A library of native and synthetic yeast core promoters driving YFP expression
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE68168 | GEO | 2015/04/23
SECONDARY ACCESSION(S): PRJNA281926
REPOSITORIES: GEO
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Project description:Mediator is an essential, broadly utilized eukaryotic transcriptional co-activator. How and what it communicates from activators to RNA polymerase II remains an open question. Here we performed genome-wide location profiling of yeast Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence (UAS), upstream of the pre-initiation complex. In the absence of Kin28/CDK7 kinase activity, or in cells where the CTD is mutated to replace Ser5 with alanines, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is quickly released from promoters upon Ser5 phosphorylation by Kin28/CDK7, which also allows for RNAPII to escape from the promoter.
2014-03-24 | GSE55402 | GEO
Project description:Despite much research, our understanding of the architecture and cis-regulatory elements of human promoters is still lacking. Here, we devised a high-throughput assay to quantify the activity of ~15,000 fully designed sequences that we integrated and expressed from a fixed location within the human genome. We used this method to investigate thousands of native promoters and pre-initiation complex (PIC) binding regions followed by in-depth characterization of the sequence motifs underlying promoter activity including core promoter elements and TF binding-sites. We find that core promoters drive transcription mostly unidirectionally, and that sequences originating from promoters exhibit stronger activity than those originating from enhancers. Testing multiple synthetic configurations of core promoter elements, we dissect the motifs that positively and negatively regulate transcription as well as the effect of their combinations and distances, including a 10bp periodicity in the optimal distance between the TATA and the Initiator. By comprehensively screening 133 TF binding-sites, we find that in contrast to core promoters, TF binding-sites maintain similar activity levels in both orientations supporting a model by which divergent transcription is driven by two distinct unidirectional core promoters sharing bidirectional TF binding-sites. Finally, we find a striking agreement between the effect of binding site multiplicity of individual TFs in our assay and their tendency to appear in homotypic clusters throughout the genome. Overall, our study systematically assays the elements that drive expression in core- and proximal- promoter regions and shed light on organization principles of regulatory regions in the human genome.
2018-08-08 | GSE118242 | GEO
Project description:Protein coding genes in the trypanosome genome are organized in polycistronic transcription units (PTUs). How RNA Polymerase II (Pol II) initiates PTU transcription has not been resolved and the current model favors initiation driven by chromatin epigenetic marks, rather than core-promoters. Here, we identify sequence-specific core promoters by functional characterization of ChIP-Seq Pol II accumulation-peaks. Sequences located between oppositely orientated PTUs contained two independent and distinct core promoters, driving unidirectional transcription. Detailed analysis of one promoter identified 75 bp, necessary and sufficient to fully drive reporter expression, and containing short functional motifs. Analysis of further promoters suggested activity and initiation is regulated and both, focused and dispersed transcription patterns were found. Thus, in contrast to the model of unregulated and promoter-independent transcription initiation, sequence-specific promoters determine the initiation of RNA Pol II transcription of protein coding genes in trypanosomes. These findings resolve the discrepancy between trypanosomes and other eukaryotes in how Pol II initiates protein-coding gene transcription.
2021-02-01 | GSE130798 | GEO
Project description:Despite extensive research, our understanding of the rules according to which cis-regulatory sequences are converted into gene expression is limited We devised a method for obtaining parallel, highly accurate gene expression measurements from thousands of designed promoters and applied it to measure the effect of systematic changes in the location, number, orientation, affinity and organization of transcription-factor binding sites and nucleosome-disfavoring sequences. Our analyses reveal a clear relationship between expression and binding-site multiplicity, as well as dependencies of expression on the distance between transcription-factor binding sites and gene starts which are transcription-factor specific, including a striking ~10-bp periodic relationship between gene expression and binding-site location. We show how this approach can measure transcription-factor sequence specificities and the sensitivity of transcription-factor sites to the surrounding sequence context, and we compare the activity of 75 yeast transcription factors. Our method can be used to study both cis and trans effects of genotype on transcriptional, post-transcriptional and translational control. Expression profiling of 6500 synthetic promoters in yeast (Saccharomyces cerevisiae). The results were achieved using a method for obtaining pooled and highly accurate expression measurements (closely related to MPRA). In short, the synthetic promoters, which contain unique barcodes, are inserted upstream to a yellow florescence reporter gene, the cells are sorted by their florescence level (using FACS) to expression bins, the promoters in each expression bin are amplified and are send to parallel sequencing (SOLiD) to determine the percent of cells of each promoter in each expression bin; finally an mean expression value is extracted from the raw results. The measurements contain two replicates of exponentially growing yeast cells in SC-Gal-URA (synthetic complete media with 2% galactose and without uracil).
