No abstract

Silencing is a universal form of transcriptional regulation in which regions of the genome are reversibly inactivated by changes in chromatin structure. Sir2 (Silent Information Regulator) protein is unique among the silencing factors in Saccharomyces cerevisiae because it silences the rDNA as well as the silent mating-type loci and telomeres. Discovery of a gene family of Homologues of Sir Two (HSTs) in organisms from bacteria to humans suggests that SIR2's silencing mechanism might be conserved. The Sir2 and Hst proteins share a core domain, which includes two diagnostic sequence motifs of unknown function as well as four cysteines of a putative zinc finger. We demonstrate by mutational analyses that the conserved core and each of its motifs are essential for Sir2p silencing. Chimeras between Sir2p and a human Sir2 homologue (hSir2Ap) indicate that this human protein's core can substitute for that of Sir2p, implicating the core as a silencing domain. Immunofluorescence studies reveal partially disrupted localization, accounting for the yeast-human chimeras' ability to function at only a subset of Sir2p's target loci. Together, these results support a model for the involvement of distinct Sir2p-containing complexes in HM/telomeric and rDNA silencing and that HST family members, including the widely expressed hSir2A, may perform evolutionarily conserved functions.

Abstract. During cell division and growth, the nucleus and chromosomes are remodeled for DNA replication and cell type-specific transcriptional control. The yeast silencing protein Sir3p functions in both chromosome structure and in transcriptional regulation. Specifically, Sir3p is critical for the maintenance of telomere structure and for transcriptional repression at both the silent mating-type loci and telomeres. We demonstrate that Sir3p becomes hyperphosphorylated in response to mating pheromone, heat shock, and starvation. Cells exposed to pheromone arrest in G1 of the cell cycle, yet G1 arrest is neither necessary nor sufficient for pheromone-induced Sir3p hyperphosphorylation. Rather, hyperphosphorylation of Sir3p requires the mitogen-activated protein (MAP) kinase pathway genes STE11, STE7, FUS3/KSS1, and STE12, indicating that an intact signal transduction pathway is crucial for this Sir3p phosphorylation event. Constitutive activation of the pheromone-response MAP kinase cascade in an STEll-4 strain leads to hyperphosphorylation of Sir3p and increased Sir3p-dependent transcriptional silencing at telomeres. Regulated phosphorylation of Sir3p may thus be a mechanistically significant means for modulating silencing. Together, these observations suggest a novel role for MAP kinase signal transduction in coordinating chromatin structure and nuclear organization for transcriptional silencing. SIGNAL transduction through mitogen-activated protein (MAP) t kinase cascades communicates extracellular signals to the nucleus and elicits distinct responses. In yeast, six different MAP kinase pathways have been described; they coordinate signals induced by such diverse stimuli as mating pheromone, heat shock, starvation, and changes in osmolarity (for reviews see Levin and Errede, 1995;Herskowitz, 1995). Induction of any of these MAP kinase pathways leads to transcriptional activation of many genes ihat are responsible for alterations in cellular structure and physiology. For example, reorganization of the nucleus may enable the cell to respond properly to extracellular signals.Chromosomal structures, like telomeres and centromeres, as well as specialized regions of transcription, such as the nucleolus, specify discrete structural and functional domains in the nucleus. Some of these nuclear domains Address all correspondence to Lorraine

The SIR1 gene product of Saccharomyces cerevisiae is one of several proteins involved in repressing transcription of the silent mating-type genes. Strains with mutations in the genes coding for these proteins are defective in mating due to derepression of the silent loci. We have found that overexpression of the SIR1 gene suppresses the mating defects of several of these mutants, including nat1 and ard1 mutants (the products of these two genes are responsible for N-terminal acetylation of a subset of yeast proteins), certain sir3 mutants, and a histone H4 mutant. The SIR1 gene has been sequenced and found to contain an open reading frame coding for a 678-amino-acid protein.