rRNA transcription in Saccharomyces cerevisiae is performed by RNA polymerase I and regulated by changes in growth conditions. During log phase,~50% of the ribosomal DNA (rDNA) genes in each cell are transcribed and maintained in an open, psoralenaccessible conformation. During stationary phase, the percentage of open rDNA genes is greatly reduced. In this study we found that the Rpd3 histone deacetylase was required to inactivate (close) individual rDNA genes as cells entered stationary phase. Even though 50% of the rDNA genes remained open during stationary phase in rpd3D mutants, overall rRNA synthesis was still reduced. Using electron microscopy of Miller chromatin spreads, we found that the number of RNA polymerases transcribing each open gene in the rpd3D mutant was signi®cantly reduced when cells grew past log phase. Bulk levels of histone H3 and H4 acetylation were reduced during stationary phase in an RPD3-dependent manner. However, histone H3 and H4 acetylation was not signi®cantly altered at the rDNA locus in an rpd3D mutant. Rpd3 therefore regulates the number of open rDNA repeats. Keywords: deacetylation/repression/RNA polymerase I/ RPD3/yeast Introduction Ribosome biogenesis is highly regulated by cellular growth conditions and the demand for protein synthesis: fast growth demands larger numbers of ribosomes and cessation of growth correlates with decreased ribosome synthesis. Ribosomes in all cells are made up of ribosomal proteins and several non-coding rRNA molecules. As a result, ribosome biogenesis is controlled primarily through the regulation of rRNA transcription by RNA polymerase I (Pol I), and ribosomal protein gene transcription by RNA polymerase II (Pol II). RNA Pol I is a multiprotein complex that is conserved in all eukaryotes. Therefore, the powerful genetic methods in the budding yeast, Saccharomyces cerevisiae, have led to its use as a model system in the study of rRNA transcriptional regulation. The~150 ribosomal DNA (rDNA) gene copies of S.cerevisiae are organized in the nucleolus as a single tandem array on chromosome XII (for a review, see Planta, 1997). Each 9.1 kb rDNA repeat contains the Pol Itranscribed 35S pre-rRNA gene and the Pol III-transcribed 5S rRNA gene (see Figure 1A). The 5S gene is located in the middle of a non-transcribed spacer (NTS), which separates each 35S transcriptional unit.Previous studies have determined that the rate of rRNA transcription is high in exponentially growing yeast cells and is repressed as cells enter stationary phase (Ju and Warner, 1994). Sogo and co-workers have developed an in vivo psoralen cross-linking assay in yeast that can distinguish between Pol I-transcribed and non-transcribed rDNA genes based on their differential accessibility to the cross-linker (Dammann et al., 1993). They determined that only a subset of rDNA repeats in each cell are transcribed at a given time. Approximately 50% of the genes are transcribed in log phase cells, but this percentage is reduced as cells enter stationary phase (Dammann et al., 1993). These r...

The ribosomal DNA (rDNA) tandem array in Saccharomyces cerevisiae induces transcriptional silencing of RNA polymerase II-transcribed genes. This SIR2-dependent form of repression (rDNA silencing) also functions to limit rDNA recombination and is involved in life span control. In this report, we demonstrate that rDNA silencing spreads into the centromere-proximal unique sequence located downstream of RNA polymerase I (Pol I) transcription, but fails to enter the upstream telomere-proximal sequences. The spreading of silencing correlates with SIR2-dependent histone H3 and H4 deacetylation and can be extended by SIR2 overexpression. Surprisingly, rDNA silencing required transcription by RNA polymerase I and the direction of spreading was controlled by the direction of Pol I transcription.

The eukaryotic genome is divided into chromosomal domains of distinct gene activities. Transcriptionally silent chromatin tends to encroach upon active chromatin. Barrier elements that can block the spread of silent chromatin have been documented, but the mechanisms of their function are not resolved. We show that the prokaryotic LexA protein can function as a barrier to the propagation of transcriptionally silent chromatin in yeast. The barrier function of LexA correlates with its ability to disrupt local chromatin structure. In accord with this, (CCGNN) n and poly(dA-dT), both of which do not favor nucleosome formation, can also act as efficient boundaries of silent chromatin. Moreover, we show that a Rap1p-binding barrier element also disrupts chromatin structure. These results demonstrate that nucleosome exclusion is one of the mechanisms for the establishment of boundaries of silent chromatin domains.Eukaryotic DNA is compacted into chromatin. The first level of packaging is the formation of nucleosomes, each consisting of a protein core of histones H2A, H2B, H3, and H4, around which 146 bp of DNA is wrapped. Higher levels of compaction involve histone H1 and/or other proteins that associate with nucleosomes (38). Based on its cytological and molecular properties, chromatin is roughly divided into condensed heterochromatin and decondensed euchromatin, which are interspersed in the genome. In general, heterochromatin inhibits gene expression whereas euchromatin allows it, leading to a position effect on gene activity. Heterochromatin formed in one part of the genome may propagate along the chromosome, consuming euchromatin in its path. This is accomplished by the spreading of heterochromatin-specific complexes that interact with nucleosomes and condense chromatin to a higher level (20,35). In addition, various covalent modifications of histones (e.g., acetylation and methylation) also play pivotal roles in establishing the state of chromatin at a particular locus (27). For instance, heterochromatin is associated with characteristic hypoacetylation of histones.In Saccharomyces cerevisiae, transcriptionally silent chromatin at HMR, HML, or telomeres is the yeast equivalent of metazoan heterochromatin that is formed through coordinated actions of cis-acting elements and trans-acting factors (41). The cis-acting elements include telomeric repeats and sites flanking each HM locus that are known as silencers, and the trans-acting proteins include Sir2p-Sir4p and silencer-or telomere-binding proteins. Silencer-or telomere-binding proteins recruit the SIR complex (Sir2p/Sir3p/Sir4p), which then propagates sequentially along an array of nucleosomes. The SIR complex is an integral part of silent chromatin, and interactions between Sir3p/Sir4p and histones H3 and H4 are key to the establishment and maintenance of silenced chromatin (41). There is evidence that Sir3p has higher affinity to unacetylated histone H4 (10). Sir2p is an NAD-dependent protein deacetylase that is likely involved in reducing histone acetylation ...

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