Measuring the buffering capacity of gene silencing in Saccharomyces cerevisiae

Gene silencing in budding yeast is mediated by Sir protein binding to unacetylated nucleosomes to form a chromatin structure that inhibits transcription. Transcriptional silencing is characterized by the high-fidelity transmission of the silent state. Despite its relative stability, the constituent parts of the silent state are in constant flux, giving rise to a model that silent loci can tolerate such fluctuations without functional consequences. However, the level of tolerance is unknown, and we developed methods to measure the threshold of histone acetylation that causes the silent chromatin state to switch to the active state as well as to measure the levels of the enzymes and structural proteins necessary for silencing. We show that loss of silencing required 50 to 75% acetyl-mimic histones, though the precise levels were influenced by silencer strength and upstream activating sequence (UAS) enhancer/promoter strength. Measurements of repressor protein levels necessary for silencing showed that reducing SIR4 gene dosage two- to threefold significantly weakened silencing, though reducing the gene copy numbers for Sir2 or Sir3 to the same extent did not significantly affect silencing suggesting that Sir4 was a limiting component in gene silencing. Calculations suggest that a mere twofold reduction in the ability of acetyltransferases to acetylate nucleosomes across a large array of nucleosomes may be sufficient to generate a transcriptionally silent domain.

A Role for Mediator Core in Limiting Coactivator Recruitment in Saccharomyces cerevisiae

Mediator is an essential, multisubunit complex that functions as a transcriptional coactivator in yeast and other eukaryotic organisms. Mediator has four conserved modules, Head, Middle, Tail, and Kinase, and has been implicated in nearly all aspects of gene regulation. The Tail module has been shown to recruit the Mediator complex to the enhancer or upstream activating sequence (UAS) regions of genes via interactions with transcription factors, and the Kinase module facilitates the transition of Mediator from the UAS/enhancer to the preinitiation complex via protein phosphorylation. Here, we analyze expression of the Saccharomyces cerevisiae HO gene using a sin4 Mediator Tail mutation that separates the Tail module from the rest of the complex; the sin4 mutation permits independent recruitment of the Tail module to promoters without the rest of Mediator. Significant increases in recruitment of the SWI/SNF and SAGA coactivators to the HO promoter UAS were observed in a sin4 mutant, along with increased gene activation. These results are consistent with recent studies that have suggested that the Kinase module functions negatively to inhibit activation by the Tail. However, we found that Kinase module mutations did not mimic the effect of a sin4 mutation on HO expression. This suggests that at HO the core Mediator complex (Middle and Head modules) must play a role in limiting Tail binding to the promoter UAS and gene activation. We propose that the core Mediator complex helps modulate Mediator binding to the UAS regions of genes to limit coactivator recruitment and ensure proper regulation of gene transcription.

Regulation of bistability in the std fimbrial operon of Salmonella enterica by DNA adenine methylation and transcription factors HdfR, StdE and StdF

Bistable expression of the Salmonella enterica std operon is controlled by an AND logic gate involving three transcriptional activators: the LysR-type factor HdfR and the StdE and StdF regulators encoded by the std operon itself. StdE activates transcription of the hdfR gene, and StdF activates std transcription together with HdfR. Binding of HdfR upstream of the std promoter is hindered by methylation of GATC sites located within the upstream activating sequence (UAS). Epigenetic control by Dam methylation thus antagonizes formation of the StdE-StdF-HdfR loop and tilts the std switch toward the StdOFF state. In turn, HdfR binding hinders methylation of the UAS, permitting activation of the StdE-StdF-HdfR loop and concomitant formation of StdON cells. Bistability is thus the outcome of competition between DNA adenine methylation and the StdE-StdF-HdfR activator loop.

Mss11, a Transcriptional Activator, Is Required for Hyphal Development in Candida albicans

ABSTRACT Candida albicans undergoes a morphological transition from yeast to hyphae in response to a variety of stimuli and growth conditions. We previously isolated a LisH domain containing transcription factor Flo8, which is essential for hyphal development in C. albicans. To search the putative binding partner of Flo8 in C. albicans, we identified C. albicans Mss11, a functional homolog of Saccharomyces cerevisiae Mss11, which also contains a LisH motif at its N terminus. C. albicans Mss11 can interact with Flo8 via the LisH motif by in vivo coimmunoprecipitation. The results of a chromatin immunoprecipitation (ChIP) assay showed that more Mss11 and Flo8 proteins bound to the upstream activating sequence region of HWP1 promoter in hyphal cells than in yeast cells, and the increased binding of each of these two proteins responding to hyphal induction was dependent on the other. Overexpression of MSS11 enhanced filamentous growth. Deletion of MSS11 caused a profound defect in hyphal development and the induction of hypha-specific genes. Our data suggest that Mss11 functions as an activator in hyphal development of C. albicans. Furthermore, overexpression of FLO8 can bypass the requirement of Mss11 in filamentous formation, whereas overexpression of MSS11 failed to promote hyphae growth in flo8 mutants. In summary, we show that the expression level of MSS11 increases during hyphal induction, and the enhanced expression of MSS11 may contribute to cooperative binding of Mss11 and Flo8 to the HWP1 promoter.

Targeted gain-of-function screening inDrosophilausingGAL4-UASand random transposon insertions

SummaryAlterations in the activity level or temporal expression of key signalling genes elicit profound patterning effects during development. Consequently, gain-of-function genetic schemes that overexpress or misexpress such loci can identify novel candidates for functions essential for a developmental process.GAL4-Upstream Activating Sequence(UAS)-targeted regulation of gene expression inDrosophilahas allowed rapid analyses of coding sequences for potential roles in specific tissues at particular developmental stages.GAL4has also been combined with randomly mobilized transposons capable ofUAS-directed misexpression or overexpression of flanking sequences. This combination has produced a genetic screening system that can uncover novel loci refractory to standard loss of function genetic approaches, such as redundant genes. Available libraries of strains with sequenced insertion sites can allow direct correlation of phenotypes to genetic function. These techniques have also been applied to genetic interaction screening, where aGAL4driver andUAS-regulated insertion collection are combined with an extant mutant genotype. In this article, we summarize studies that have utilizedGAL4-UASoverexpression or misexpression of random loci to screen for candidates involved in specific developmental processes.

