TOF1 and RRM3 reveal a link between gene silencing and the pausing of replication forks.

Eukaryotic DNA replication is accompanied by the disassembly and reassembly of nucleosomes and the transmission of epigenetic marks to the newly assembled chromatids. Several histone chaperones, including CAF-1 and Asf1p, are central to these processes. On the other hand, replication forks pause at numerous positions throughout the genome, but it is not known if and how this pausing affects the reassembly and maintenance of chromatin structures. Here, we applied drug-free gene silencing assays to analyze the genetic interactions between CAC1, ASF1, and two genes that regulate the stability of the paused replisome (TOF1) and the resumption of elongation (RRM3). Our results show that TOF1 and RRM3 differentially interact with CAF-1 and ASF1 and that the deletions of TOF1 and RRM3 lead to reduced silencing and increased frequency of epigenetic conversions at three loci in the genome of S. cerevisiae. Our study adds details to the known activities of CAF-1 and Asf1p and suggests that the pausing of the replication fork can lead to epigenetic instability.

Evaluation of drug-free methods for the detection of gene silencing in Saccharomyces cerevisiae.

Multiple studies in Saccharomyces cerevisiae have measured the levels of gene silencing by inserting the URA3 gene at various loci and selecting against URA3-expressing cells by 5-flouroorotic acid (5-FOA). However, 5-FOA affects the cellular pools of dNTPs and can produce side effects. To circumvent this issue, we and others have introduced drug-free techniques to detect silent and active gene states. In this study, we compared three drug-free methods based on the expression of fluorescent reporters in the VIIL telomere of S. cerevisiae. Our results point out that only one of these methods is suitable for large-scale drug-free analyses of gene silencing.

Dbf4-Dependent Kinase: DDK-ated to post-initiation events in DNA replication.

Dbf4-Dependent Kinase (DDK) has a well-established essential role at origins of DNA replication, where it phosphorylates and activates the replicative MCM helicase. It also acts in the response to mutagens and in DNA repair as well as in key steps during meiosis. Recent studies have indicated that, in addition to the MCM helicase, DDK phosphorylates several substrates during the elongation stage of DNA replication or upon replication stress. However, these activities of DDK are not essential for viability. Dbf4-Dependent Kinase is also emerging as a key factor in the regulation of genome-wide origin firing and in replication-coupled chromatin assembly. In this review, we summarize recent progress in our understanding of the diverse roles of DDK.

Variation, Variegation and Heritable Gene Repression in S. cerevisiae.

Phenotypic heterogeneity provides growth advantages for a population upon changes of the environment. In S. cerevisiae, such heterogeneity has been observed as "on/off" states in the expression of individual genes in individual cells. These variations can persist for a limited or extended number of mitotic divisions. Such traits are known to be mediated by heritable chromatin structures, by the mitotic transmission of transcription factors involved in gene regulatory circuits or by the cytoplasmic partition of prions or other unstructured proteins. The significance of such epigenetic diversity is obvious, however, we have limited insight into the mechanisms that generate it. In this review, we summarize the current knowledge of epigenetically maintained heterogeneity of gene expression and point out similarities and converging points between different mechanisms. We discuss how the sharing of limiting repression or activation factors can contribute to cell-to-cell variations in gene expression and to the coordination between short- and long- term epigenetic strategies. Finally, we discuss the implications of such variations and strategies in adaptation and aging.

Gene repression in S. cerevisiae-looking beyond Sir-dependent gene silencing.

Gene silencing by the SIR (Silent Information Region) family of proteins in S. cerevisiae has been extensively studied and has served as a founding paradigm for our general understanding of gene repression and its links to histone deacetylation and chromatin structure. In recent years, our understanding of other mechanisms of gene repression in S.cerevisiae was significantly advanced. In this review, we focus on such Sir-independent mechanisms of gene repression executed by various Histone Deacetylases (HDACs) and Histone Methyl Transferases (HMTs). We focus on the genes regulated by these enzymes and their known mechanisms of action. We describe the cooperation and redundancy between HDACs and HMTs, and their involvement in gene repression by non-coding RNAs or by their non-histone substrates. We also propose models of epigenetic transmission of the chromatin structures produced by these enzymes and discuss these in the context of gene repression phenomena in other organisms. These include the recycling of the epigenetic marks imposed by HMTs or the recycling of the complexes harboring HDACs.

Dysfunctional CAF-I reveals its role in cell cycle progression and differential regulation of gene silencing.

