Combinatorial control of gene expression by the three yeast repressors Mig1, Mig2 and Mig3

<p>Abstract</p> <p>Background</p> <p>Expression of a large number of yeast genes is repressed by glucose. The zinc finger protein Mig1 is the main effector in glucose repression, but yeast also has two related proteins: Mig2 and Mig3. We have used microarrays to study global gene expression in all possible combinations of <it>mig1</it>, <it>mig2 </it>and <it>mig3 </it>deletion mutants.</p> <p>Results</p> <p>Mig1 and Mig2 repress a largely overlapping set of genes on 2% glucose. Genes that are upregulated in a <it>mig1 mig2 </it>double mutant were grouped according to the contribution of Mig2. Most of them show partially redundant repression, with Mig1 being the major repressor, but some genes show complete redundancy, and some are repressed only by Mig1. Several redundantly repressed genes are involved in phosphate metabolism. The promoters of these genes are enriched for Pho4 sites, a novel GGGAGG motif, and a variant Mig1 site which is absent from genes repressed only by Mig1. Genes repressed only by Mig1 on 2% glucose include the hexose transporter gene <it>HXT4</it>, but Mig2 contributes to <it>HXT4 </it>repression on 10% glucose. <it>HXT6 </it>is one of the few genes that are more strongly repressed by Mig2. Mig3 does not seem to overlap in function with Mig1 and Mig2. Instead, Mig3 downregulates the <it>SIR2 </it>gene encoding a histone deacetylase involved in gene silencing and the control of aging.</p> <p>Conclusion</p> <p>Mig2 fine-tunes glucose repression by targeting a subset of the Mig1-repressed genes, and by responding to higher glucose concentrations. Mig3 does not target the same genes as Mig1 and Mig2, but instead downregulates the <it>SIR2 </it>gene.</p

Histone modifications influence mediator interactions with chromatin

The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. Genome wide localization studies have demonstrated that Mediator occupancy not only correlates with high levels of transcription, but that the complex also is present at transcriptionally silenced locations. We provide evidence that Mediator localization is guided by an interaction with histone tails, and that this interaction is regulated by their post-translational modifications. A quantitative, high-density genetic interaction map revealed links between Mediator components and factors affecting chromatin structure, especially histone deacetylases. Peptide binding assays demonstrated that pure wild-type Mediator forms stable complexes with the tails of Histone H3 and H4. These binding assays also showed Mediator—histone H4 peptide interactions are specifically inhibited by acetylation of the histone H4 lysine 16, a residue critical in transcriptional silencing. Finally, these findings were validated by tiling array analysis that revealed a broad correlation between Mediator and nucleosome occupancy in vivo, but a negative correlation between Mediator and nucleosomes acetylated at histone H4 lysine 16. Our studies show that chromatin structure and the acetylation state of histones are intimately connected to Mediator localization

Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome

Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV effects reflect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specific miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49-96%) corresponded to rRNA-, coding-and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics.Peer reviewe

Gis1 and Rph1 Regulate Glycerol and Acetate Metabolism in Glucose Depleted Yeast Cells

Aging in organisms as diverse as yeast, nematodes, and mammals is delayed by caloric restriction, an effect mediated by the nutrient sensing TOR, RAS/cAMP, and AKT/Sch9 pathways. The transcription factor Gis1 functions downstream of these pathways in extending the lifespan of nutrient restricted yeast cells, but the mechanisms involved are still poorly understood. We have used gene expression microarrays to study the targets of Gis1 and the related protein Rph1 in different growth phases. Our results show that Gis1 and Rph1 act both as repressors and activators, on overlapping sets of genes as well as on distinct targets. Interestingly, both the activities and the target specificities of Gis1 and Rph1 depend on the growth phase. Thus, both proteins are associated with repression during exponential growth, targeting genes with STRE or PDS motifs in their promoters. After the diauxic shift, both become involved in activation, with Gis1 acting primarily on genes with PDS motifs, and Rph1 on genes with STRE motifs. Significantly, Gis1 and Rph1 control a number of genes involved in acetate and glycerol formation, metabolites that have been implicated in aging. Furthermore, several genes involved in acetyl-CoA metabolism are downregulated by Gis1