Regulation of phospholipid synthesis in yeast by zinc

The yeast Saccharomyces cerevisiae has the ability to cope with a variety of stress conditions (e.g. zinc deficiency) by regulating the expression of enzyme activities including those involved with phospholipid synthesis. Zinc is an essential mineral required for the growth and metabolism of S. cerevisiae. Depletion of zinc from the growth medium of wild-type cells results in alterations in phospholipid composition including an increase in PI (phosphatidylinositol) and a decrease in phosphatidylethanolamine. These changes can be attributed to an increase in PIS1-encoded PI synthase activity and a decrease in the activities of several CDP-diacylglycerol pathway enzymes including the CHO1-encoded PS (phosphatidylserine) synthase. The reduction in PS synthase in response to zinc depletion is due to a repression mechanism that involves the UASINO (inositol upstream activating sequence) element in the CHO1 promoter and the negative transcription factor Opi1p. These factors are also responsible for the inositol-mediated repression of CHO1. This regulation may play an important role in allowing cells to adapt to zinc deficiency given the essential roles that phospholipids play in the structure and function of cellular membranes.

Global Regulation by the Yeast Spt10 Protein Is Mediated through Chromatin Structure and the Histone Upstream Activating Sequence Elements

ABSTRACT The yeast SPT10 gene encodes a putative histone acetyltransferase (HAT) implicated as a global transcription regulator acting through basal promoters. Here we address the mechanism of this global regulation. Although microarray analysis confirmed that Spt10p is a global regulator, Spt10p was not detected at any of the most strongly affected genes in vivo. In contrast, the presence of Spt10p at the core histone gene promoters in vivo was confirmed. Since Spt10p activates the core histone genes, a shortage of histones could occur in spt10Δ cells, resulting in defective chromatin structure and a consequent activation of basal promoters. Consistent with this hypothesis, the spt10Δ phenotype can be rescued by extra copies of the histone genes and chromatin is poorly assembled in spt10Δ cells, as shown by irregular nucleosome spacing and reduced negative supercoiling of the endogenous 2μm plasmid. Furthermore, Spt10p binds specifically and highly cooperatively to pairs of upstream activating sequence elements in the core histone promoters [consensus sequence, (G/A)TTCCN6TTCNC], consistent with a direct role in histone gene regulation. No other high-affinity sites are predicted in the yeast genome. Thus, Spt10p is a sequence-specific activator of the histone genes, possessing a DNA-binding domain fused to a likely HAT domain.

The seven E. coli ribosomal RNA operon upstream regulatory regions differ in structure and transcription factor binding efficiencies

Ribosomal RNAs inE. coliare transcribed from seven operons, which are highly conserved in their organization and sequence. However, the upstream regulatory DNA regions differ considerably, suggesting differences in regulation. We have therefore analyzed the conformation of all seven DNA elements located upstream of the majorE. colirRNA P1 promoters. As judged by temperature-dependent gel electrophoresis with isolated DNA fragments comprising the individual P1 promoters and the complete upstream regulatory regions, all seven rRNA upstream sequences are intrinsically curved. The degree of intrinsic curvature was highest for therrnBandrrnDfragments and less pronounced for therrnAandrrnEoperons. Comparison of the experimentally determined differences in curvature with programs for the prediction of DNA conformation revealed a generally high degree of conformity. Moreover, the analysis showed that the center of curvature is located at about the same position in all fragments. The different upstream regions were analyzed for their capacity to bind the transcription factors FIS and H-NS, which are known as antagonists in the regulation of rRNA synthesis. Gel retardation experiments revealed that both proteins interact with the upstream promoter regions of all seven rDNA fragments, with the affinities of the different DNA fragments for FIS and H-NS and the structure of the resulting complexes deviating considerably. FIS binding was non-cooperative, and at comparable protein concentrations the occupancy of the different DNA fragments varied between two and four binding sites. In contrast, H-NS was shown to bind cooperatively and intermediate states of occupancy could not be resolved for each fragment. The different gel electrophoretic mobilities of the individual DNA/protein complexes indicate variable structures and topologies of the upstream activating sequence regulatory complexes. Our results are highly suggestive of differential regulation of the individual rRNA operons.

Evidence that the Elongation Factor TFIIS Plays a Role in Transcription Initiation at GAL1 in Saccharomyces cerevisiae

ABSTRACT TFIIS is a transcription elongation factor that has been extensively studied biochemically. Although the in vitro mechanisms by which TFIIS stimulates RNA transcript cleavage and polymerase read-through have been well characterized, its in vivo roles remain unclear. To better understand TFIIS function in vivo, we have examined its role during Gal4-mediated activation of the Saccharomyces cerevisiae GAL1 gene. Surprisingly, TFIIS is strongly associated with the GAL1 upstream activating sequence. In addition, TFIIS recruitment to Gal4-binding sites is dependent on Gal4, SAGA, and Mediator but not on RNA polymerase II (Pol II). The association of TFIIS is also necessary for the optimal recruitment of TATA-binding protein and Pol II to the GAL1 promoter. These results provide strong evidence that TFIIS plays an important role in the initiation of transcription at GAL1 in addition to its well-characterized roles in transcription elongation.