Transcriptionally silent chromatin localizes to the nuclear periphery, which provides a special microenvironment for gene repression. A variety of nuclear membrane proteins interact with repressed chromatin, yet the functional role of these interactions remains poorly understood. Here, we show that, in Schizosaccharomyces pombe, the nuclear membrane protein Lem2 associates with chromatin and mediates silencing and heterochromatin localization. Unexpectedly, we found that these functions can be separated and assigned to different structural domains within Lem2, excluding a simple tethering mechanism. Chromatin association and tethering of centromeres to the periphery are mediated by the N-terminal LEM (LAP2-Emerin-MAN1) domain of Lem2, whereas telomere anchoring and heterochromatin silencing require exclusively its conserved C-terminal MSC (MAN1-Src1 C-terminal) domain. Particularly, silencing by Lem2 is epistatic with the Snf2/HDAC (histone deacetylase) repressor complex SHREC at telomeres, while its necessity can be bypassed by deleting Epe1, a JmjC protein with anti-silencing activity. Furthermore, we found that loss of Lem2 reduces heterochromatin association of SHREC, which is accompanied by increased binding of Epe1. This reveals a critical function of Lem2 in coordinating these antagonistic factors at heterochromatin. The distinct silencing and localization functions mediated by Lem2 suggest that these conserved LEM-containing proteins go beyond simple tethering to play active roles in perinuclear silencing.

In nature, Saccharomyces yeasts manifest a number of adaptive responses to overcome adverse environments such as filamentation, invasive growth, flocculation and adherence to solid surfaces. Certain Saccharomyces wild yeasts, namely ''flor yeasts,'' have also acquired the ability to form a buoyant biofilm at the broth surface. Here we report that mutations in a single gene, identified as FLO11, separate these ''floating'' yeasts from their nonfloating relatives. We have determined that the capability to form a self-supporting biofilm at the liquid surface is largely dependent on two changes in the FLO11 gene. First, we identified a 111-nt deletion within a repression region of the FLO11 promoter that significantly increases FLO11 gene expression. Secondly, we found rearrangements within the central tandem repeat domain of the coding region that yield a more hydrophobic Flo11p variant. Together, these mutations result in dramatic increase in cell surface hydrophobicity, which in turn confers these yeasts the ability to float by surface tension, an adaptive mechanism to gain direct access to oxygen within oxygen-poor liquid environments.adaptive mechanism ͉ buoyant biofilm ͉ yeast hydrophobicity

Adhesins play a central role in the cellular response of eukaryotic microorganisms to their host environment. In pathogens such as Candida spp. and other fungi, adhesins are responsible for adherence to mammalian tissues, and in Saccharomyces spp. yeasts also confer adherence to solid surfaces and to other yeast cells. The analysis of FLO11, the main adhesin identified in Saccharomyces cerevisiae, has revealed complex mechanisms, involving both genetic and epigenetic regulation, governing the expression of this critical gene. We designed a genomewide screen to identify new regulators of this pivotal adhesin in budding yeasts. We took advantage of a specific FLO11 allele that confers very high levels of FLO11 expression to wild ''flor'' strains of S. cerevisiae. We screened for mutants that abrogated the increased FLO11 expression of this allele using the loss of the characteristic fluffy-colony phenotype and a reporter plasmid containing GFP controlled by the same FLO11 promoter. Using this approach, we isolated several genes whose function was essential to maintain the expression of FLO11. In addition to previously characterized activators, we identified a number of novel FLO11 activators, which reveal the pH response pathway and chromatin-remodeling complexes as central elements involved in FLO11 activation.

Heterochromatin plays important roles in eukaryotic genome regulation. However, the repressive nature of heterochromatin combined with its propensity to self-propagate necessitates robust mechanisms to contain heterochromatin within defined boundaries and thus prevent silencing of expressed genes. Here we show that loss of the PAF complex (PAFc) component Leo1 compromises chromatin boundaries, resulting in invasion of heterochromatin into flanking euchromatin domains. Similar effects are seen upon deletion of other PAFc components, but not other factors with related functions in transcription-associated chromatin modification, indicating a specific role for PAFc in heterochromatin regulation. Loss of Leo1 results in reduced levels of H4K16 acetylation at boundary regions, while tethering of the H4K16 acetyltransferase Mst1 to boundary chromatin suppresses heterochromatin spreading in leo1Δ cells, suggesting that Leo1 antagonises heterochromatin spreading by promoting H4K16 acetylation. Our findings reveal a previously undescribed role for PAFc in regulating global heterochromatin distribution.

