The silent mating loci and chromosomal regions adjacent to telomeres of S. cerevisiae have features similar to heterochromatin of more complex eukaryotes. Transcriptional repression at these sites depends on the silent information regulators SIR3 and SIR4 as well as histones H3 and H4. We show here that the SIR3 and SIR4 proteins interact with specific silencing domains of the H3 and H4 N-termini in vitro. Certain mutations in these factors, which affect their silencing functions in vivo, also disrupt their interactions in vitro. Immunofluorescence studies with antibodies against RAP1 and SIR3 demonstrate that the H3 and H4 N-termini are required for the association of SIR3 with telomeric chromatin and the perinuclear positioning of yeast telomeres. Based on these interactions, we propose a model for heterochromatin-mediated transcriptional silencing in yeast, which may serve as a paradigm for other eukaryotic organisms as well.

Yeast core telomeric heterochromatin can silence adjacent genes and requires RAP1, SIR2, SIR3, and SIR4 and histones H3 and H4 for this telomere position effect. SIR3 overproduction can extend the silenced domain. We examine here the nature of these multiprotein complexes. SIR2 and SIR4 were immunoprecipitated from whole-cell extracts. In addition, using formaldehyde cross-linking we have mapped SIR2, SIR4, and RAP1 along telomeric chromatin before and after SIR3 overexpression. Our data demonstrate that SIR2 and SIR4 interact in a protein complex and that SIR2, SIR3, SIR4, and RAP1 map to the same sites along telomeric heterochromatin in wild-type cells. However, when overexpressed, SIR3 spreads along the chromosome and its interactions are dominant to those of SIR4 and especially SIR2, whose detection is decreased in extended heterochromatin. RAP1 binding at the core region is unaffected by SIR3 overproduction and RAP1 shows no evidence of spreading. Thus, we propose that the structure of core telomeric heterochromatin differs from that extended by SIR3.[Key Words: Heterochromatin; telomeres; silencing; SIR proteins; RAP1] Received September 25, 1996; revised version accepted November 15, 1996.Heterochromatin was cytologically defined as that fraction of the eukaryotic genome that is constitutively condensed throughout the cell cycle (Heitz 1928). Such regions, often found near centromeres or telomeres, can repress adjacent genes epigenetically. For example, euchromatic genes placed adjacent to centromeric heterochromatin in Drosophila melanogaster are repressed in some but not all cells. This silencing is inherited clonally, resulting in a mosaic phenotype that is referred to as position effect variegation (PEV; for review, see Henikoff 1990). PEV has provided a tool for the identification of a number of suppressors or enhancers of variegation that exhibit dosage effects. As a result, it has been proposed that heterochromatin involves the nucleation of multimeric protein complexes that can then spread into adjacent euchromatic regions (Locke et al. 1988).However, despite the identification of a variety of transacting factors that affect PEV (for review, see Weiler and Wakimoto 1995), the molecular basis for heterochromatin formation and propagation in Drosophila has been elusive.The yeasts also have chromosomal regions with feaPresent addresses: 1Lehrstuhl fiir Zellbiologie und Pfalanzenphysiologie,

Telomeric genes and the HM loci in saccharomyces cerevisiae are transcriptionally repressed and adopt a heterochromatin-like structure. The trans-acting factors RAP1, SIR3 and SIR4 are required for telomeric and HM silencing, and are thought to be chromosomal, but how they contribute to histone-dependent repression of adjacent chromatin is unclear. SIR3 suppresses silencing defects in histones, is limiting for silencing adjacent to telomeres, and interacts with the H3 and H4 amino termini in vitro. Here we show that SIR3 co-immunoprecipitates SIR4, RAP1 and histones from cellular extracts, suggesting the presence of large chromatin-associated protein complexes. Crosslinking experiments show that SIR3 is present at HMRa, HMLalpha and telomeres in vivo, and that is spreads from telomeric regions into adjacent chromatin when overexpressed. Thus SIR3 is a structural component of yeast heterochromatin, repressing adjacent genes as it spreads along the chromosome.

Wnt growth factors regulate a variety of developmental processes by altering specific gene expression patterns. In vertebrates β‐catenin acts as transcriptional activator, which is needed to overcome target gene repression by Groucho/TLE proteins, and to permit promoter activation as the final consequence of Wnt signaling. However, the molecular mechanisms of transcriptional activation by β‐catenin are only poorly understood. Here we demonstrate that the closely related acetyltransferases p300 and CBP potentiate β‐catenin‐mediated activation of the siamois promoter, a known Wnt target. β‐catenin and p300 also synergize to stimulate a synthetic reporter gene construct, whereas activation of the cyclin D1 promoter by β‐catenin is refractory to p300 stimulation. Axis formation and activation of the β‐catenin target genes siamois and Xnr‐3 in Xenopus embryos are sensitive to the E1A oncoprotein, a known inhibitor of p300/CBP. The C‐terminus of β‐catenin interacts directly with a region overlapping the CH‐3 domain of p300. p300 could participate in alleviating promoter repression imposed by chromatin structure and in recruiting the basal transcription machinery to promoters of particular Wnt target genes.

Signal transduction within the canonical Wnt/␤-catenin pathway drives development and carcinogenesis through programmed or unprogrammed changes in gene transcription. Although the upstream events linked to signal-induced activation of ␤-catenin in the cytoplasm have been deciphered in considerable detail, much less is known regarding the mechanism by which ␤-catenin stimulates target gene transcription in the nucleus. Here, we show that ␤-catenin physically and functionally targets the MED12 subunit in Mediator to activate transcription. The ␤-catenin transactivation domain bound directly to isolated MED12 and intact Mediator both in vitro and in vivo, and Mediator was recruited to Wnt-responsive genes in a ␤-catenin-dependent manner. Disruption of the ␤-catenin/MED12 interaction through dominant-negative interference-or RNA interference-mediated MED12 suppression inhibited ␤-catenin transactivation in response to Wnt signaling. This study thus identifies the MED12 interface within Mediator as a new component and a potential therapeutic target in the Wnt/␤-catenin pathway.