2012-05-20 | E-GEOD-37851 | biostudies-arrayexpress
Project description:The RNA polymerase II (Pol II) core promoter is the strategic site of convergence of the signals that lead to the initiation of DNA transcription, but the downstream core promoter in humans has been difficult to understand. Here we analyse the human Pol II core promoter and use machine learning to generate predictive models for the downstream core promoter region (DPR) and the TATA box. We developed a method termed HARPE (high-throughput analysis of randomized promoter elements) to create hundreds of thousands of DPR (or TATA box) variants, each with known transcriptional strength. We then analysed the HARPE data by support vector regression (SVR) to provide comprehensive models for the sequence motifs, and found that the SVR-based approach is more effective than a consensus-based method for predicting transcriptional activity. These results show that the DPR is a functionally important core promoter element that is widely used in human promoters. Notably, there appears to be a duality between the DPR and the TATA box, as many promoters contain one or the other element. More broadly, these findings show that functional DNA motifs can be identified by machine learning analysis of a comprehensive set of sequence variants.
2020-06-04 | GSE139635 | GEO
Project description:The preinitiation complex (PIC) assembles on promoters of protein-coding genes to position RNA polymerase II (Pol II) for transcription initiation. Previous structural studies revealed the PIC on different promoters, but did not address how the PIC assembles in chromatin. In the yeast Saccharomyces cerevisiae, PIC assembly occurs adjacent to the +1 nucleosome that is positioned downstream of the core promoter. Here we present the cryo-EM structure of the yeast PIC bound to promoter DNA and the +1 nucleosome at a resolution of 3.4–3.9 Å. The general transcription factor TFIIH forms four contacts with the nucleosome, indicating how PIC assembly can be assisted by the +1 nucleosome. The TFIIH subunit Ssl2 (XPB in human TFIIH) forms a wedge between promoter DNA and the nucleosome, stabilizing two turns of DNA that are detached from the nucleosome. During subsequent scanning for the transcription start site (TSS), three additional turns of DNA can be detached before the partially unraveled nucleosome may counteract further scanning, explaining why TSSs are located at a distance of ~60 bp from the dyad of the +1 nucleosome in yeast.
2023-03-11 | PXD029840 | Pride
Project description:Genetically identical cells exhibit large variability (noise) in gene expression, with important consequences for cellular function. Although the amount of noise decreases with and is thus partly determined by the mean expression level, the extent to which different promoter sequences can deviate away from this trend is not known. Here, we study how different noise levels are encoded by the promoter sequence using massively parallel noise measurements of thousands of synthetically designed promoters. We find that the noise levels of promoters with similar mean expression levels can vary over more than one order of magnitude, with nucleosome-disfavoring sequences resulting in lower noise and more transcription factor binding sites resulting in higher noise. We devised a computational model that can accurately predict the mean-independent component of the noise from DNA sequence alone. Our model suggests that the effect of promoters on noise is partly mediated by the combination of non-specific DNA binding and one-dimensional sliding along the DNA that occurs when transcription factors search for their target sites. Overall, our results demonstrate that small changes in the DNA sequence of promoters can allow tuning of noise levels in a manner that is largely predictable and partly decoupled from effects on the mean expression levels. These insights may assist in designing promoters with desired noise levels. Expression measurements of a collection of synthetic promoters collection that was published in Sharon et al. Nature Biotechnology 2012(doi: 10.1038/nbt.2205). Two replicates of the promoter library integrated into a plasmid in yeast were measured in SC-Glu-URA medium. The promoter library was measured as described in Sharon et. al.