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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 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. 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 sir3
MAT
strains, suggesting
restoration of silencing at HMR in a sir3
mutant background.
*
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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