SIR2-Induced Inviability Is Suppressed by Histone H4 Overexpression

Corresponding author: Scott G. Holmes, Hall-Atwater Laboratories, Wesleyan University, Middletown, CT 06459-0175., sholmes{at}wesleyan.edu (E-mail)

Communicating editor: M. JOHNSTON

	ABSTRACT

TOP ABSTRACT LITERATURE CITED

We have identified histone H4 as a high-expression suppressor of Sir2-induced inviability in yeast cells. Overexpression of histone H3 does not suppress Sir2-induced lethality, nor does overexpression of histone H4 alleles associated with silencing defects. These results suggest a direct and specific interaction between Sir2 and H4 in the silencing mechanism.

THE Sir2 protein is required to mediate transcription silencing at the HM loci, telomeres, and rDNA repeats in yeast (see GARTENBERG 2000 ; SHORE 2000 ; MOAZED 2001 for reviews). Several observations suggest that Sir2-mediated deacetylation of histones may have an important role in the silencing mechanism. Silenced sequences are specifically associated with deacetylated histone H3 and H4, and overexpression of Sir2 leads to a global decrease of histone acetylation in vivo (BRAUNSTEIN et al. 1993 ). Histone H4 also serves as a substrate for Sir2's deacetylase activity in vitro (TANNY et al. 1999 ; IMAI et al. 2000 ; SMITH et al. 2000 ), with specificity for a lysine shown by genetic studies to play a key role in silencing (IMAI et al. 2000 ). Mutations in Sir2 that decrease its deacetylase activity in vitro cause defects in silencing in vivo (IMAI et al. 2000 ). Finally, while experiments designed to test Sir2's ability to bind histones have yielded negative results (HECHT et al. 1995 ; GHIDELLI et al. 2001 ), protein complexes purified from yeast that contain Sir2 interact with nucleosomes in vitro (GHIDELLI et al. 2001 ). However, thus far there is no direct evidence that histones are a relevant substrate for Sir2 in vivo. To identify the in vivo substrates and regulators of Sir2 we have isolated suppressors of the inviability caused by Sir2p overexpression (Table 1).

Plasmid pSS3 is a high-copy plasmid in which the SIR2 gene is under control of the GAL10 promoter. Yeast strains containing pSS3 are unable to form colonies when plated on galactose media (Fig 1). A H364Y mutation that eliminates the silencing and catalytic activity of Sir2p (TANNY et al. 1999 ; IMAI et al. 2000 ) also completely suppresses overexpression lethality when introduced into pSS3 (plasmid pMM11; see Fig 1), suggesting that the lethality depends on Sir2's enzymatic function. We transformed a yeast strain carrying pSS3 with a high-expression yeast cDNA library (LIU et al. 1992 ) and screened for transformants that could grow on galactose plates. We reasoned that such suppressors might include substrates of Sir2p that titrate excess Sir2 protein or regulators that inhibit Sir2's catalytic function.

View larger version (83K): In this window In a new window Download PPT slide   Figure 1. (A) SIR2-induced inviabilty. Strain YSH278 was transformed with plasmids containing galactose-inducible SIR2 alleles and grown overnight in raffinose medium lacking leucine. The H364Y mutation causes substitution of a tyrosine for a histidine at amino acid 364 in the Sir2 protein. Sets of 10-fold serial dilutions from these cultures were spotted on glucose medium lacking leucine (-LEU) and on galactose medium lacking leucine (-LEU GAL). Photographs were taken after 2–4 days growth at 30°. (B) Suppressors of SIR2 inviability. Serial dilutions of cells bearing the indicated plasmids were made on glucose medium lacking leucine (-LEU -URA) and on galactose medium lacking leucine (-LEU -URA GAL). The "control" row cells contain YEp51 (LEU2 vector) and pRS416 (URA3 vector). The "vector" row cells contain pSS3 (GAL-SIR2) and pRS416. All others contain pSS3 and a library plasmid containing the indicated gene fused to the GAL1 promoter. (C) Histone H4 overexpression does not alter Sir2 protein levels. Steady-state levels of Sir2 protein were determined by Western blotting in the indicated conditions. Lanes 1 and 2 contain protein from cells bearing the single wild-type copy of the SIR2 gene. Lanes 3–8 contain protein from cells bearing plasmid pSS3 (GAL-SIR2). Cells also contain either the GAL-HHF2 plasmid (+) or a vector control (-). All cells were grown in media containing galactose. Equal amounts of protein were loaded in lanes 1 and 2 and independently in lanes 3 and 6. The indicated dilutions of the lane 3 sample were made in lanes 4 and 5; dilutions of the lane 6 sample were made in lanes 7 and 8. Lanes 1 and 2 were exposed to film for a longer period than lanes 3–8.

Genes specific for their ability to suppress Sir2-induced lethality are shown in Fig 1. Notable among this group of suppressors is HHF2, the gene for histone H4, which was isolated multiple times in this screen. We found that H4 overexpression does not affect the steady-state levels of Sir2 protein (Fig 1C). A straightforward hypothesis for our result is that H4 protein is a direct substrate for Sir2 and that excess histone H4 in the cell is titrating Sir2, allowing the cells to live. To characterize the Sir2-histone interaction further we conducted suppression experiments with additional histone alleles. We first determined if overexpression of histone H3 could suppress Sir2 inviability. While H3 is also an in vitro substrate for Sir2 (IMAI et al. 2000 ; LANDRY et al. 2000 ), substitution of the potential lysine substrates in H3 cause only slight silencing defects in vivo compared to comparable mutations in H4 (THOMPSON et al. 1994 ). We found that H3 overexpression does not permit growth in cells expressing high levels of Sir2. If overexpression of specific histone alleles is lethal to yeast, then this could appear in our experiments as an inability to suppress. We found that the failure to observe suppression with H3 or with the H4 alleles described below is not due to histone-induced inviability, as no change in growth is observed when any of the histone alleles we tested are overexpressed in the absence of high Sir2 levels (not shown).

