Esc1, a nuclear periphery protein required for Sir4-based plasmid anchoring and partitioning

A targeted silencing screen was performed to identify yeast proteins that, when tethered to a telomere, suppress a telomeric silencing defect caused by truncation of Rap1. A previously uncharacterized protein, Esc1 (establishes silent chromatin), was recovered, in addition to well-characterized proteins Rap1, Sir1, and Rad7. Telomeric silencing was slightly decreased in Deltaesc1 mutants, but silencing of the HM loci was unaffected. On the other hand, targeted silencing by various tethered proteins was greatly weakened in Deltaesc1 mutants. Two-hybrid analysis revealed that Esc1 and Sir4 interact via a 34-amino-acid portion of Esc1 (residues 1440 to 1473) and a carboxyl-terminal domain of Sir4 known as PAD4 (residues 950 to 1262). When tethered to DNA, this Sir4 domain confers efficient partitioning to otherwise unstable plasmids and blocks the ability of bound DNA segments to rotate freely in vivo. Here, both phenomena were shown to require ESC1. Sir protein-mediated partitioning of a telomere-based plasmid also required ESC1. Fluorescence microscopy of cells expressing green fluorescent protein (GFP)-Esc1 showed that the protein localized to the nuclear periphery, a region of the nucleus known to be functionally important for silencing. GFP-Esc1 localization, however, was not entirely coincident with telomeres, the nucleolus, or nuclear pore complexes. Our data suggest that Esc1 is a component of a redundant pathway that functions to localize silencing complexes to the nuclear periphery.     

A SUMO-like domain protein, Esc2, is required for genome integrity and sister chromatid cohesion in Saccharomyces cerevisiae

The ESC2 gene encodes a protein with two tandem C-terminal SUMO-like domains and is conserved from yeasts to humans. Previous studies have implicated Esc2 in gene silencing. Here, we explore the functional significance of SUMO-like domains and describe a novel role for Esc2 in promoting genome integrity during DNA replication. This study shows that esc2Delta cells are modestly sensitive to hydroxyurea (HU) and defective in sister chromatid cohesion and have a reduced life span, and these effects are enhanced by deletion of the RRM3 gene that is a Pif1-like DNA helicase. esc2Delta rrm3Delta cells also have a severe growth defect and accumulate DNA damage in late S/G(2). In contrast, esc2Delta does not enhance the HU sensitivity or sister chromatid cohesion defect in mrc1Delta cells, but rather partially suppresses both phenotypes. We also show that deletion of both Esc2 SUMO-like domains destabilizes Esc2 protein and functionally inactivates Esc2, but this phenotype is suppressed by an Esc2 variant with an authentic SUMO domain. These results suggest that Esc2 is functionally equivalent to a stable SUMO fusion protein and plays important roles in facilitating DNA replication fork progression and sister chromatid cohesion that would otherwise impede the replication fork in rrm3Delta cells.     

Tethered Sir3p nucleates silencing at telomeres and internal loci in Saccharomyces cerevisiae.

Rap1p binds to sites embedded within the Saccharomyces cerevisiae telomeric TG1-3 tract. Previous studies have led to the hypothesis that Rap1p may recruit Sir3p and Sir3p-associating factors to the telomere. To test this, we tethered Sir3p adjacent to the telomere via LexA binding sites in the rap1-17 mutant that truncates the Rap1p C-terminal 165 amino acids thought to contain sites for Sir3p association. Tethering of LexA-Sir3p adjacent to the telomere is sufficient to restore telomeric silencing, indicating that Sir3p can nucleate silencing at the telomere. Tethering of LexA-Sir3p or the LexA-Sir3p(N2O5) gain-of-function protein to a telomeric LexA site hyperrepresses an adjacent ADE2 gene in wild-type cells. Hence, Sir3p recruitment to the telomere is limiting in telomeric silencing. In addition, LexA-Sir3p(N2O5) hyperrepresses telomeric silencing when tethered to a subtelomeric site 3.6 kb from the telomeric tract. This hyperrepression is dependent on the C terminus of Rap1p, suggesting that subtelomeric LexA-Sir3p(N205) can interact with Rap1p-associated factors at the telomere. We also demonstrate that LexA-Sir3p or LexA-Sir3p(N205) tethered in cis with a short tract of telomeric TG1-3 sequences is sufficient to confer silencing at an internal chromosomal position. Internal silencing is enhanced in rap1-17 strains. We propose that sequestration of silencing factors at the telomere limits the efficiency of internal silencing.     