Neurophysiological Defects and Neuronal Gene Deregulation in Drosophila mir-124 Mutants

miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, we deleted its single mir-124 locus and found that it is dispensable for gross aspects of neural specification and differentiation. On the other hand, we detected a variety of mutant phenotypes that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124–expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology

Genome-wide Studies of Transcriptional Regulation in Yeast

In this thesis, nutrient signalling in yeast is used as a model to study several features of gene regulation, such as combinatorial gene regulation, the role of motif context and chromatin modifications. The nutrient signalling system in yeast consists of several pathways that transmit signals about the availability of key nutrients, and regulate the transcription of a large part of the genome. Some of the signalling pathways are also conserved in other eukaryotic species where they are implicated in processes such as aging and in human disease.   Combinatorial gene regulation is examined in papers I and II. In paper I, the role of Mig1, Mig2 and Mig3 is studied. To elucidate how the three proteins contribute to the control of gene expression, we used microarrays to study the expression of all yeast genes in the wild type and in all seven possible combinations of mig1, mig2 and mig3 deletions. In paper II, a similar strategy is used to investigate Gis1 and Rph1, two related transcription factors. Our results reveal that Rph1 is involved in nutrient signalling together with Gis1, and we find that both the activities and the target specificities of Gis1 and Rph1 depend on the growth phase.   Paper III describes ContextFinder, a program for identifying constraints on sequence motif locations and orientations. ContextFinder was used to analyse over 300 cases of motifs that are enriched in experimentally selected groups of yeast promoters. Our results suggest that motif context frequently is important for stable DNA binding and/or regulatory activity of transcription factors.   In paper IV, we investigated how gene expression changes resulting from nitrogen starvation are accompanied by chromatin modifications. Activation of gene expression is concentrated to specific genomic regions. It is associated with nucleosome depletion (in both promoters and coding regions) and increased levels of H3K9ac (but not H4K5ac)

Local search methods in gene expression analysis

The aim of this project was to investigate a combinatorial optimization problem stated by Global Genomics AB. The problem has its background in genomics, and is part of a new method for measuring gene expression levels. This method involves experiments as well as post processing of the experimental data. The experiments take a cell sample as input, and the output is incomplete information about the genes expressed in the sample. To estimate the gene expression levels from this information, it has to be matched with a gene database. Since the experimental data is incomplete, there are many possible matchings. The problem investigated in this project is to find the best matching between the experimental data and the gene database, using some different local search techniques

GENKOMB project report

The aim of the GENKOMB project was to find and analyse transcription factor binding sites in the human genome, by correlating expression data for a set of genes with the nucleotide sequences in their upstream regions. This document is a technical description of tools that have been developed and results that have been obtained in the GENKOMB project. Apart from detecting transcription factor binding sites, this project has resulted in programs for matching weight matrices to DNA sequences and multiple alignment of short nucleotide sequences

Global gene expression analysis by combinatorial optimization

Generally, there is a trade-off between methods of gene expression analysis that are precise but labor-intensive, e.g. RT-PCR, and methods that scale up to global coverage but are not quite as quantitative, e.g. microarrays. In the present paper, we show how how a known method of gene expression profiling (K. Kato, Nucleic Acids Research 23, 3685-3690 (1995)), which relies on a fairly small number of steps, can be turned into a global gene expression measurement by advanced data post-processing, with potentially little loss of accuracy. Post-processing here entails solving an ancillary combinatorial optimization problem. Validation is performed on in silico experiments generated from the FANTOM data base of full-length mouse cDNA. We present two variants of the method. One uses state-of-the-art commercial software for solving problems of this kind, the other a code developed by us specifically for this purpose, released in the public domain under GPL license