Analysis of epigenetic stability and conversions in Saccharomyces cerevisiae reveals a novel role of CAF-I in position-effect variegation

Abstract

Position-effect variegation (PEV) phenotypes are characterized by the robust multigenerational repression of a gene located at a certain locus (often called gene silencing) and occasional conversions to fully active state. Consequently, the active state then persists with occasional conversions to the repressed state. These effects are mediated by the establishment and maintenance of heterochromatin or euchromatin structures, respectively. In this study, we have addressed an important but often neglected aspect of PEV: the frequency of conversions at such loci. We have developed a model and have projected various PEV scenarios based on various rates of conversions. We have also enhanced two existing assays for gene silencing in Saccharomyces cerevisiae to measure the rate of switches from repressed to active state and vice versa. We tested the validity of our methodology in Δsir1 cells and in several mutants with defects in gene silencing. The assays have revealed that the histone chaperone Chromatin Assembly Factor I is involved in the control of epigenetic conversions. Together, our model and assays provide a comprehensive methodology for further investigation of epigenetic stability and position effects.

Totipotency in the absence of CAF-I: unhindered choices when the chaperone is out

“…In the absence of CAF-I these chromatin conversions are impeded and the cells remain in 2C-like state. Such an interpretation is in agreement with the proposed role of CAF-I as a regulator of epigenetic conversions 8 and can be supported by the findings in. 4,5 A very important implication of this interpretation is that CAF-I is generally involved epigenetic conversions in eukaryotic cells.…”

“…10,11 However, a recent study has pointed out that the loss of telomeric gene silencing in Dcac1 cells should be attributed to low frequency of conversions between the active and silenced states rather than to overall reduced silencing. 8 Hence, this paper suggested that in S.cerevisiae CAF-I is involved in epigenetic conversions.…”

“…7 In S.cerevisiae the destruction of the Cac1p subunit of CAF-I (the yeast homolog of the mammalian p150CAF-I subunit) leads to massive de-repression of subtelomeric genes. 8,9 These genes normally alternate between a fully silenced or fully active state of expression. This interesting epigenetic phenomenon is referred to as Telomere Position Effect.…”

CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

“…28 These assays showed the all S!A mutants reduced the repression of a telomeric URA3 reporter 2-to 9-fold relative to wild type cells ( Fig. 7) with S503A and S94A showing the weakest and strongest effect, respectively.…”

“…The deletion of CAC1 is known to cause sensitivity to DNA damaging agents, 7,27 loss of telomeric gene silencing, 4,5 decrease in the frequency of epigenetic conversions at telomeres 28 and impaired in vitro replication-coupled nucleosome assembly. 5,29 However, point mutations in Cac1p that affect its interaction with PCNA do not phenocopy the loss of CAC1.…”