RPD3 is required for the inactivation of yeast ribosomal DNA genes in stationary phase

Abstract

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...

Microspherule Protein 1, Mi-2β, and RET Finger Protein Associate in the Nucleolus and Up-regulate Ribosomal Gene Transcription

“…Human cells contain about 400 ribosomal genes in tandem repeats at nucleolus organizer regions encoded on five pairs of chromosomes 13, 14, 15, 21, and 22. Previous studies have indicated that only 30 -50% of rRNA genes display an open accessible chromatin structure (3,4).…”

“…Human diploid cells contain about 400 ribosomal genes organized as tandem repeats at nucleolus organizer regions encoded on chromosomes 13, 14, 15, 21, and 22 (1,2). rRNA gene transcription varies according to the demand for ribosome production and protein synthesis, and in yeast and murine cells, only 30 -50% of the rRNA genes are in an open structure that facilitates active transcription (3,4). Transcription of rRNA is extensively regulated by a large number of proteins such as growth factors, CBP (cAMPresponse element-binding protein (CREB)-binding protein), Rb, p53, and Myc (5)(6)(7)(8)(9)(10).…”

Sir2 Represses Endogenous Polymerase II Transcription Units in the Ribosomal DNA Nontranscribed Spacer

“…At the HM loci and telomeres, hypoacetylated H3 and H4 promote the interaction of Sir3 and Sir4 with nucleosomes, thereby facilitating the formation and spread of silent chromatin (Braunstein et al, 1993(Braunstein et al, , 1996Hecht et al, 1995;Wu and Grunstein, 2000;Carmen et al, 2002;Liou et al, 2005). Hypoacetylated histones are present at the rDNA (Bryk et al, 2002;Buck et al, 2002;Sandmeier et al, 2002;Huang and Moazed, 2003), although how they contribute to rDNA silencing remains unclear.…”

“…At the HM loci and telomeres, hypoacetylated H3 and H4 promote the interaction of Sir3 and Sir4 with nucleosomes, thereby facilitating the formation and spread of silent chromatin (Braunstein et al, 1993(Braunstein et al, , 1996Hecht et al, 1995;Wu and Grunstein, 2000;Carmen et al, 2002;Liou et al, 2005). Hypoacetylated histones are present at the rDNA (Bryk et al, 2002;Buck et al, 2002;Sandmeier et al, 2002;Huang and Moazed, 2003), although how they contribute to rDNA silencing remains unclear.Cells lacking K4-methylated histone H3 exhibit defects in transcriptional silencing at the rDNA and telomeres (Nislow et al, 1997;Briggs et al, 2001;Bryk et al, 2002;Nagy et al, 2002;Mueller et al, 2006). Set1 is the catalytic subunit of the COMPASS complex that is required for mono-, di-, and trimethylation of histone H3 on K4.…”

Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states

“…1A), accounting for ;50% of all rRNA genes, is silenced via epigenetic mechanisms that include changes in DNA methylation and histone modification (Earley et al 2006(Earley et al , 2010Pontvianne et al 2010Pontvianne et al , 2012. Chromatin modifications mediate rRNA gene dosage control in yeast, mouse, and human cells as well (Sandmeier et al 2002;McStay and Grummt 2008;Grummt and Langst 2013).…”