[Abstract unavailable

Most studies of eukaryotic gene regulation have been done looking at mature mRNA levels. Nevertheless, the steady-state mRNA level is the result of two opposing factors: transcription rate (TR) and mRNA degradation. Both can be important points to regulate gene expression. Here we show a new method that combines the use of nylon macroarrays and in vivo radioactive labeling of nascent RNA to quantify TRs, mRNA levels, and mRNA stabilities for all the S. cerevisiae genes. We found that during the shift from glucose to galactose, most genes undergo drastic changes in TR and mRNA stability. However, changes in mRNA levels are less pronounced. Some genes, such as those encoding mitochondrial proteins, are coordinately regulated in mRNA stability behaving as decay regulons. These results indicate that, although TR is the main determinant of mRNA abundance in yeast, modulation of mRNA stability is a key factor for gene regulation.

Chromatin remodeling can facilitate the recruitment of RNA polymerase II (Pol II) to targeted promoters, as well as enhancing the level of transcription. Here, we describe a further key role for chromatin remodeling in transcriptional termination. Using a genetic screen in S. pombe, we identified the CHD-Mi2 class chromatin remodeling ATPase, Hrp1, as a termination factor. In S. cerevisiae, we show that transcriptional termination and chromatin structure at the 3' ends of three genes all depend on the activity of the Hrp1 homolog, Chd1p, either alone or redundantly with the ISWI ATPases, Isw1p, and Isw2p. We suggest that chromatin remodeling of termination regions is a necessary prelude to efficient Pol II termination.

The nucleolus is the site of ribosome synthesis in the nucleus, whose integrity is essential. Epigenetic mechanisms are thought to regulate the activity of the ribosomal RNA (rRNA) gene copies, which are part of the nucleolus. Here we show that human cells lacking DNA methyltransferase 1 (Dnmt1), but not Dnmt33b, have a loss of DNA methylation and an increase in the acetylation level of lysine 16 histone H4 at the rRNA genes. Interestingly, we observed that SirT1, a NAD+-dependent histone deacetylase with a preference for lysine 16 H4, interacts with Dnmt1; and SirT1 recruitment to the rRNA genes is abrogated in Dnmt1 knockout cells. The DNA methylation and chromatin changes at ribosomal DNA observed are associated with a structurally disorganized nucleolus, which is fragmented into small nuclear masses. Prominent nucleolar proteins, such as Fibrillarin and Ki-67, and the rRNA genes are scattered throughout the nucleus in Dnmt1 deficient cells. These findings suggest a role for Dnmt1 as an epigenetic caretaker for the maintenance of nucleolar structure.

BackgroundYeast viability and vitality are essential for different industrial processes where the yeast Saccharomyces cerevisiae is used as a biotechnological tool. Therefore, the decline of yeast biological functions during aging may compromise their successful biotechnological use. Life span is controlled by a variety of molecular mechanisms, many of which are connected to stress tolerance and genomic stability, although the metabolic status of a cell has proven a main factor affecting its longevity. Acetic acid and ethanol accumulation shorten chronological life span (CLS), while glycerol extends it.ResultsDifferent age-related gene classes have been modified by deletion or overexpression to test their role in longevity and metabolism. Overexpression of histone deacetylase SIR2 extends CLS and reduces acetate production, while overexpression of SIR2 homolog HST3 shortens CLS, increases the ethanol level, and reduces acetic acid production. HST3 overexpression also enhances ethanol tolerance. Increasing tolerance to oxidative stress by superoxide dismutase SOD2 overexpression has only a moderate positive effect on CLS. CLS during grape juice fermentation has also been studied for mutants on several mRNA binding proteins that are regulators of gene expression at the posttranscriptional level; we found that NGR1 and UTH4 deletions decrease CLS, while PUF3 and PUB1 deletions increase it. Besides, the pub1Δ mutation increases glycerol production and blocks stress granule formation during grape juice fermentation. Surprisingly, factors relating to apoptosis, such as caspase Yca1 or apoptosis-inducing factor Aif1, play a positive role in yeast longevity during winemaking as their deletions shorten CLS.ConclusionsManipulation of regulators of gene expression at both transcriptional (i.e., sirtuins) and posttranscriptional (i.e., mRNA binding protein Pub1) levels allows to modulate yeast life span during its biotechnological use. Due to links between aging and metabolism, it also influences the production profile of metabolites of industrial relevance.