Yeast Silent Mating Type Loci Form Heterochromatic Clusters through Silencer Protein-Dependent Long-Range Interactions

The organization of eukaryotic genomes is characterized by the presence of distinct euchromatic and heterochromatic sub-nuclear compartments. In Saccharomyces cerevisiae heterochromatic loci, including telomeres and silent mating type loci, form clusters at the nuclear periphery. We have employed live cell 3-D imaging and chromosome conformation capture (3C) to determine the contribution of nuclear positioning and heterochromatic factors in mediating associations of the silent mating type loci. We identify specific long-range interactions between HML and HMR that are dependent upon silencing proteins Sir2p, Sir3p, and Sir4p as well as Sir1p and Esc2p, two proteins involved in establishment of silencing. Although clustering of these loci frequently occurs near the nuclear periphery, colocalization can occur equally at more internal positions and is not affected in strains deleted for membrane anchoring proteins yKu70p and Esc1p. In addition, appropriate nucleosome assembly plays a role, as deletion of ASF1 or combined disruption of the CAF-1 and HIR complexes abolishes the HML-HMR interaction. Further, silencer proteins are required for clustering, but complete loss of clustering in asf1 and esc2 mutants had only minor effects on silencing. Our results indicate that formation of heterochromatic clusters depends on correctly assembled heterochromatin at the silent loci and, in addition, identify an Asf1p-, Esc2p-, and Sir1p-dependent step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions.     

The polycomb group mutant esc leads to augmented levels of paused Pol II in the Drosophila embryo

Many developmental control genes contain paused RNA polymerase II (Pol II) and are thereby "poised" for rapid and synchronous activation in the early Drosophila embryo. Evidence is presented that Polycomb group (PcG) repressors can influence paused Pol II. ChIP-Seq and GRO-Seq assays were used to determine the genome-wide distributions of Pol II, H3K27me3, and H3K4me3 in extra sex combs (esc) mutant embryos. ESC is a key component of the Polycomb repressive complex 2 (PRC2), which mediates H3K27me3 modification. Enhanced Pol II occupancy is observed for thousands of genes in esc mutant embryos, including genes not directly regulated by PRC2. Thus, it would appear that silent genes lacking promoter-associated paused Pol II in wild-type embryos are converted into "poised" genes with paused Pol II in esc mutants. We suggest that this conversion of silent genes into poised genes might render differentiated cell types susceptible to switches in identity in PcG mutants.     

A histone variant, Htz1p, and a Sir1p-like protein, Esc2p, mediate silencing at HMR

Silencing at HMR requires silencers, and one of the roles of the silencer is to recruit Sir proteins. This work focuses on the function of Sir1p once it is recruited to the silencer. We have generated mutants of Sir1p that are recruited to the silencer but are unable to silence, and we have utilized these mutants to identify four proteins, Sir3p, Sir4p, Esc2p, and Htz1p, that when overexpressed, restored silencing. The isolation of Sir3p and Sir4p validated this screen. Molecular analysis suggested that Esc2p contributed to silencing in a manner similar to Sir1p and probably helped recruit or stabilize the other Sir proteins, while Htz1p present at HMR assembled a specialized chromatin structure necessary for silencing.     

The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae.

