Two distinct repressive mechanisms for histone 3 lysine 4 methylation through promoting 3'-end antisense transcription

International audienceHistone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3'-end, indicating that repression is coupled to 3'-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3'-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3'-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3'-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3'-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3'-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms

Yeast glucose pathways converge on the transcriptional regulation of trehalose biosynthesis

<p>Abstract</p> <p>Background</p> <p>Cellular glucose availability is crucial for the functioning of most biological processes. Our understanding of the glucose regulatory system has been greatly advanced by studying the model organism <it>Saccharomyces cerevisiae</it>, but many aspects of this system remain elusive. To understand the organisation of the glucose regulatory system, we analysed 91 deletion mutants of the different glucose signalling and metabolic pathways in <it>Saccharomyces cerevisiae</it> using DNA microarrays.</p> <p>Results</p> <p>In general, the mutations do not induce pathway-specific transcriptional responses. Instead, one main transcriptional response is discerned, which varies in direction to mimic either a high or a low glucose response. Detailed analysis uncovers established and new relationships within and between individual pathways and their members. In contrast to signalling components, metabolic components of the glucose regulatory system are transcriptionally more frequently affected. A new network approach is applied that exposes the hierarchical organisation of the glucose regulatory system.</p> <p>Conclusions</p> <p>The tight interconnection between the different pathways of the glucose regulatory system is reflected by the main transcriptional response observed. Tps2 and Tsl1, two enzymes involved in the biosynthesis of the storage carbohydrate trehalose, are predicted to be the most downstream transcriptional components. Epistasis analysis of <it>tps2</it>Δ double mutants supports this prediction. Although based on transcriptional changes only, these results suggest that all changes in perceived glucose levels ultimately lead to a shift in trehalose biosynthesis.</p

A consensus of core protein complex compositions for Saccharomyces cerevisiae

Analyses of biological processes would benefit from accurate definitions of protein complexes. High-throughput mass spectrometry data offer the possibility of systematically defining protein complexes; however, the predicted compositions vary substantially depending on the algorithm applied. We determine consensus compositions for 409 core protein complexes from Saccharomyces cerevisiae by merging previous predictions with a new approach. Various analyses indicate that the consensus is comprehensive and of high quality. For 85 out of 259 complexes not recorded in GO, literature search revealed strong support in the form of coprecipitation. New complexes were verified by an independent interaction assay and by gene expression profiling of strains with deleted subunits, often revealing which cellular processes are affected. The consensus complexes are available in various formats, including a merge with GO, resulting in 518 protein complex compositions. The utility is further demonstrated by comparison with binary interaction data to reveal interactions between core complexe

A high-resolution gene expression atlas of epistasis between gene-specific transcription factors exposes potential mechanisms for genetic interactions

BACKGROUND: Genetic interactions, or non-additive effects between genes, play a crucial role in many cellular processes and disease. Which mechanisms underlie these genetic interactions has hardly been characterized. Understanding the molecular basis of genetic interactions is crucial in deciphering pathway organization and understanding the relationship between genotype, phenotype and disease. RESULTS: To investigate the nature of genetic interactions between gene-specific transcription factors (GSTFs) in Saccharomyces cerevisiae, we systematically analyzed 72 GSTF pairs by gene expression profiling double and single deletion mutants. These pairs were selected through previously published growth-based genetic interactions as well as through similarity in DNA binding properties. The result is a high-resolution atlas of gene expression-based genetic interactions that provides systems-level insight into GSTF epistasis. The atlas confirms known genetic interactions and exposes new ones. Importantly, the data can be used to investigate mechanisms that underlie individual genetic interactions. Two molecular mechanisms are proposed, "buffering by induced dependency" and "alleviation by derepression". CONCLUSIONS: These mechanisms indicate how negative genetic interactions can occur between seemingly unrelated parallel pathways and how positive genetic interactions can indirectly expose parallel rather than same-pathway relationships. The focus on GSTFs is important for understanding the transcription regulatory network of yeast as it uncovers details behind many redundancy relationships, some of which are completely new. In addition, the study provides general insight into the complex nature of epistasis and proposes mechanistic models for genetic interactions, the majority of which do not fall into easily recognizable within- or between-pathway relationships

COMPASS repression is mediated through 3′-end antisense transcriptional gene silencing.

