A Genetic Screen for Ribosomal DNA Silencing Defects Identifies Multiple DNA Replication and Chromatin-Modulating Factors

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Molecular and Cellular Biology, April 1999, p. 3184-3197, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

A Genetic Screen for Ribosomal DNA Silencing Defects Identifies Multiple DNA Replication and Chromatin-Modulating Factors

Jeffrey S. Smith, Emerita Caputo, and Jef D. Boeke^*

Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received 20 October 1998/Returned for modification 3 December 1998/Accepted 21 December 1998

Transcriptional silencing in Saccharomyces cerevisiae occurs at several genetic loci, including the ribosomal DNA (rDNA).Silencing at telomeres (telomere position effect [TPE]) and thecryptic mating-type loci (HML and HMR) depends on the silent informationregulator genes, SIR1, SIR2, SIR3, and SIR4. However, silencingof polymerase II-transcribed reporter genes integrated withinthe rDNA locus (rDNA silencing) requires only SIR2. The mechanismof rDNA silencing is therefore distinct from TPE and HM silencing.Few genes other than SIR2 have so far been linked to the rDNAsilencing process. To identify additional non-Sir factors thataffect rDNA silencing, we performed a genetic screen designedto isolate mutations which alter the expression of reporter genesintegrated within the rDNA. We isolated two classes of mutants:those with a loss of rDNA silencing (lrs) phenotype and thosewith an increased rDNA silencing (irs) phenotype. Using transposonmutagenesis, lrs mutants were found in 11 different genes, andirs mutants were found in 22 different genes. Surprisingly, wedid not isolate any genes involved in rRNA transcription. Instead,multiple genes associated with DNA replication and modulationof chromatin structure were isolated. We describe these two geneclasses, and two previously uncharacterized genes, LRS4 and IRS4.Further characterization of the lrs and irs mutants revealed thatmany had alterations in rDNA chromatin structure. Several lrsmutants, including those in the cdc17 and rfc1 genes, caused lengthenedtelomeres, consistent with the hypothesis that telomere lengthmodulates rDNA silencing. Mutations in the HDB (RPD3) histonedeacetylase complex paradoxically increased rDNA silencing bya SIR2-dependent, SIR3-independent mechanism. Mutations in rpd3also restored mating competence selectively to sir3 $Delta$ MAT $alpha$ strains,suggesting restoration of silencing at HMR in a sir3 mutantbackground.

^* Corresponding author. Mailing address: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine,725 N. Wolfe St., 617 Hunterian Building, Baltimore, MD 21205.Phone: (410) 955-2481. Fax: (410) 614-2987. E-mail: jboeke{at}jhmi.edu.

Molecular and Cellular Biology, April 1999, p. 3184-3197, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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