Acetaldehyde is a toxic compound produced by Saccharomyces cerevisiae cells under several growth conditions. The adverse effects of this molecule are important, as significant amounts accumulate inside the cells. By means of global gene expression analyses, we have detected the effects of acetaldehyde addition in the expression of about 400 genes. Repressed genes include many genes involved in cell cycle control, cell polarity, and the mitochondrial protein biosynthesis machinery. Increased expression is displayed in many stress response genes, as well as other families of genes, such as those encoding vitamin B1 biosynthesis machinery and proteins for aryl alcohol metabolism. The induction of genes involved in sulfur metabolism is dependent on Met4p and other well-known factors involved in the transcription of MET genes under nonrepressing conditions of sulfur metabolism. Moreover, the deletion of MET4 leads to increased acetaldehyde sensitivity. TPO genes encoding polyamine transporters are also induced by acetaldehyde; in this case, the regulation is dependent on the Haa1p transcription factor. In this paper, we discuss the connections between acetaldehyde and the processes affected by this compound in yeast cells with reference to the microarray data.

One of the stress conditions that yeast may encounter is the presence of acetaldehyde. In a previous study we identified that, in response to this stress, several HSP genes are induced that are also involved in the response to other forms of stress. Aldehyde dehydrogenases (ALDH) play an important role in yeast acetaldehyde metabolism (e.g. when cells are growing in ethanol). In this work we analyse the expression of the genes encoding these enzymes (ALD) and also the corresponding enzymatic activities under several growth conditions. We investigate three kinds of yeast strains: laboratory strains, strains involved in the alcoholic fermentation stage of wine production and flor yeasts (responsible for the biological ageing of sherry wines). The latter are very important to consider because they grow in media containing high ethanol concentrations, and produce important amounts of acetaldehyde. Under several growth conditions, further addition of acetaldehyde or ethanol in flor yeasts induced the expression of some ALD genes and led to an increase in ALDH activity. This result is consistent with their need to obtain energy from ethanol during biological ageing processes. Our data also suggest that post-transcriptional and/or post-translational mechanisms are involved in regulating the activity of these enzymes. Finally, analyses indicate that the Msn2/4p and Hsf1p transcription factors are necessary for HSP26, ALD2/3 and ALD4 gene expression under acetaldehyde stress, while PKA represses the expression of these genes.

Transcriptional termination of the GAL10 gene in Saccharomyces cerevisiae depends on the efficiency of polyadenylation. Either cis mutations in the poly(A) signal or trans mutations of mRNA 3 end cleavage factors result in GAL10 read-through transcripts into the adjacent GAL7 gene and inactivation (occlusion) of the GAL7 promoter. Herein, we present a molecular explanation of this transcriptional interference phenomenon. In vivo footprinting data reveal that GAL7 promoter occlusion is associated with the displacement of Gal4p transcription factors from the promoter. Interestingly, overexpression of Gal4p restores promoter occupancy, activates GAL7 expression, and rescues growth on the otherwise toxic galactose substrate. Our data therefore demonstrate a precise balance between transcriptional interference and initiation.T ranscriptional termination of RNA polymerase II (Pol II) is linked to mRNA 3Ј processing, which is a two-step reaction consisting of endonucleolytic cleavage of the RNA precursor and subsequent polyadenylation (1). Mutation of poly(A) signals results in increased transcription beyond the poly(A) site of a gene (1) and recently has been shown to depend on the activity of 3Ј end processing factors (2). Some RNA processing factors are associated with elongating Pol II, indicating a tight link between transcription and RNA processing (3, 4). In contrast to Pol I (5) and Pol III, termination of Pol II occurs at variable, ill-defined positions downstream of the poly(A) site of a gene (1). These findings suggest that transcriptional termination can be a random process.In some instances, however, termination of transcription must occur efficiently, because enhanced transcriptional read-through can result in inhibition of an adjacent, downstream promoter and also perturbs origin of replication and centromere function (6-9). Closely spaced genes are particularly prone to promoter occlusion, especially when they are expressed at the same time, as in the case of the GAL10 and GAL7 genes of Saccharomyces cerevisiae. These two genes are separated by 600 bp and are activated simultaneously to high levels by galactose (gal). We have shown previously that deletion of the GAL10 poly(A) signal leads to enhanced GAL10 read-through transcription and formation of GAL10-7 bicistronic transcripts, which in turn results in inhibition of the downstream GAL7 promoter (ref. 10; Fig. 1A). Gal7p (gal uridyl transferase) catalyzes conversion of the metabolic intermediate gal 1-phosphate, which is otherwise toxic. Inhibition of Gal7p expression by transcriptional readthrough into the GAL7 promoter renders cells gal sensitive and results in a Gal Ϫ phenotype, emphasizing the importance of termination in this system.Transcription of the GAL genes depends on the transcription factor Gal4p, which binds to 17-bp sequence elements within the promoter regions (11, 12). The binding affinity of Gal4p for these sites depends on both the sequence and spacing between the critical outer nucleotide triplets (CGG. . . GGC), which are conta...