It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres. This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci. Here, we show that a null mutation of the DNA repair gene RAD6 reduces silencing of the HM loci and lowers the mating efficiency of MATa strains. Likewise, rad6-delta reduces silencing of the telomere-located RNAP II-transcribed genes URA3 and ADE2. We also show that the RNAP III-transcribed tyrosyl tRNA gene, SUP4-o, is subject to position effect variegation when located near a telomere and that this silencing requires the RAD6 and SIR genes. Neither of the two known Rad6 binding factors, Rad18 and Ubr1, is required for telomeric silencing. Since Ubrl is the recognition component of the N-end rule-dependent protein degradation pathway, this suggests that N-end rule-dependent protein degradation is not involved in telomeric silencing. Telomeric silencing requires the amino terminus of Rad6. Two rad6 point mutations, rad6(C88A) and rad6(C88S), which are defective in ubiquitin-conjugating activity fail to complement the silencing defect, indicating that the ubiquitin-conjugating activity of RAD6 is essential for full telomeric silencing.     

Compensatory Interactions between Sir3p and the Nucleosomal LRS Surface Imply Their Direct Interaction

The previously identified LRS (Loss of rDNA Silencing) domain of the nucleosome is critically important for silencing at both ribosomal DNA and telomeres. To understand the function of the LRS surface in silencing, we performed an EMS mutagenesis screen to identify suppressors of the H3 A75V LRS allele. We identified dominant and recessive mutations in histones H3, H4, and dominant mutations in the BAH (Bromo Adjacent Homology) domain of SIR3. We further characterized a surface of Sir3p critical for silencing via the LRS surface. We found that all alleles of the SIR3 BAH domain were able to 1) generally suppress the loss of telomeric silencing of LRS alleles, but 2) could not suppress SIN (Swi/Snf Independent) alleles or 3) could not suppress the telomeric silencing defect of H4 tail alleles. Moreover, we noticed a complementary trend in the electrostatic changes resulting from most of the histone mutations that gain or lose silencing and the suppressor alleles isolated in SIR3, and the genes for histones H3 and H4. Mutations in H3 and H4 genes that lose silencing tend to make the LRS surface more electronegative, whereas mutations that increase silencing make it less electronegative. Conversely, suppressors of LRS alleles in either SIR3, histone H3, or H4 also tend to make their respective surfaces less electronegative. Our results provide genetic evidence for recent data suggesting that the Sir3p BAH domain directly binds the LRS domain. Based on these findings, we propose an electrostatic model for how an extensive surface on the Sir3p BAH domain may regulate docking onto the LRS surface.     

Genetic Analysis of Rap1p/Sir3p Interactions in Telomeric and Hml Silencing in Saccharomyces Cerevisiae

We have identified three SIR3 suppressors of the telomeric silencing defects conferred by missense mutations within the Rap1p C-terminal tail domain (aa 800-827). Each SIR3 suppressor was also capable of suppressing a rap1 allele (rap1-21), which deletes the 28 aa C-terminal tail domain, but none of the suppressors restored telomeric silencing to a 165 amino acid truncation allele. These data suggest a Rap1p site for Sir3p association between the two truncation points (aa 664-799). In SIR3 suppressor strains lacking the Rap1p C-terminal tail domain, the presence of a second intragenic mutation within the rap1s domain (aa 727-747), enhanced silencing 30-300-fold. These data suggest a competition between Sir3p and factors that interfere with silencing for association in the rap1(s) domain. rap1-21 strains containing both wild-type Sir3p and either of the Sir3 suppressor proteins displayed a 400-4000-fold increase in telomeric silencing over rap1-21 strains carrying either Sir3p suppressor in the absence of wild-type Sir3p. We propose that this telomere-specific synergism is mediated in part through stabilization of Rap1p/Sir3p telomeric complexes by Sir3p-Sir3p interactions.     

