Charles S. Hoffman

Stepwise chromatin remodelling by a cascade of transcription initiation of non-coding RNAs

Recent transcriptome analyses using high-density tiling arrays and data from large-scale analyses of full-length complementary DNA libraries by the FANTOM3 consortium demonstrate that many transcripts are non-coding RNAs (ncRNAs). These transcriptome analyses indicate that many of the non-coding regions, previously thought to be functionally inert, are actually transcriptionally active regions with various features. Furthermore, most relatively large (∼several kilobases) polyadenylated messenger RNA transcripts are transcribed from regions harbouring little coding potential. However, the function of such ncRNAs is mostly unknown and has been a matter of debate. Here we show that RNA polymerase II (RNAPII) transcription of ncRNAs is required for chromatin remodelling at the fission yeast Schizosaccharomyces pombe fbp1+ locus during transcriptional activation. The chromatin at fbp1+ is progressively converted to an open configuration, as several species of ncRNAs are transcribed through fbp1+. This is coupled with the translocation of RNAPII through the region upstream of the eventual fbp1+ transcriptional start site. Insertion of a transcription terminator into this upstream region abolishes both the cascade of transcription of ncRNAs and the progressive chromatin alteration. Our results demonstrate that transcription through the promoter region is required to make DNA sequences accessible to transcriptional activators and to RNAPII.

Glucose sensing via the protein kinase A pathway in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe primarily detects glucose via a cAMP-signalling pathway. Components of this pathway include the Git3 G-protein-coupled receptor and a heterotrimeric G-protein, from which the Gpa2 Galpha subunit activates adenylate cyclase (Git2/Cyr1). Three additional proteins, Git1, Git7 and Git10 are required to generate a cAMP response even in a strain expressing an activated form of Gpa2, which is capable of bypassing the loss of the GPCR and Gbetagamma dimer. Therefore, Git1, Git7 and Git10 either act in a G-protein-independent manner or are required to stabilize or assemble a functional signalling complex. Although prior data suggested that the Cgs2 cAMP phosphodiesterase (PDE) does not regulate the cAMP response, we now have evidence that along with adenylate cyclase regulation, PDE activation is important for limiting the response to glucose. Finally, regulation of protein kinase A activation appears to involve both traditional post-translational regulation of the function of the components of the cAMP pathway and glucose-dependent transcriptional regulation of some of these cAMP pathway genes.

Preparation of Yeast DNA

Molecular studies in yeast often require the isolation of both plasmid and chromosomal yeast DNA. Plasmid DNA is used in the transformation of E. coli, whereas chromosomal DNA is used for Southern hybridization analysis, in vitro amplification by the polymerase chain reaction (PCR), or cloning of integrated plasmids. This unit presents two variations of the “smash and grab” protocol that produce suitable DNA for all these applications. These protocols work for both Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Except in Every Detail: Comparing and Contrasting G-Protein Signaling in Saccharomyces cerevisiae and Schizosaccharomyces pombe

When asked to explain the difference between the mechanisms controlling mating type switching in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe , Brandeis Universitys Jim Haber once replied, “Its exactly the same, except in every detail.” He may as

Cloning and manipulation of the Schizosaccharomyces pombe his7+gene as a new selectable marker for molecular genetic studies

We have cloned the his7+ gene of the fission yeast Schizosaccharomyces pombe by complementation of the recessive mutant allele his7-366. The his7+ gene is able to complement a mutation of the Escherichia coli hisI gene, suggesting that his7+ encodes a phosphoribosyl-AMP cyclohydrase. Subcloning experiments localize the gene to a 1.9-kb XbaI-BglII fragment. We describe the construction of plasmids to facilitate the use of his7+ as a selectable marker in S. pombe studies. Plasmid pEA2 carries his7+ cloned into the pUC18 polylinker. From either pEA2 or the original his7+ clone, pMN1, fragments carrying his7+ can be isolated using a variety of restriction enzymes for the construction of gene disruptions. Plasmid pEA500 is a cloning vector that carries his7+ and ars1, yet retains the ability to use the blue/white color screen to identify recombinants.

Protein Kinase A and Mitogen-Activated Protein Kinase Pathways Antagonistically Regulate Fission Yeast fbp1 Transcription by Employing Different Modes of Action at Two Upstream Activation Sites

ABSTRACT A significant challenge to our understanding of eukaryotic transcriptional regulation is to determine how multiple signal transduction pathways converge on a single promoter to regulate transcription in divergent fashions. To study this, we have investigated the transcriptional regulation of theSchizosaccharomyces pombe fbp1 gene that is repressed by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway and is activated by a stress-activated mitogen-activated protein kinase (MAPK) pathway. In this study, we identified and characterized twocis-acting elements in the fbp1 promoter required for activation of fbp1 transcription. Upstream activation site 1 (UAS1), located approximately 900 bp from the transcriptional start site, resembles a cAMP response element (CRE) that is the binding site for the atf1-pcr1 heterodimeric transcriptional activator. Binding of this activator to UAS1 is positively regulated by the MAPK pathway and negatively regulated by PKA. UAS2, located approximately 250 bp from the transcriptional start site, resembles a Saccharomyces cerevisiae stress response element. UAS2 is bound by transcriptional activators and repressors regulated by both the PKA and MAPK pathways, although atf1 itself is not present in these complexes. Transcriptional regulation offbp1 promoter constructs containing only UAS1 or UAS2 confirms that the PKA and MAPK regulation is targeted to both sites. We conclude that the PKA and MAPK signal transduction pathways regulatefbp1 transcription at UAS1 and UAS2, but that the antagonistic interactions between these pathways involve different mechanisms at each site.