In Saccharomyces yeasts, the FLO11 gene encodes an adhesin involved in filamentation, invasive growth, flocculation and adherence to solid surfaces. In wild Saccharomyces flor yeasts, a particularly expanded FLO11 allele also confers to these yeasts the ability to float under stressing liquid environments. We report here that, under optimal laboratory conditions, the repeats domain of the FLO11 gene in these wild yeasts is extremely unstable. Changes in length in the FLO11 coding repeats domain affected Flo11p-associated functions but, interestingly, some of these functions were affected more than others. Therefore, length variations in this single gene provide a combinatorial diversity, which may contribute to a very rapid adaptation to fluctuating environments. Functional analysis of contracted alleles indicated that buoyancy was not associated to FLO11 length. In contrast, this property depended on the different types of repetitive units found in this gene. Thus, not only variations in the number of intragenic repeats but also the abundance and/or distribution of the different repetitive units may have phenotypic and evolutionary implications.

Fungal pathogenesis depends on accurate secretion and location of virulence factors which drive host colonization. Protein glycosylation is a common posttranslational modification of cell wall components and other secreted factors, typically required for correct protein localization, secretion and function. Thus, the absence of glycosylation is associated with animal and plant pathogen avirulence. While the relevance of protein glycosylation for pathogenesis has been well established, the main glycoproteins responsible for the loss of virulence observed in glycosylation-defective fungi have not been identified. Here, we devise a proteomics approach to identify such proteins and use it to demonstrate a role for the highly conserved protein disulfide isomerase Pdi1 in virulence. We show that efficient Pdi1 N-glycosylation, which promotes folding into the correct protein conformation, is required for full pathogenic development of the corn smut fungus Ustilago maydis. Remarkably, the observed virulence defects are reminiscent of those seen in glycosylation-defective cells suggesting that the N-glycosylation of Pdi1 is necessary for the full secretion of virulence factors. All these observations, together with the fact that Pdi1 protein and RNA expression levels rise upon virulence program induction, suggest that Pdi1 glycosylation is important for normal pathogenic development in U. maydis. Our results provide new insights into the role of glycosylation in fungal pathogenesis.

Protection of euchromatin from invasion by gene-repressive heterochromatin is critical for cellular health and viability. In addition to constitutive loci such as pericentromeres and subtelomeres, heterochromatin can be found interspersed in gene-rich euchromatin, where it regulates gene expression pertinent to cell fate. While heterochromatin and euchromatin are globally poised for mutual antagonism, the mechanisms underlying precise spatial encoding of heterochromatin containment within euchromatic sites remain opaque. We investigated ectopic heterochromatin invasion by manipulating the fission yeast mating type locus boundary using a single-cell spreading reporter system. We found that heterochromatin repulsion is locally encoded by Set1/COMPASS on certain actively transcribed genes and that this protective role is most prominent at heterochromatin islands, small domains interspersed in euchromatin that regulate cell fate specifiers. Sensitivity to invasion by heterochromatin, surprisingly, is not dependent on Set1 altering overall gene expression levels. Rather, the gene-protective effect is strictly dependent on Set1's catalytic activity. H3K4 methylation, the Set1 product, antagonizes spreading in two ways: directly inhibiting catalysis by Suv39/Clr4 and locally disrupting nucleosome stability. Taken together, these results describe a mechanism for spatial encoding of euchromatic signals that repel heterochromatin invasion.

Cell adhesion and biofilm formation are critical processes in the pathogenicity of fungi and are mediated through a family of adhesin proteins conserved throughout yeasts and fungi. In Saccharomyces cerevisiae, Flo11 is the main adhesin involved in cell adhesion and biofilm formation, making the study of its function and regulation in this nonpathogenic budding yeast highly relevant. The S. cerevisiae FLO11 gene is driven by a TATA-box-containing promoter that is regulated through one of the longest regulatory upstream regions (3 kb) in yeast. We reported recently that two chromatin cofactor complexes, the Rpd3L deacetylase and the Swi/Snf chromatin-remodeling complexes, contribute significantly to the regulation of FLO11. Here, we analyze directly how these complexes impact on FLO11 promoter chromatin structure and dissect further the interplay between histone deacetylases, chromatin remodeling, and the transcriptional repressor Sfl1. We show that the regulation of chromatin structure represents an important layer of control in the highly complex regulation of the FLO11 promoter.