Double-stranded DNA binding, an unusual property of DNA polymerase epsilon, promotes epigenetic silencing in Saccharomyces cerevisiae

We have previously shown that DNA polymerase epsilon (Pol epsilon)of Saccharomyces cerevisiae binds stably to double-stranded DNA (dsDNA), a property not generally associated with DNA polymerases. Here, by reconstituting Pol epsilon activity from Pol2p-Dpb2p and Dpb3p-Dpb4p, its two component subassemblies, we report that Dpb3p-Dpb4p, a heterodimer of histone-fold motif-containing subunits, is responsible for the dsDNA binding. Substitution of specific lysine residues in Dpb3p, highlighted by homology modeling of Dpb3p-Dpb4p based on the structure of the histone H2A-H2B dimer, indicated that they play roles in binding of dsDNA by Dpb3p-Dpb4p, in a manner similar to the histone-DNA interaction. The lysine-substituted dpb3 mutants also displayed reduced telomeric silencing, whose degree paralleled that of the dsDNA-binding activity of Pol epsilon in the corresponding dpb3 mutants. Furthermore, additional amino acid substitutions to lysines in Dpb4p, to compensate for the loss of positive charges in the Dpb3p mutants, resulted in simultaneous restoration of dsDNA-binding activity by Pol epsilon and telomeric silencing. We conclude that the dsDNA-binding property of Pol epsilon is required for epigenetic silencing at telomeres.     

New mutations of Saccharomyces cerevisiae that partially relieve both glucose and galactose repression activate the protein kinase Snf1

We isolated from Saccharomyces cerevisiae two mutants, esc1-1 and ESC3-1, in which genes FBP1, ICL1 or GDH2 were partially derepressed during growth in glucose or galactose. The isolation was done starting with a triple mutant pyc1 pyc2 mth1 unable to grow in glucose-ammonium medium and selecting for mutants able to grow in the non-permissive medium. HXT1 and HXT2 which encode glucose transporters were expressed at high glucose concentrations in both esc1-1 and ESC3-1 mutants, while derepression of invertase at low glucose concentrations was impaired. REG1, cloned as a suppressor of ESC3-1, was not allelic to ESC3-1. Two-hybrid analysis showed an increased interaction of the protein kinase Snf1 with Snf4 in the ESC3-1 mutant; this was not due to mutations in SNF1 or SNF4. ESC3-1 did not bypass the requirement of Snf1 for derepression. We hypothesize that ESC3-1 either facilitates activation of Snf1 or interferes with its glucose-dependent inactivation.     

Esc4/Rtt107 and the control of recombination during replication

When replication forks stall during DNA synthesis, cells respond by assembling multi-protein complexes to control the various pathways that stabilize the replication machinery, repair the replication fork, and facilitate the reinitiation of processive DNA synthesis. Increasing evidence suggests that cells have evolved scaffolding proteins to orchestrate and control the assembly of these repair complexes, typified in mammalian cells by several BRCT-motif containing proteins, such as Brca1, Xrcc1, and 53BP1. In Saccharomyces cerevisiae, Esc4 contains six such BRCT domains and is required for the most efficient response to a variety of agents that damage DNA. We show that Esc4 interacts with several proteins involved in the repair and processing of stalled or collapsed replication forks, including the recombination protein Rad55. However, the function of Esc4 does not appear to be restricted to a Rad55-dependent process, as we observed an increase in sensitivity to the DNA alkylating agent methane methylsulfonate (MMS) in a esc4Deltarad55Delta mutant, as well as in double mutants of esc4Delta and other recombination genes, compared to the corresponding single mutants. In addition, we show that Esc4 forms multiple nuclear foci in response to treatment with MMS. Similar behavior is also observed in the absence of damage when either of the S-phase checkpoint proteins, Tof1 or Mrc1, is deleted. Thus, we propose that Esc4 associates with ssDNA of stalled forks and acts as a scaffolding protein to recruit and/or modulate the function of other proteins required to reinitiate DNA synthesis.