Chromatin Assembly Factor I (CAF-I) plays a central role in the reassembly of H3/H4 histones during DNA replication. In S. cerevisiae CAF-I is not essential and its loss is associated with reduced gene silencing at telomeres and increased sensitivity to DNA damage. Two kinases, Cyclin Dependent Kinase (CDK) and Dbf4-Dependent Kinase (DDK), are known to phosphorylate the Cac1p subunit of CAF-I, but their role in the regulation of CAF-I activity is not well understood. In this study we systematically mutated the phosphorylation target sites of these kinases. We show that concomitant mutations of the CDK and DDK target sites of Cac1p lead to growth retardation and significant cell cycle defects, altered cell morphology and increased sensitivity to DNA damage. Surprisingly, some mutations also produced flocculation, a phenotype that is lost in most laboratory strains, and displayed elevated expression of FLO genes. None of these effects is observed upon the destruction of CAF-I. In contrast, the mutations that caused flocculation did not affect gene silencing at the mating type and subtelomeric loci. We conclude that dysfunctional CAF-I produces severe phenotypes, which reveal a possible role of CAF-I in the coordination of DNA replication, chromatin reassembly and cell cycle progression. Our study highlights the role of phosphorylation of Cac1p by CDK and a putative role for DDK in the transmission and re-assembly of chromatin during DNA replication.

Histone chaperones and the Rrm3p helicase regulate flocculation in S. cerevisiae.

BACKGROUND: Biofilm formation or flocculation is a major phenotype in wild type budding yeasts but rarely seen in laboratory yeast strains. Here, we analysed flocculation phenotypes and the expression of FLO genes in laboratory strains with various genetic backgrounds. RESULTS: We show that mutations in histone chaperones, the helicase RRM3 and the Histone Deacetylase HDA1 de-repress the FLO genes and partially reconstitute flocculation. We demonstrate that the loss of repression correlates to elevated expression of several FLO genes, to increased acetylation of histones at the promoter of FLO1 and to variegated expression of FLO11. We show that these effects are related to the activity of CAF-1 at the replication forks. We also demonstrate that nitrogen starvation or inhibition of histone deacetylases do not produce flocculation in W303 and BY4742 strains but do so in strains compromised for chromatin maintenance. Finally, we correlate the de-repression of FLO genes to the loss of silencing at the subtelomeric and mating type gene loci. CONCLUSIONS: We conclude that the deregulation of chromatin maintenance and transmission is sufficient to reconstitute flocculation in laboratory yeast strains. Consequently, we propose that a gain in epigenetic silencing is a major contributing factor for the loss of flocculation phenotypes in these strains. We suggest that flocculation in yeasts provides an excellent model for addressing the challenging issue of how epigenetic mechanisms contribute to evolution.

Cellular immune responses to recombinant Mycobacterium bovis BCG constructs expressing major antigens of region of difference 1 of Mycobacterium tuberculosis.

Besides being the most widely used vaccine directed against tuberculosis (TB) worldwide, Mycobacterium bovis BCG is also the most controversial vaccine in current use. Its protective efficacy varies widely in different parts of the world. One approach to improving the current BCG vaccine might be to produce recombinant BCG strains that express major antigens encoded by genes that are present in the M. tuberculosis-specific region of difference 1 (RD1), such as pe35, cfp10, and esat6. In this study, pe35, cfp10, and esat6 genes were cloned into shuttle plasmid pDE22 to generate the recombinant plasmids PDE22-PE35, PDE22-CFP10, and PDE22-ESAT6, which were electroporated into BCG to generate recombinant BCGs (rBCGs). The cellular immune responses (antigen-induced proliferation and secretion of selected T helper 1 [Th1], Th2, and anti-inflammatory cytokines, i.e., gamma interferon [IFN-γ], interleukin 5 [IL-5], and IL-10, respectively) that are specific to the proteins of cloned genes were studied by using spleen cells from mice immunized with native BCGs and rBCGs and synthetic peptides covering the protein sequence of the cloned genes. The results showed that the spleen cells did not secrete IL-5, whereas IL-10 was secreted in response to peptides of all three proteins from mice immunized with rBCGs only, suggesting expression of the cloned genes and in vivo priming of spleen cells to the expressed proteins. However, in Th1 cell assays that correlate with protective cellular immune responses, i.e., antigen-induced proliferation and IFN-γ secretion, only mice immunized with rBCG-pDE22-PE35 yielded positive responses to the peptides of PE35. These results suggest that rBCG-PDE22-PE35 is the only one of the three vaccines used in this work that is worthy of consideration as a new vaccine candidate against TB.