Academic journal article Genetics

Pnc1p-Mediated Nicotinamide Clearance Modifies the Epigenetic Properties of rDNA Silencing in Saccharomyces Cerevisiae

Academic journal article Genetics

Pnc1p-Mediated Nicotinamide Clearance Modifies the Epigenetic Properties of rDNA Silencing in Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

The histone deacetylase activity of Sir2p is dependent on NAD^sup +^ 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^sup +^ and NAM levels. We have determined that high concentrations of NAM in the growth medium elevate the intracellular NAD^sup +^ concentration through a mechanism that is partially dependent on NPT1, an important gene in the Preiss-Handler NAD^sup +^ salvage pathway. Overexpression of the nicotinamidase, Pnc1p, prevents inhibition of Sir2p by the excess NAM while maintaining the elevated NAD^sup +^ 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^sup +^ salvage pathway. Exogenous nicotinamide can therefore have negative or positive impacts on rDNA silencing, depending on the PNC1 expression level.

IN the budding yeast, Saccharomyces cerevisiae, there are three general locations that are silenced in the genome, the silent-mating type loci HML and HMR, the telomeres, and the ribosomal DNA (rDNA) (see Rusche et al. 2003 for review). Silencing at these locations is dependent on the silent information regulator genes, SIR1-SIR4. All four SIR genes are required for the efficient establishment, maintenance, and inheritance of silent chromatin structure at the HM loci (Pillus and Rine 1989). SIR2, SIR3, and SIR4 are critical for silencing at telomeres (Aparicio et al. 1991), but only SIR2 is required for silencing and suppression of recombination at the rDNA (Gottlieb and Esposito 1989; Bryk et al. 1997; Fritze et al. 1997; Smith and Boeke 1997). SIR2 encodes a highly conserved NAD^sup +^-dependent histone deacetylase that is predominantly localized in the nucleolus and in perinuclear foci that harbor the telomeres (Gotta et al. 1997). In the nucleolus, Sir2p is a subunit of the multiprotein deacetylase complex known as RENT. This complex also contains Net1p and Cdc14p, and functions in rDNA silencing and regulation of the exit from mitosis (Shou et al. 1999; Straight et al. 1999), as well as rDNA transcription by RNA polymerase I (Shou et al. 2001). At the telomeres and HM loci, Sir2p is a subunit of the SIR complex, which minimally consists of Sir2p and Sir4p (Ghidelli et al. 2001; Tanny et al. 2004). Sir3p can also be part of the complex, which is a heterotrimer when purified from insect cells (Cubizolles et al. 2006). The sharing of Sir2p by all forms of yeast silencing leads to competition between the various compartments for a limiting amount of Sir2p (Smith et al. 1998). rDNA silencing is especially sensitive to changes in Sir2p levels, as SIR2 overexpression dramatically strengthens silencing at this locus (Fritze et al. 1997; Smith et al. 1998), and extends replicative life span through the suppression of rDNA recombination (Gottlieb and Esposito 1989; Kaeberlein et al. 1999). Similarly, deletion of SIR4 releases Sir2p from the telomeres and HM loci, causing it to accumulate in the nucleolus and strengthen rDNA silencing (Kennedy et al. 1997; Smith et al. 1998).

The Sir2p family of protein deacetylases (collectively known as sirtuins) utilize NAD^sup +^ as a cosubstrate. For every lysine that is deacetylated, one molecule of NAD^sup +^ is hydrolyzed, yielding one molecule each of nicotinamide (NAM) and O-acetyl-ADP-ribose (AAR) (Landry et al. 2000; TannyandMoazed 2001). The consumption of NAD^sup +^ implies there is a constant need for NAD^sup +^ production in the cell if sirtuins are to remain active. …

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