Dependency of Heterochromatin Domains on Replication Factors

Abstract

Chromatin structure regulates both genome expression and dynamics in eukaryotes, where large heterochromatic regions are epigenetically silenced through the methylation of histone H3K9, histone deacetylation, and the assembly of repressive complexes. Previous genetic screens with the fission yeast Schizosaccharomyces pombe have led to the identification of key enzymatic activities and structural constituents of heterochromatin. We report here on additional factors discovered by screening a library of deletion mutants for silencing defects at the edge of a heterochromatic domain bound by its natural boundary-the IR-R⁺ element-or by ectopic boundaries. We found that several components of the DNA replication progression complex (RPC), including Mrc1/Claspin, Mcl1/Ctf4, Swi1/Timeless, Swi3/Tipin, and the FACT subunit Pob3, are essential for robust heterochromatic silencing, as are the ubiquitin ligase components Pof3 and Def1, which have been implicated in the removal of stalled DNA and RNA polymerases from chromatin. Moreover, the search identified the cohesin release factor Wpl1 and the forkhead protein Fkh2, both likely to function through genome organization, the Ssz1 chaperone, the Fkbp39 proline cis-trans isomerase, which acts on histone H3P30 and P38 in Saccharomyces cerevisiae, and the chromatin remodeler Fft3. In addition to their effects in the mating-type region, to varying extents, these factors take part in heterochromatic silencing in pericentromeric regions and telomeres, revealing for many a general effect in heterochromatin. This list of factors provides precious new clues with which to study the spatiotemporal organization and dynamics of heterochromatic regions in connection with DNA replication.

Keywords: DNA replication; chromatin remodeling; cohesins; fission yeast; gene silencing; genetic screen; heterochromatin; mating-type region; nuclear organization; proline isomerase.

Figure 1

Experimental design. (A) Mating-type region of query strains, showing the (EcoRV)::ade6⁺ reporter used to monitor heterochromatic silencing close to the wild-type boundary element IR-R⁺ or ectopic boundaries. Silencing of (EcoRV)::ade6⁺ results in red colony formation on the medium used, while expression results in white colonies. The euchromatic (XmnI):ura4⁺ gene was used to select the mating-type region in crosses with the Bioneer collection. (B) ORF deletions allowing (EcoRV)::ade6⁺ expression were identified in three rounds of screening as depicted. Candidates were retested as described in the text. ORF, open reading frame; RT-QPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 2

Factors identified in the screens include histone modifiers and readers, and their regulators. (A) Bulk progeny of crosses combining the indicated boundary elements and ORF deletions, replicated onto medium lacking adenine [NBA (yeast nitrogen base) supplemented with leucine] to assay (EcoRV)::ade6⁺ expression. Light colors reflect (EcoRV)::ade6⁺ expression. Control: an ORF deletion with no effect on (EcoRV)::ade6⁺ expression (V3-P25-45, SPAC15E1.07c). (B) Schematic representation of factors and complexes shown in (A). HDACs remove acetyl groups (in green); CLRC methylates H3K9 (in red); CRL4^Cdt2, positively regulated by Csn1, destabilizes the antisilencing factor Epe1. HDACs, histone deacetylases; ORF, open reading frame.

Figure 3

Overview of other factors identified in the screen. Purified ade6-DN/N strains combining the indicated boundary elements and open reading frame deletions were propagated on medium lacking adenine [NBA (yeast nitrogen base) supplemented with leucine] to assay (EcoRV)::ade6⁺ expression. Spot tests for these strains are presented in Figures S9–S11 in File S1.

Figure 4

Effects of selected factors on (EcoRV)::ade6⁺ in the presence of the WT boundary IR-R⁺. (A) Mating-type region in this experiment. (B) Growth assays with 10-fold serial dilutions of indicated deletion strains. Strains LJ99, PG3950, LJ102, LJ100, LJ220, LJ203, LJ104, LJ103, LJ199, LJ105, PM20, and PM24 were used. AA -ade, medium lacking adenine; WT, wild-type; YE, medium with limited adenine.

Figure 5

Effects of selected factors on (EcoRV)::ade6⁺ transcript in the presence of the WT boundary IR-R⁺, assayed by RT-QPCR. The strains are the same as in Figure 4. The means of three biological replicates are displayed. (EcoRV)::ade6⁺ RNA level was normalized to act1⁺ in each strain and is expressed as % expression in IR-RΔ cells. The y-axis labeling on the left applies to the three strains on that side that have the highest (EcoRV)::ade6⁺ expression. The labeling on the right applies to the other strains. Errors bars represent the SE of the mean. RT-QPCR, reverse transcription-quantitative polymerase chain reaction; WT, wild-type.

Figure 6

Effects of selected factors on (EcoRV)::ura4⁺ in the presence of the WT boundary IR-R⁺. (A) Mating-type region in this experiment. (B) Growth assays with 10-fold serial dilutions of indicated deletion strains. Strains used for spot tests: LJ177, PG1899, LJ176, LJ178, LJ179, and LJ175. AA-ura, medium lacking uracil; FOA, fluoroorotic acid; WT, wild-type.

Figure 7

Effects of Mcl1 and Wpl1 on heterochromatic silencing. (A) The mating-type regions used in this experiment featured two ectopic boundaries, STAR2 and the 85 bp STAR2-S element (Toteva et al. 2017), in addition to wild-type IR-R⁺. (B) mcl1Δ and wpl1Δ mutants with the indicated boundaries were spotted on low adenine to compare (EcoRV)::ade6⁺ expression with the wild-type background. mcl1Δ confers a pronounced growth defect; however, the formed (EcoRV)::ade6⁺ mcl1Δ colonies are Ade⁺. wpl1Δ induces strong variegation. The strains were wild-type: PG3947, PG3950, PC152, and PT600; mcl1Δ: PA71, PA72, PA73, and PA74; and wpl1Δ: PA82, PA83, PA84, and PA85. YE, medium with limited adenine.

Figure 8

Effects of selected factors on telomeric and centromeric silencing. Serial dilutions (10-fold) of cell suspensions were spotted. (A) Effects on the m23::ura4⁺-Tel[72] telomeric reporter gene. Derepression of the reporter improves plating efficiency on AA-ura and decreases white colony formation on FOA medium with low adenine concentration (where only cells with a repressed reporter can form white colonies). Strains used: LJ171, FY520, LJ163, LJ172, LJ173, LJ162, LJ174, LJ165, LJ170, LJ168, LJ161, and LJ166. (B) Effects on centromeric otr1(SphI)::ura4⁺ reporter gene. Strains used: LJ185, FY648, LJ180, LJ186, LJ190, LJ192, LJ187, LJ183, LJ181, LJ191, and LJ189. AA-ura, medium lacking uracil; ade, adenine; Chr, chromosome; FOA, fluoroorotic acid; WT, wild-type.

Figure 9

Silencing factors identified in this screen have roles in DNA replication. The drawing schematizes a replication fork. Factors identified in the screen are in color, while other subunits of the represented complexes are in gray.