Drosophila melanogaster
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA314298 | ENA |
REPOSITORIES: ENA
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Project description:Transcription start site (TSS) selection is a key step in gene expression and occurs at many promoter positions over a wide range of efficiencies. Here, we develop a massively parallel reporter assay to quantitatively dissect contributions of promoter sequence, NTP substrate levels, and RNA polymerase II (Pol II) activity to TSS selection by "promoter scanning" in Saccharomyces cerevisiae (Pol II MAssively Systematic Transcript End Readout, "Pol II MASTER"). Using Pol II MASTER, we measure the efficiency of Pol II initiation at 1,000,000 individual TSS sequences in a defined promoter context. Pol II MASTER confirms proposed critical qualities of S. cerevisiae TSS -8, -1, and +1 positions quantitatively in a controlled promoter context. Pol II MASTER extends quantitative analysis to surrounding sequences and determines that they tune initiation over a wide range of efficiencies. These results enabled the development of a predictive model for initiation efficiency based on sequence. We show that genetic perturbation of Pol II catalytic activity alters initiation efficiency mostly independently of TSS sequence, but selectively modulates preference for initiating nucleotide. Intriguingly, we find that Pol II initiation efficiency is directly sensitive to GTP levels at the first five transcript positions and to CTP and UTP levels at the second position genome wide. These results suggest individual NTP levels can have transcript-specific effects on initiation, representing a cryptic layer of potential regulation at the level of Pol II biochemical properties. The results establish Pol II MASTER as a method for quantitative dissection of transcription initiation in eukaryotes.
2023-11-26 | GSE185290 | GEO
Project description:Chromatin connectivity maps reveal dynamic promoter-enhancer long-range associations
| PRJNA189721 | ENA
Project description:The core promoter is the regulatory sequence to which RNA polymerase is recruited and where it acts to initiate transcription. Here, we present the first comprehensive study of yeast core promoters, providing massively parallel measurements of core promoter activity and of TSS locations and relative usage for thousands of native and designed sequences. We found core promoter activity to be highly correlated to the activity of the entire promoter, and that sequence variation in different core promoter regions substantially tunes its activity in a predictable way. We also show that location, orientation and flanking bases critically affect TATA element function, that transcription initiation in highly active core promoters is focused within a narrow region, that poly(dA:dT) orientation has functional consequence at the 3' end of promoters, and that orthologous core promoters across yeast species have conserved activities. Our results demonstrate the importance of core promoters in the quantitative study of gene regulation.
2015-04-23 | GSE68168 | GEO
Project description:We report that TAF3, a TBP-associated core promoter factor, is highly enriched in ES cells. In addition to its role in the core promoter recognition complex TFIID, genome-wide binding studies reveal that TAF3 localizes to chromosomal regions bound by CTCF and cohesin. Enrichment for TAF3/CTCF/cohesin bound regions distinguishes TAF3-activated from TAF3-repressed genes. Our findings support a new role of TAF3 in mediating long-range chromatin regulatory interactions to safeguard the finely-balanced transcriptional programs that give rise to pluripotency. Genome-wide binding of TAF3, TAF1, TBP and PolII assayed by ChIP-Seq.
2011-09-02 | E-GEOD-31270 | biostudies-arrayexpress
Project description:Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a Precision nuclear Run-On and sequencing assay (PRO-seq) to map the genome-wide distribution of transcriptionally-engaged Pol II at base-pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3' poly-adenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing . We test this ‘Complex Interaction’ model with insertional mutagenesis of the Drosophila Hsp70 core promoter. Identification of RNA polymerase active sites in Drosophila S2 cell line using PRO-seq method. Identification of transcription initiation sites in Drosophila S2 cell line using PRO-cap method. Identification of changes in RNA polymerase active sites on transgenic Hsp70 promoters upon disruption of DNA sequence elements in 3 transgenic fly lines using PRO-seq method.
2013-02-01 | E-GEOD-42117 | biostudies-arrayexpress
Project description:Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a Precision nuclear Run-On and sequencing assay (PRO-seq) to map the genome-wide distribution of transcriptionally-engaged Pol II at base-pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3' poly-adenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing . We test this ‘Complex Interaction’ model with insertional mutagenesis of the Drosophila Hsp70 core promoter.
2013-02-01 | GSE42117 | GEO
Project description:We report that TAF3, a TBP-associated core promoter factor, is highly enriched in ES cells. In addition to its role in the core promoter recognition complex TFIID, genome-wide binding studies reveal that TAF3 localizes to chromosomal regions bound by CTCF and cohesin. Enrichment for TAF3/CTCF/cohesin bound regions distinguishes TAF3-activated from TAF3-repressed genes. Our findings support a new role of TAF3 in mediating long-range chromatin regulatory interactions to safeguard the finely-balanced transcriptional programs that give rise to pluripotency.
2011-09-02 | GSE31271 | GEO
Project description:We report that TAF3, a TBP-associated core promoter factor, is highly enriched in ES cells. In addition to its role in the core promoter recognition complex TFIID, genome-wide binding studies reveal that TAF3 localizes to chromosomal regions bound by CTCF and cohesin. Enrichment for TAF3/CTCF/cohesin bound regions distinguishes TAF3-activated from TAF3-repressed genes. Our findings support a new role of TAF3 in mediating long-range chromatin regulatory interactions to safeguard the finely-balanced transcriptional programs that give rise to pluripotency.
2011-09-02 | GSE31270 | GEO
Project description:Previous methods to systematically characterize sequence-intrinsic activity of promoters have been limited by relatively low throughput and the length of sequences that could be tested. Here we present Survey of Regulatory Elements (SuRE), a method to assay more than 10^8 DNA fragments, each 0.2-2kb in size, for their ability to drive transcription autonomously. In SuRE, a plasmid library is constructed of random genomic fragments upstream of a 20bp barcode and decoded by paired-end sequencing. This library is then transfected into cells and transcribed barcodes are quantified in the RNA by high throughput sequencing. When applied to the human genome, we achieved a 55-fold genome coverage, allowing us to map autonomous promoter activity genome-wide. By computational modeling we delineated subregions within promoters that are relevant for their activity. For instance, we show that antisense promoter transcription is generally dependent on the sense core promoter sequences, and that most enhancers and several families of repetitive elements act as autonomous transcription initiation sites.
2016-11-10 | GSE78709 | GEO
Project description:Genome-wide quantitative enhancer activity maps identified by STARR-seq
| PRJNA175267 | ENA