SubtelomericACS-containing Proto-silencers Act as Antisilencers in Replication Factors Mutants inSaccharomyces cerevisiae

Abstract

Subtelomeric genes are either fully active or completely repressed and can switch their state about once per 20 generations. This meta-stable telomeric position effect is mediated by strong repression signals emitted by the telomere and relayed/enhanced by weaker repressor elements called proto-silencers. In addition, subtelomeric regions contain sequences with chromatin partitioning and antisilencing activities referred to as subtelomeric antisilencing regions. Using extensive mutational analysis of subtelomeric elements, we show that ARS consensus sequence (ACS)-containing protosilencers convert to antisilencers in several replication factor mutants. We point out the significance of the B1 auxiliary sequence next to ACS in mediating these effects. In contrast, an origin-derived ACS does not convert to antisilencer in mutants and its B1 element has little bearing on silencing. These results are specific for the analyzed ACS and in addition to the effects of each mutation (relative to wild type) on global silencing. Another line of experiments shows that Mcm5p possesses antisilencing activity and is recruited to telomeres in an ACS-dependent manner. Mcm5p persists at this location at the late stages of S phase. We propose that telomeric ACS are not static proto-silencers but conduct finely tuned silencing and antisilencing activities mediated by ACS-bound factors. INTRODUCTIONGenes positioned close to telomeres are either fully active or completely repressed. They switch their state of expression once per 20 generations on average. This meta-stable telomeric position effect (TPE) is governed by strong repression signals emitted by the telomeres (Fourel et al., 2002a;Tham and Zakian, 2002;Rusche et al., 2003). Weaker repressor elements in the core X-and YЈ-subtelomeric elements, called proto-silencers, can relay and enhance repression signals away from telomeres (Fourel et al., 2002a). To date, protosilencer activity has been demonstrated for ARS consensus sites (ACS) and for the binding sites for Rap1p and Abf1p (Boscheron et al., 1996;Fourel et al., 1999;Pryde and Louis, 1999;Lebrun et al., 2001). In addition, subtelomeric core X-and YЈ-elements contain sequences, which display chromatin partitioning or antisilencing activities and are referred to as subtelomeric antisilencing regions (STARS) (Fourel et al., 1999(Fourel et al., , 2004Pryde and Louis, 1999). The state of gene expression at individual telomeres is determined by the complex interplay between the telomere, proto-silencers, and antisilencers (Fourel et al., 1999;Pryde and Louis, 1999;Lebrun et al., 2001;Fourel et al., 2002aFourel et al., , 2004Rehman et al., 2006); however, it is not clear whether and how these elements contribute to the variegated nature of TPE.ACS is a conserved 11-base pair sequence found in all yeast origins of replication, in subtelomeric core X-and YЈ-elements and in the silencers of the mating type loci. ACS binds origin recognition complex (ORC) and is essential for replication initiation at origins and for silencing at the...

Replication Timing of Human Telomeres Is Chromosome Arm–Specific, Influenced by Subtelomeric Structures and Connected to Nuclear Localization

“…Our results suggest that the links between replication timing and high-order genome organization, also observed in other organisms, may have been conserved throughout evolution [28], [41], [75], [76].…”

Sub-Telomeric core X and Y' Elements in S.cerevisiae Suppress Extreme Variations in Gene Silencing

“…URA3-tel, GF2, GF3, GF6, GF9, GF44, GF46, GF61, GF6ΔSTAR, GF6ΔACS and GF44ΔACS showed very similar levels of %FOA R in BY4742 cells as compared to the previously used W303 strain [4], [24], [25]. In addition, the prototype URA3-tel construct recapitulated the silencing defects observed in sas2, sas3, orc2-1, orc5-1, mcm5-461, cdc6-1, cdc45-1 and cdc7-1(sas1) (Fig.…”

“…The insertions between URA3 and the telomeric repeat add 145-900 base pairs in different constructs as compared to URA3-tel. Several studies have shown that the telomeric silencing for these and other constructs does not directly correlate to the distance from the telomeres [2], [3], [4], [24], [25]. Instead, silencing is discontinuous and is strongly influenced by the nature and the positions of different regulatory elements [2], [26].…”

GCN5 Is a Positive Regulator of Origins of DNA Replication in Saccharomyces cerevisiae

“…Cross-linked chromatin was sheared, extracted and immunoprecipitated with a mixture of anti-MCM2 and anti-MCM5 antibodies (Santa Cruz). We have found that this mixture of anti-peptide antibodies is more efficient in recovery of origin DNA as compared to the individual antibodies used alone [40], [41]. Finally, the immunoprecipitated DNA was analyzed by quantitative PCR with primers for ARS1 and ARS305 .…”