Multiple studies in Saccharomyces cerevisiae have measured the levels of gene silencing by inserting the URA3 gene at various loci and selecting against URA3-expressing cells by 5-flouroorotic acid (5-FOA). However, 5-FOA affects the cellular pools of dNTPs and can produce side effects. To circumvent this issue, we and others have introduced drug-free techniques to detect silent and active gene states. In this study, we compared three drug-free methods based on the expression of fluorescent reporters in the VIIL telomere of S. cerevisiae. Our results point out that only one of these methods is suitable for large-scale drug-free analyses of gene silencing.

No abstract

No abstract

Phenotypic heterogeneity provides growth advantages for a population upon changes of the environment. In S. cerevisiae, such heterogeneity has been observed as “on/off” states in the expression of individual genes in individual cells. These variations can persist for a limited or extended number of mitotic divisions. Such traits are known to be mediated by heritable chromatin structures, by the mitotic transmission of transcription factors involved in gene regulatory circuits or by the cytoplasmic partition of prions or other unstructured proteins. The significance of such epigenetic diversity is obvious, however, we have limited insight into the mechanisms that generate it. In this review, we summarize the current knowledge of epigenetically maintained heterogeneity of gene expression and point out similarities and converging points between different mechanisms. We discuss how the sharing of limiting repression or activation factors can contribute to cell-to-cell variations in gene expression and to the coordination between short- and long- term epigenetic strategies. Finally, we discuss the implications of such variations and strategies in adaptation and aging.

Background Classical studies on position effect variegation in Drosophila have demonstrated the existence of bi-modal Active/Silent state of the genes juxtaposed to heterochromatin. Later studies with irreversible methods for the detection of gene repression have revealed a similar phenomenon at the telomeres of Saccharomyces cerevisiae and other species. In this study, we used dual reporter constructs and a combination of reversible and non-reversible methods to present evidence for the existence of different states of gene repression at the sub-telomeres of S. cerevisiae. Results We show position dependent transient repression or bimodal expression of reporter genes at the VIIL sub-telomere. We also show that mutations in the replicative clamp POL30 (PCNA) or the destruction of the histone chaperone CAF1 or the RRM3 helicase lead to transient de-repression, while the destruction of the histone chaperone ASF1 causes a shift from transient de-repression to a bi-modal state of repression. We analyze the physical interaction of CAF1 and RRM3 with PCNA and discuss the implications of these findings for our understanding of the stability and transmission of the epigenetic state of the genes. Conclusions There are distinct modes of gene silencing, bi-modal and transient, at the sub-telomeres of S. cerevisiae. We characterise the roles of CAF1, RRM3 and ASF1 in these modes of gene repression. We suggest that the interpretations of past and future studies should consider the existence of the dissimilar states of gene silencing.