Genetic evidence for an interaction between SIR3 and histone H4 in the repression of the silent mating loci in Saccharomyces cerevisiae.

Abstract

Repression of transcription from the silent mating loci (HMLa and HMRa) is essential for mating ability in Saccharomyces cerevisiae. This silencing is known to require at least five proteins (SIR1, SIR2, SIR3, SIR4, and histone H4) and is accompanied by a change in chromatin structure. We show here that four positions of histone H4 (N-terminal residues 16, 17, 18, and 19) are crucial to silencing. HMLa and HMRa are efficiently repressed when these positions are occupied by basic amino acids but are derepressed when substituted with glycine. These results suggest that acetylation of would lead to derepression of the silent mating loci. Three strong extragenic suppressors of the latter H4 mutations were isolated and determined to be located in SIR3. These suppressors allow high mating efficiencies in cells expressing either wild-type H4 or H4 containing single amino acid substitutions. They did not allow efficient mating in a strain that contained an H4 N-terminal deletion. These results indicate that the SIR3 mutations do not bypass the requirement for the H4 N terminus but, rather, allow repression in the presence of a less than optimal H4 N terminus. This provides a link between one of the SIR proteins and a component of chromatin.In Saccharomyces cerevisiae, the determination of mating type relies on the permanent repression of genetic information. How these proteins and DNA sequences interact to inhibit transcription in this region is yet unclear. Although there are chromatin structure differences between MAT and the silent loci (6), it is unknown whether they are the cause or result of expression at MAT. Recently, deletions in the N terminus of histone H4, a component of the nucleosome, were found to lead to transcriptional derepression of the silent loci (7). This was, to our knowledge, the first direct evidence that a change in chromatin structure resulted in a change in expression at HMLa and HMRa. We have extended these studies to investigate which residues in the N terminus of H4 are important for silencing. The H4 deletion, removing amino acids 4-14, reduces mating efficiency only 3-fold, whereas deletion of amino acids 4-19 reduces mating efficiency >4 orders of magnitude. This larger deletion removes all four acetylatable lysines (located at positions 5, 8, 12, and 16) as well as a highly basic region extending from amino acids 16 to 20. Is the reversible acetylation of the lysine residues crucial for repression of the mating loci or is the highly basic region the only requirement? Is one or more of the SIR proteins functioning through H4 to modulate chromatin structure? To address these questions, we have made single amino acid changes in H4 and found four residues that are essential for repression. In addition, three independent suppressors ofthese mutations were found to be SIR3 mutations, suggesting that an interaction between histone H4 and SIR3 is important for repression of the silent mating loci. METHODSPlasmid Constructions. The plasmid pLJ438T was constructed by cloning a BamHI-Hin...

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Nucleoprotein Structure Influences the Response of the Mouse Mammary Tumor Virus Promoter to Activation of the Cyclic AMP Signalling Pathway

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