<p>(A) Hierarchical clustering of the 69 COMPASS-repressed genes in the <i>rrp6</i>Δ, <i>set1</i>Δ and the <i>set1</i>Δ <i>rrp6</i>Δ strains. <i>PHO84</i> is marked by P and <i>AMS1</i>, <i>YGR110W</i>, <i>ARG1</i>, <i>SPR3</i> and <i>OYE3</i> are marked by 1 to 5, respectively. The black bar marks the subset of genes where the two mutations are epistatic. Three quarters of these genes are related to phosphate metabolism. (B) Sense and antisense RNA levels analyzed by qPCR in indicated backgrounds. The schematic representation of the follow-up genes shows the relative positions of the primers used for strand-specific reverse transcription reactions in arrows, while the black box indicates the location of the DNA fragment produced during the qPCR. Error bars reflect standard deviations of an average signal obtained from at least two independent experiments. The significance of the difference in expression changes observed between the mutant cells and the corresponding background strain (rrp6<i>Δ</i> in the case of <i>spp1Δrrp6Δ</i> and <i>set1Δrrp6Δ</i>, <i>wt for the others</i>), was evaluated using Student's <i>t</i>-test (^*<i>P</i> 0.01–0.05; ^**<i>P</i> 0.001–0.01; ^***<i>P</i><0.001).</p

Set1 represses sense transcription through promotion of antisense transcription.

<p>(A) Scheme showing <i>YGR110W</i> and <i>YGR111W</i> genes before (YGR) and after (YGR-ingdel) deletion of their intergenic region. The sense <i>YGR110W</i> transcript (<i>sYGR110W</i>), as well as the longer sense transcript in the YGR-ingdel strain are shown in dark grey, while the antisense transcript (SUT557) is shown in light grey. The position and 5′-3′ direction of the strand-specific probes used to detect the transcripts are also shown. (B) Autoradiographs of Northern blots hybridized with the strand-specific DNA probes designed to detect the sense (<i>sYGR110W</i>) or antisense (<i>asYGR110W</i>) transcripts of <i>YGR110W</i> in the YGR and YGR-ingdel strains with wild-type (<i>SET1</i>) or deleted <i>SET1</i> (<i>set1Δ</i>). An autoradiograph of the same blot hybridized with a tubulin probe (<i>TUB1</i>) was used as loading control. Quantitation of the bands are shown below each panel relative to the wt (SET1 YGR) strain for TUB1 and relative to the wt (SET1 YGR) strain and the loading control for the <i>sYGR110W</i> and <i>asYGR110W</i> panels.</p

COMPASS-repressed genes have aberrant H3K4 methylation patterns, indicative of 3′-end antisense transcription.

<p>(A) Heatmaps of enrichment of H3K4me2(left) and H3K4me3(right) over H3 in the gene body and flanking regions of the 69 COMPASS repressed genes, based on <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002952#pgen.1002952-Kirmizis1" target="_blank">[53]</a>. The enrichments are rescaled for each individual gene with blue and white corresponding to the highest and lowest enrichment, respectively. <i>PHO84</i> is marked by P. (B) The average enrichment of H3K4me1 (blue), H3K4me2 (red) and H3K4me3 (grey) over H3, for the set of 47 COMPASS-repressed genes that show at least a two-fold enrichment of H3K4me2 or H3K4me3 somewhere across the gene or flanking region <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002952#pgen.1002952-Kirmizis1" target="_blank">[53]</a>.</p

Loss of H3K4 di- and trimethylation results in upregulation of a subset of genes.

<p>(A) Commassie-stained gel of purified histones from the indicated strains (top) and western blots with antibodies directed against H3 carboxy-terminus and the different H3K4 methylation states (bottom). (B) Hierarchical clustering of all genes with significantly changed mRNA expression (p-value less than 0.01 and fold-change versus wild-type more than 1.7) in at least two COMPASS mutants. Fold-change of mRNA expression in mutant versus wild-type is indicated by the colour bar as log₂ values. Number of genes below each heatmap correspond to the genes called significant in each mutant. (C) Genes depicted in the same order as in B for the H3K4R point mutant and <i>bre1</i>Δ.</p