(Sharon et al. 2012), except for the differences below. Briefly, a large collection of synthetic promoter reporter gene strains was generated by a pooled ligation of 6500 fully designed DNA oligos (obtained by synthesis on a microarray(LeProust et al. 2010) by Agilent Technologies, Santa Clara, California). The oligos were ligated upstream to a yellow fluorescent protein (YFP) gene with a short (100 bp) core promoter sequence taken from HIS3 gene promoter and into a low copy plasmid which also contains a TEF2 promoter deriving red fluorescent protein (mCherry). The resulting plasmids were then transformation into yeast (S. cerevisiae). Next, the pool of cells was grown in amino acid starvation condition (SCD without amino acid except Histidine), and sorted according to their YFP expression level into 32 expression bins (mCherry was used for gating one plasmid copy cells and for normalization). The DNA of the promoters in each bin were then amplified and sent to multiplexed parallel sequencing. Each sequencing result was mapped to a specific promoter and expression bin, resulting in a distribution of cells that contain each promoter across all expression bins. The following differences were applied relative to the description in Sharon et. al.(Sharon et al. 2012). The medium used both for growing the cells and for their sorting was SC-Glu-URA (synthetic complete media with 2% glucose and without uracil) medium without amino acids, except for Histidine. In order to achieve expression distributions with high resolution that would allow good assessment of expression noise, the library cells were sorted into 32 bins according to their ratio of YFP and mCherry expression level, thereby normalizing for extrinsic noise effects. Each of the two extreme expression bins contained 2% of the library cells and each of the remaining 30 bins contained 3.2%. We collected a total of 10,000,000 cells. As previously described, the mapping of cells to bins involves parallel sequencing of the amplified promoter regions. For this purpose, Illumina Hi-Seq 2000 was used to obtain >30,000,000 mapped reads. The two replicates were separately generated from the ssDNA oligo library and separately measured as described above.
2014-07-12 | E-GEOD-55346 | biostudies-arrayexpress
Project description:Three general classes of yeast protein-coding genes are distinguished by their dependence on the transcription cofactors TFIID, SAGA and Mediator (MED) Tail, but little is known about whether this dependence is determined by the core promoter, Upstream activation sites (UAS), or other gene features. It is also unclear whether UASs can broadly activate transcription from the different promoter classes or whether efficient transcription requires matching UASs and promoters of similar gene class. Here we measure transcription and cofactor specificity for tens of thousands of UAS-core promoter combinations. We find that few UASs display strong core promoter specificity while most UASs can broadly activate promoters regardless of regulatory class. However, we find that matching UASs and promoters from the same gene class is generally important for optimal expression. We find that MED Tail and SAGA are dependent on the identity of both UAS and promoter while dependence on TFIID localizes to only the core promoter.
2023-03-17 | GSE227006 | GEO
Project description:Three general classes of yeast protein-coding genes are distinguished by their dependence on the transcription cofactors TFIID, SAGA and Mediator (MED) Tail, but little is known about whether this dependence is determined by the core promoter, Upstream activation sites (UAS), or other gene features. It is also unclear whether UASs can broadly activate transcription from the different promoter classes or whether efficient transcription requires matching UASs and promoters of similar gene class. Here we measure transcription and cofactor specificity for tens of thousands of UAS-core promoter combinations. We find that few UASs display strong core promoter specificity while most UASs can broadly activate promoters regardless of regulatory class. However, we find that matching UASs and promoters from the same gene class is generally important for optimal expression. We find that MED Tail and SAGA are dependent on the identity of both UAS and promoter while dependence on TFIID localizes to only the core promoter.
2023-03-17 | GSE217227 | GEO
Project description:Three general classes of yeast protein-coding genes are distinguished by their dependence on the transcription cofactors TFIID, SAGA and Mediator (MED) Tail, but little is known about whether this dependence is determined by the core promoter, Upstream activation sites (UAS), or other gene features. It is also unclear whether UASs can broadly activate transcription from the different promoter classes or whether efficient transcription requires matching UASs and promoters of similar gene class. Here we measure transcription and cofactor specificity for tens of thousands of UAS-core promoter combinations. We find that few UASs display strong core promoter specificity while most UASs can broadly activate promoters regardless of regulatory class. However, we find that matching UASs and promoters from the same gene class is generally important for optimal expression. We find that MED Tail and SAGA are dependent on the identity of both UAS and promoter while dependence on TFIID localizes to only the core promoter.
2023-03-17 | GSE217229 | GEO