Pro-Aging Effects of Glucose Signaling through a G Protein-Coupled Glucose Receptor in Fission Yeast

Glucose is the preferred carbon and energy source in prokaryotes, unicellular eukaryotes, and metazoans. However, excess of glucose has been associated with several diseases, including diabetes and the less understood process of aging. On the contrary, limiting glucose (i.e., calorie restriction) slows aging and age-related diseases in most species. Understanding the mechanism by which glucose limits life span is therefore important for any attempt to control aging and age-related diseases. Here, we use the yeast Schizosaccharomyces pombe as a model to study the regulation of chronological life span by glucose. Growth of S. pombe at a reduced concentration of glucose increased life span and oxidative stress resistance as reported before for many other organisms. Surprisingly, loss of the Git3 glucose receptor, a G protein-coupled receptor, also increased life span in conditions where glucose consumption was not affected. These results suggest a role for glucose-signaling pathways in life span regulation. In agreement, constitutive activation of the Gα subunit acting downstream of Git3 accelerated aging in S. pombe and inhibited the effects of calorie restriction. A similar pro-aging effect of glucose was documented in mutants of hexokinase, which cannot metabolize glucose and, therefore, are exposed to constitutive glucose signaling. The pro-aging effect of glucose signaling on life span correlated with an increase in reactive oxygen species and a decrease in oxidative stress resistance and respiration rate. Likewise, the anti-aging effect of both calorie restriction and the Δgit3 mutation was accompanied by increased respiration and lower reactive oxygen species production. Altogether, our data suggest an important role for glucose signaling through the Git3/PKA pathway to regulate S. pombe life span.

Properties of the type B histone acetyltransferase HAT1: H4 tail interaction, site preference, and involvement in DNA repair

The Hat1 histone acetyltransferase catalyzes the acetylation of H4 at lysines 5 and 12, the same sites that are acetylated in newly synthesized histone H4. By performing histone acetyltransferase (HAT) assays on various synthetic H4 N-terminal peptides, we have examined the interactions between Hat1 and the H4 tail domain. It was found that acetylation requires the presence of positively charged amino acids at positions 8 and 16 of H4, positions that are normally occupied by lysine; however, lysine per se is not essential and can be replaced by arginine. In contrast, replacing Lys-8 and -16 of H4 with glutamines reduces acetylation to background levels. Similarly, phosphorylation of Ser-1 of the H4 tail depresses acetylation by both yeast Hat1p and the human HAT-B complex. These results strongly support the model proposed by Ramakrishnan and colleagues for the interaction between Hat1 and the H4 tail (Dutnall, R. N., Tafrov, S. T., Sternglanz, R., and Ramakrishnan, V. (1998) Cell 94, 427–438) and may have implications for the genetic analysis of histone acetylation. It was also found that Lys-12 of H4 is preferentially acetylated by human HAT-B, in further agreement with the proposed model of H4 tail binding. Finally, we have demonstrated that deletion of the hat1 gene from the fission yeast Schizosaccharomyces pombe causes increased sensitivity to the DNA-damaging agent methyl methanesulfonate in the absence of any additional mutations. This is in contrast to results obtained with a Saccharomyces cerevisiae hat1Δ strain, which must also carry mutations of the acetylatable lysines of H3 for heightened methyl methanesulfonate sensitivity to be observed. Thus, although the role of Hat1 in DNA damage repair is evolutionarily conserved, the ability of H3 acetylation to compensate for Hat1 deletion appears to be more variable.

An Ancient Yeast for Young Geneticists: A Primer on the Schizosaccharomyces pombe Model System

The fission yeast Schizosaccharomyces pombe is an important model organism for the study of eukaryotic molecular and cellular biology. Studies of S. pombe, together with studies of its distant cousin, Saccharomyces cerevisiae, have led to the discovery of genes involved in fundamental mechanisms of transcription, translation, DNA replication, cell cycle control, and signal transduction, to name but a few processes. However, since the divergence of the two species approximately 350 million years ago, S. pombe appears to have evolved less rapidly than S. cerevisiae so that it retains more characteristics of the common ancient yeast ancestor, causing it to share more features with metazoan cells. This Primer introduces S. pombe by describing the yeast itself, providing a brief description of the origins of fission yeast research, and illustrating some genetic and bioinformatics tools used to study protein function in fission yeast. In addition, a section on some key differences between S. pombe and S. cerevisiae is included for readers with some familiarity with budding yeast research but who may have an interest in developing research projects using S. pombe.

Retrotransposon Tf1 is targeted to pol II promoters by transcription activators

The LTR-retrotransposon Tf1 preserves the coding capacity of its host Schizosaccharomyces pombe by integrating upstream of open reading frames (ORFs). To determine which features of the target sites were recognized by the transposon, we introduced plasmids containing candidate insertion sites into S. pombe and mapped the positions of integration. We found that Tf1 was targeted specifically to the promoters of Pol II-transcribed genes. A detailed analysis of integration in plasmids that contained either ade6 or fbp1 revealed insertions occurred in the promoters at positions where transcription factors bound. Further experiments revealed that the activator Atf1p and its binding site were required for directing integration to the promoter of fbp1. An interaction between Tf1 integrase and Atf1p was observed, indicating that integration at fbp1 was mediated by the activator bound to its promoter. Surprisingly, we found Tf1 contained sequences that activated transcription, and these substituted for elements of the ade6 promoter disrupted by integration.