RESUMO
RNA polymerase II (Pol II)-transcribed genes embedded within the yeast rDNA locus are repressed through a Sir2-dependent process called 'rDNA silencing'. Sir2 is recruited to the rDNA promoter through interactions with RNA polymerase I (Pol I), and to a pair of DNA replication fork block sites (Ter1 and Ter2) through interaction with Fob1. We utilized a reporter gene (mURA3) integrated adjacent to the leftmost rDNA gene to investigate localized Pol I and Fob1 functions in silencing. Silencing was attenuated by loss of Pol I subunits or insertion of an ectopic Pol I terminator within the adjacent rDNA gene. Silencing left of the rDNA array is naturally attenuated by the presence of only one intact Fob1 binding site (Ter2). Repair of the 2nd Fob1 binding site (Ter1) dramatically strengthens silencing such that it is no longer impacted by local Pol I transcription defects. Global loss of Pol I activity, however, negatively affects Fob1 association with the rDNA. Loss of Ter2 almost completely eliminates localized silencing, but is restored by artificially targeting Fob1 or Sir2 as Gal4 DNA binding domain fusions. We conclude that Fob1 and Pol I make independent contributions to establishment of silencing, though Pol I also reinforces Fob1-dependent silencing.
Assuntos
DNA Ribossômico/genética , Proteínas de Ligação a DNA/genética , RNA Polimerase I/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Gênica , Sítios de Ligação , Inativação Gênica , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Sirtuína 2/genéticaRESUMO
The histone deacetylase activity of Sir2p is dependent on NAD(+) and inhibited by nicotinamide (NAM). As a result, Sir2p-regulated processes in Saccharomyces cerevisiae such as silencing and replicative aging are susceptible to alterations in cellular NAD(+) and NAM levels. We have determined that high concentrations of NAM in the growth medium elevate the intracellular NAD(+) concentration through a mechanism that is partially dependent on NPT1, an important gene in the Preiss-Handler NAD(+) salvage pathway. Overexpression of the nicotinamidase, Pnc1p, prevents inhibition of Sir2p by the excess NAM while maintaining the elevated NAD(+) concentration. This growth condition alters the epigenetics of rDNA silencing, such that repression of a URA3 reporter gene located at the rDNA induces growth on media that either lacks uracil or contains 5-fluoroorotic acid (5-FOA), an unusual dual phenotype that is reminiscent of telomeric silencing (TPE) of URA3. Despite the similarities to TPE, the modified rDNA silencing phenotype does not require the SIR complex. Instead, it retains key characteristics of typical rDNA silencing, including RENT and Pol I dependence, as well as a requirement for the Preiss-Handler NAD(+) salvage pathway. Exogenous nicotinamide can therefore have negative or positive impacts on rDNA silencing, depending on the PNC1 expression level.
Assuntos
DNA Ribossômico/genética , Inativação Gênica , Niacinamida/metabolismo , Nicotinamidase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , DNA Ribossômico/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , NAD/metabolismo , Nicotinamidase/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2 , Sirtuínas/genética , Sirtuínas/metabolismo , Telômero/metabolismoRESUMO
The silent information regulator (Sir2) family of protein deacetylases (Sirtuins) are nicotinamide adenine dinucleotide (NAD)(+)-dependent enzymes that hydrolyze one molecule of NAD(+) for every lysine residue that is deacetylated. The Sirtuins are phylogenetically conserved in eukaryotes, prokaryotes, and Archeal species. Prokaryotic and Archeal species usually have one or two Sirtuin homologs, whereas eukaryotes typically have multiple versions. The founding member of this protein family is the Sir2 histone deacetylase of Saccharomyces cerevisiae, which is absolutely required for transcriptional silencing in this organism. Sirtuins in other organisms often have nonhistone substrates and in eukaryotes, are not always localized in the nucleus. The diversity of substrates is reflected in the various biological activities that Sirtuins function, including development, metabolism, apoptosis, and heterochromatin formation. This review emphasizes the great diversity in Sirtuin function and highlights its unusual catalytic properties.
Assuntos
Sirtuínas/fisiologia , Sequência de Aminoácidos , Histona Desacetilases/fisiologia , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/fisiologia , Sirtuína 2RESUMO
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.