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

Abstract

The histone deacetylase activity of Sir2p is dependent on NAD 1 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 1 and NAM levels. We have determined that high concentrations of NAM in the growth medium elevate the intracellular NAD 1 concentration through a mechanism that is partially dependent on NPT1, an important gene in the Preiss-Handler NAD 1 salvage pathway. Overexpression of the nicotinamidase, Pnc1p, prevents inhibition of Sir2p by the excess NAM while maintaining the elevated NAD 1 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 1 salvage pathway. Exogenous nicotinamide can therefore have negative or positive impacts on rDNA silencing, depending on the PNC1 expression level.

Isonicotinamide Enhances Sir2 Protein-mediated Silencing and Longevity in Yeast by Raising Intracellular NAD+ Concentration

“…Supplementation of cells with NA or NR leads to increased NAD ϩ concentration because they are precursors utilized by the salvage pathways, but they do not activate sirtuin activity in vitro. Another NAD ϩ precursor, NAM, shares a functional property of INAM in its ability to raise the NAD ϩ concentration in yeast (38), although it acts as a strong sirtuin inhibitor (22). Only when PNC1 is overexpressed to clear the excess NAM (15) does the increase in NAD ϩ concentration induced by high NAM concentrations have any positive effect on silencing (38).…”

“…Therefore, we also utilized a strain in which the mURA3 reporter gene was stably integrated into unique chromosome XII sequence, 50 bp left of the rDNA array (YSB348) (37). Silencing of mURA3 at this position results in poor growth on SC-Ura but is not strong enough to induce growth on FOA unless SIR2 is overexpressed or silencing is improved by other means (38). As shown in Fig.…”

Nicotinamide Suppresses the DNA Damage Sensitivity of Saccharomyces cerevisiae Independently of Sirtuin Deacetylases

“…We suggest that nicotinamide simultaneously both promotes and inhibits MMS resistance via different pathways. In pnc1D cells, the inhibitory effect could be mediated by a failure to properly clear exogenously added nicotinamide and convert it to NAD+ (McClure et al 2008). This could negatively regulate functions of sirtuins that promote DNA repair.…”

“…Nicotinamide functions to inhibit sirtuins in a negative feedback loop (Sauve et al 2001; Bitterman et al 2002;Jackson et al 2003;Yuan and Marmorstein 2012). It is normally cleared by the nicotinamide salvage pathway, which also contributes to the maintenance of NAD+ levels within the cell (Gallo et al 2004;Sauve et al 2005;McClure et al 2008). NAD+ can also be produced de novo from tryptophan in a pathway requiring Bna2 (Panozzo et al 2002).…”

“…Exogenously added nicotinamide is used to inhibit sirtuins at concentrations of 5-25 mM (Bitterman et al 2002;Tsuchiya et al 2006). In yeast, nicotinamide and/or salvage pathway intermediates and enzymes can impact transcriptional silencing (Belenky et al 2007;McClure et al 2008), replicative lifespan (Belenky et al 2007), and the clearing of protein aggregates (Ocampo et al 2013). Nicotinamide and other regulators of sirtuins have been proposed for use in the treatment of cancer and other diseases (Demarin et al 2004;Lara et al 2009;Chopra et al 2012;Chen et al 2015;Cheon et al 2015), but little is known about the specificity of these drugs or the consequences of inhibiting multiple sirtuins simultaneously.…”