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. 1999 Apr;19(4):3184-97.
doi: 10.1128/MCB.19.4.3184.

A genetic screen for ribosomal DNA silencing defects identifies multiple DNA replication and chromatin-modulating factors

J S Smith  1 , E Caputo, J D Boeke
Affiliations

A genetic screen for ribosomal DNA silencing defects identifies multiple DNA replication and chromatin-modulating factors (VSports手机版)

J S Smith et al. Mol Cell Biol. 1999 Apr.

Abstract

Transcriptional silencing in Saccharomyces cerevisiae occurs at several genetic loci, including the ribosomal DNA (rDNA). Silencing at telomeres (telomere position effect [TPE]) and the cryptic mating-type loci (HML and HMR) depends on the silent information regulator genes, SIR1, SIR2, SIR3, and SIR4. However, silencing of polymerase II-transcribed reporter genes integrated within the rDNA locus (rDNA silencing) requires only SIR2. The mechanism of rDNA silencing is therefore distinct from TPE and HM silencing. Few genes other than SIR2 have so far been linked to the rDNA silencing process. To identify additional non-Sir factors that affect rDNA silencing, we performed a genetic screen designed to isolate mutations which alter the expression of reporter genes integrated within the rDNA. We isolated two classes of mutants: those with a loss of rDNA silencing (lrs) phenotype and those with an increased rDNA silencing (irs) phenotype. Using transposon mutagenesis, lrs mutants were found in 11 different genes, and irs mutants were found in 22 different genes. Surprisingly, we did not isolate any genes involved in rRNA transcription. Instead, multiple genes associated with DNA replication and modulation of chromatin structure were isolated. We describe these two gene classes, and two previously uncharacterized genes, LRS4 and IRS4. Further characterization of the lrs and irs mutants revealed that many had alterations in rDNA chromatin structure. Several lrs mutants, including those in the cdc17 and rfc1 genes, caused lengthened telomeres, consistent with the hypothesis that telomere length modulates rDNA silencing VSports手机版. Mutations in the HDB (RPD3) histone deacetylase complex paradoxically increased rDNA silencing by a SIR2-dependent, SIR3-independent mechanism. Mutations in rpd3 also restored mating competence selectively to sir3Delta MATalpha strains, suggesting restoration of silencing at HMR in a sir3 mutant background. .

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Figures (VSports注册入口)