The proposed lysine substrates of histone H4 are restricted to the N terminus of the protein. If Sir2 interacts directly with the N terminus of histone H4, then we reasoned that overexpression of an H4 allele lacking the N terminus may fail to suppress. As shown in Fig 2, this prediction is borne out: overexpression of an H4 allele lacking amino acids 4–20 of the N terminus fails to suppress the lethal effects of Sir2p. Sir2p shows specificity for deacetylating lysine 16 of histone H4 in vitro (IMAI et al. 2000 ). Mutations in this residue also cause silencing defects in vivo; when replaced by arginine, silencing is moderately reduced, while if an uncharged glutamine residue is placed at this position, silencing is abolished (JOHNSON et al. 1990 ; MEGEE et al. 1990 ; PARK and SZOSTAK 1990 ). As an additional test of specificity we tested H4 alleles containing these substitutions for the ability to suppress Sir2 overexpression. We found in this case that the ability to promote silencing correlates with the ability to suppress; the arginine substitution causes a significant drop in suppression, while overexpressing an H4 allele in which glutamine replaced the lysine at position 16 eliminates suppression (see Fig 2). To determine if lysines subject to acetylation are required to observe suppression we constructed an H4 allele in which all four lysines are replaced by glutamines. This allele of H4 fails to support silencing when expressed as the only source of H4 (MEGEE et al. 1990 ). However, we find that this allele can suppress Sir2 overexpression (Fig 2). Finally, we show that the level of suppression in this allele can be improved further by restoring the lysine at position 16, although we note that in no case is suppression equivalent to the level seen with wild-type H4.

View larger version (64K): In this window In a new window Download PPT slide   Figure 2. Specificity of the Sir2-histone interaction. Serial dilutions of strains containing pSS3 (GAL-SIR2) and various galactose-inducible histone alleles were plated on the indicated media. The "control" row cells contain YEp51 (LEU2 vector) and pRS416 (URA3 vector). The "vector" row cells contain pSS3 and pRS416. All others contain pSS3 and a plasmid containing the indicated histone allele fused to the GAL promoter. Alleles of the H3 and H4 genes were cloned in a position identical to the wild-type H4 allele that we initially identified. Mutated HHF2 (histone H4) alleles are listed in shorthand; the identity of the amino acid at positions 5, 8, 12, and 16 is given (all are lysines in wild-type histone H4). K, lysine; R, arginine; Q, glutamine.

Thus far we have not characterized Sir2's interactions with the remaining suppressors. However, we note that TDH3 codes for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which catalyzes a key step in the Krebs cycle and requires NAD as a cofactor. Sir2's catalytic function also requires NAD. If the level of GAPDH is increased, then the concentration of NAD in the cell may fall, depressing the effects of excess Sir2 protein. Alternatively, in some eukaryotic cells GAPDH has been shown to be the enzyme most sensitive to NAD levels (GOODWIN et al. 1978 ; D'AMOURS et al. 1999 ). Overexpression of Sir2 may reduce cellular NAD, limiting GAPDH activity and contributing to cell inviability; increasing the levels of GAPDH may overcome this.

It is possible that suppression of Sir2 lethality by histone H4 overexpression is an indirect effect; for instance, H4 overexpression is known to specifically alter the expression of a number of genes in yeast (WYRICK et al. 1999 ). An alternative, straightforward model for our results is that excess Sir2 protein is titrated away by increasing its natural substrate, histone H4. This model is supported by the specificity we have observed in our experiments. The lysine at position 16 of histone H4 is a key determinant of silencing in vivo and the preferred substrate for Sir2 in vitro. We found that the ability to suppress excess Sir2 is heavily influenced by the lysine at position 16 of histone H4. We also found that an allele of H4 containing glutamine substitutions of the four lysines found in the N terminus of H4, which is unlikely to be a substrate for Sir2's deacetylase activity, can also suppress. If the titration model is correct, this indicates Sir2 binding is not dependent on the ability of the histone protein to act as a substrate; however, as glutamine mimics the neutral, acetylated state it may promote increased Sir2 binding compared to alleles with positively charged residues (BRAUNSTEIN et al. 1996 ). Deletions of the histone H4 N-terminal tail increase the lethal effects of Sir2 overexpression (HOLMES et al. 1997 ). This may indicate that the absence of a primary substrate (due to loss of the H4 tail) frees Sir2 to act on a secondary substrate, causing lethality. Finally, we show that overexpression of H3 fails to suppress Sir2 overexpression in vivo. Prior genetic results indicated a differential effect on silencing of keeping H4 in a particular acetylated state compared to H3 (THOMPSON et al. 1994 ); our results may indicate an actual preference of substrate in vivo. Overall, our results support models in which Sir2 mediates silencing via a direct interaction with histone H4.

	ACKNOWLEDGMENTS

We thank M. Mitchell Smith and James Broach for providing strains and plasmids, and Jason Tanny, Danesh Moazed, and Katrina Catron for helpful discussions. This study was supported by research program grant (RPG-98-351-01-MGO) from the American Cancer Society to S.G.H.

Manuscript received June 7, 2002; Accepted for publication July 9, 2002.

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