYC1:KanMX:loxP:yEGFP:tADH1, can1- To insert the swi2∆10R allele into the SWI2 locus, a sequence to create a guide RNA to target cleavage of SWI2 by Cas9 was first cloned into the BsmBI sites of pYTK050 (M.E.…”