FIG. 1
FIG. 1
Genetic screen for rDNA silencing mutants. (A) Schematic representation of the rDNA structure of strains JS306 and JS311, showing the positions of each reporter gene. (B) Schematic drawing showing the transposon mutagenesis strategy used in the screen. NotI yeast genomic DNA fragments disrupted with the transposon mTn3::lacZ::LEU2 (13), were transformed into strains JS306 and JS311. Homologous recombination resulted in the replacement of a specific segment of yeast chromosome (gene X), with an identical DNA fragment which was disrupted with the transposon. Transposon-disruption mutants are selected for the presence of LEU2 by growth on SC−Leu medium. (C) Flow chart describing the screening procedure and number of isolates at each stage. Sequences for 20 of the 65 of the 2° irs isolates were not recovered.
FIG. 2
FIG. 2
rDNA silencing phenotypes of selected mutants. (A) Quantitative growth assays measuring the silencing of mURA3 and HIS3 within the rDNA. Fivefold serial dilutions of freshly grown cells were plated onto either SC (Complete), SC−Ura (−Ura), or SC−His (−His) medium. Strains shown are WT (JS306 or JS311), top1 (M122), cac1 (M179), cac1Δ (JS400), rif1 (M158), rif1Δ (JS418), lrs4Δ (JS574), sir2Δ (JS576), rpd3 (M480), rpd3Δ (JS490), sin3Δ (JS493), sap30 (M475), hir3 (M489), tup1 (M419 and M432), and irs4 (M469). The photographs were taken for the SC−Ura plates at day 3 for the lrs mutant series (top of panel) and day 4 for the irs series (bottom of panel). (B) Quantitative growth assay measuring silencing of MET15 in NTS2. Fivefold serial dilutions of cells were plated onto SC and SC−Met −Cys medium. Strains were the same as for panel A.
FIG. 3
FIG. 3
Qualitative colony color assay showing the lrs and irs phenotypes of selected mutants. Freshly grown cells were scraped from YPD medium and streaked onto MLA medium for single colonies. Cells were grown 5 days before photographs were taken. lrs mutants produce white colonies and often display a hypersectoring phenotype; irs mutants produce a darker colony color, indicative of reduced MET15 expression compared to WT. lrs strains shown are WT (JS306), top1 (M122), rif1 (M158), cac1 (M179), sir2Δ (JS576), and lrs4Δ (JS574); irs strains shown are WT (JS311), hir3 (M487), rpd3 (M480), sin3Δ (JS493), sap30, M475, irs4 (M469), and tup1 (M419).
FIG. 4
FIG. 4
Effect of SIR2 overexpression on rDNA silencing mutants. (A) The high-copy-number empty vector pRS424 or the SIR2 vector pJSS70-9 was transformed into the WT strain JS306, several lrs mutants including cac1 (M179), and the top1 mutant (M154). Fivefold serial dilutions were spotted onto SC−Trp, which selects for the plasmid, SC−Trp−Ura to measure silencing of mURA3, and SC−Trp −His to measure silencing of HIS3. (B) Fivefold serial dilutions of irs mutants containing a high-copy-number empty vector or a high-copy-number SIR2 vector.
FIG. 5
FIG. 5
A class of rDNA silencing mutants with abnormally long telomeres. (A) The DNA replication mutants cdc17 and rfc1, and the telomere regulation mutant rif1, were each isolated as lrs mutants. Each mutant was crossed to a sir4Δ::HIS3 mutant of the opposite mating type to produce a heterozygous diploid which was sporulated and dissected for tetrads. The resulting haploid strains were grown on MLA medium and assayed for loss of silencing of MET15 as measured by colony color. Strains shown are WT (JS333), sir4Δ (JS337), cdc17 (JS432), cdc17 sir4Δ (JS436), rfc1 (JS443), rfc1 sir4Δ (JS445), rif1 (JS422), and rif1 sir4Δ (JS426). (B) Steady-state telomere length of lrs mutants in combination with sir3Δ and sir4Δ mutations. Genomic DNA was isolated from two isolates of each mutant listed, digested with XhoI, and separated on a 0.7% agarose gel. The transferred DNA was detected with a C1–3A-specific DNA probe, which on this blot will detect typical Y′ telomeres (shown schematically at bottom). The variable-length telomeres are visualized as a smear (brackets). In addition to the mutants described in panel A, there are congenic sir3Δ (JS335), cdc17 sir3Δ (JS434), rif1 sir3Δ (JS424), rif2Δ (JS495), and rif2Δ rif1 (JS497) strains.
FIG. 6
FIG. 6
rDNA chromatin accessibility of mutants as measured by in vivo psoralen cross-linking. (A) Log-phase cultures of lrs mutants were UV cross-linked with psoralen in vivo. Isolated DNA was digested with EcoRI and separated on a 1.3% agarose gel, and the transferred DNA was detected with a probe specific for the NTS of the rDNA (C). The sir2Δ strain (JS343) acted as a positive control for increased accessibility to psoralen cross-linking. Other strains shown are WT (JS306), top1 (M122), rif1 (M158), and cac1Δ (JS400). (B) An identical cross-linking procedure using the same NTS-specific probe was carried out on a subset of the irs mutants. The strains tested are WT (JS311), rpd3Δ (JS490), sin3Δ (JS493), sap30 (M475), hir3 (M411), and tup1 (M419). The control lanes show the 2.5-kb EcoRI fragment which is observed when the strains are not cross-linked. (C) Schematic drawing of the EcoRI rDNA fragment detected from this assay. ARS, autonomously replicating sequence.
FIG. 7
FIG. 7
Epistasis analysis between rpd3 and lrs mutations for rDNA silencing phenotypes. (A) Silencing reporter strains were generated with combinations of rpd3 mutations with either sir2Δ, cac1Δ, or rif1 mutations. The resulting strains were assayed for rDNA silencing strength in Ura+ and His+ growth assays. Fivefold serial dilutions of each haploid strain were spotted on indicator plates. A plus sign indicates the strain is WT for a particular gene, and a minus sign indicates a mutation of a particular gene. (B) The same strains were plated onto MLA medium and tested for silencing strength in the colony color assay. Each column represents strains grown on the same plate.
FIG. 8
FIG. 8
Mating phenotypes of rpd3 sir double-mutation combinations. The rpd3::mTn3 mutation was combined with either sir2Δ, sir3Δ, or sir4Δ mutation to determine the effect on mating ability. Strains were patched onto YPD, grown for 24 h, and then mated for 6 h with a lawn of MATa or MATα tester strains. Diploids were selected on SD minimal medium by growth for 18 h. The original YPD master plate was replica plated to SC medium as a nonselective growth control. The double-mutation combinations were tested in both MATα and MATa genetic backgrounds. Mating is measured by the efficiency of diploid formation on SD medium.
FIG. 9
FIG. 9
Model of multiple pathways to rDNA silencing. Sir2p is a central factor in rDNA silencing. Cdc17p and Rfc1p inhibit formation of extended telomeres, limiting the impact of telomere-rDNA competition for Sir2p, which is mediated by Sir4p. Cdc17p and Rfc1p may also positively act on rDNA silencing through their DNA replication functions. Silencing in most lrs and irs mutants can be increased by overexpression of Sir2p. CAF-I and Rif1p could be upstream of Sir2p, act in cooperation with Sir2p, or be completely separate inputs. Rpd3p counteracts rDNA silencing mostly through a Sir2p-dependent mechanism but also through a SIR-independent mechanism. Topoisomerase I may influence rDNA silencing by a mechanism that is partially independent of Sir2p.
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References (VSports最新版本)

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