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Saccharomyces cerevisiae
6 Downloadable Samples
Illumina Genome Analyzer IIx
Description
Anti-sense non-coding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical and genetic approaches in yeast to show that polymerases transcribing opposite DNA strands cannot bypass each other. RNAPII stops, but does not dissociate upon head-to-head collision in vitro, suggesting that opposing polymerases represent insurmountable obstacles for each other. Head-to-head collision in vivo results in RNAPII stopping as well, and removal of collided RNAPII from the DNA template can be achieved via ubiquitylation-directed proteolysis. Indeed, in cells lacking efficient RNAPII poly-ubiquitylation, the half-life of collided polymerases increases, so that these can be detected between convergent genes by ChIP-Seq. These results provide new insight into fundamental mechanisms of gene traffic control, and point to an unexplored effect of anti-sense transcription on gene regulation via polymerase collision. Overall design: Total RNA was extracted from WT or Elongin C deletion mutant (elc1?) cells and strand-specific RNA-Seq was performed. Three biological replicates were performed for WT and elc1?.
Publication Title
RNA polymerase II collision interrupts convergent transcription.
Sample Metadata Fields
Cell line, Subject
View Samples- Homo sapiens
- 45 Downloadable Samples
- Illumina Genome Analyzer IIx
Description
Global Run-On has been performed on WT or KD for RECQL5 cells after release from DRB. When RECQL5 is knocked-down the transcriptional wave front is more advanced, suggesting that transcription is faster. Overall design: Constitutive knock-down cell lines expressing or not endogenous levels of shRNA resistant RECQL5 under a Doxycycline inducible promoter were treated with high doses of DRB to block transcription. Upon release into fresh medium we were able to follow how much and how fast the RNA Pol II progresses through genes by mapping nascent RNA by Run-On. The experiment was performed in two cell line clones.
Publication Title
RECQL5 controls transcript elongation and suppresses genome instability associated with transcription stress.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Homo sapiens
- 9 Downloadable Samples
Affymetrix Human Exon 1.0 ST Array [transcript (gene) version (huex10st)
Description
Alternative mRNA splicing is the main reason vast mammalian proteomic complexity can be achieved with a limited number of genes. Splicing is physically and functionally coupled to transcription and the rate of transcript elongation has a profound effect on splicing. As the nascent pre-mRNA emerges from transcribing RNA polymerase II (RNAPII), it is assembled into a messenger ribonucleoprotein (mRNP) particle that represents its functional form, and the composition of which determines the fate of the mature transcript4. However, factors that connect the transcribing polymerase with the mRNP particle and help integrate transcript elongation with mRNA splicing remain obscure. Here, we characterized the interactome of chromatin-associated mRNP particles and thereby identified Deleted in Breast Cancer 1 (DBC1) and a protein we named ZIRD. These proteins are subunits of a novel protein complex, named DBIRD, which binds directly to RNAPII. DBIRD regulates alternative splicing of a large set of exons embedded in A/T-rich DNA, and is present at the affected exons. RNAi-mediated DBIRD depletion results in region-specific decreases in transcript elongation, particularly across areas encompassing affected exons. These data indicate that DBIRD complex acts at the interface between mRNP particles and RNAPII, integrating transcript elongation with regulation of alternative splicing.
Publication Title
DBIRD complex integrates alternative mRNA splicing with RNA polymerase II transcript elongation.
Sample Metadata Fields
Cell line
View Samples- Homo sapiens
- 6 Downloadable Samples
- Illumina HiSeq 4000
Description
Connections between RNA polymerase II (RNAPII) transcription stress, R-loops, and genome instability have been established however, the underlying mechanisms remain poorly understood. Here we used a mutant version of elongation factor TFIIS (TFIISmut) to specifically induce increased levels of RNAPII pausing, arrest, and/or backtracking in human cells. TFIISmut expression results in slower elongation rates, relative depletion of polymerases from the end of genes, and increased levels of stopped RNAPII. It affects mRNA splicing and termination as well. Remarkably, however, TFIISmut expression also dramatically increases R-loops, which may form at the anterior end of backtracked RNAPII and trigger genome instability, including DNA strand breaks. These results shed new light on the relationship between transcription stress and R-loops, and suggest that different classes of R-loops exist, potentially with distinct consequences for genome instability. Overall design: To study RNAPII backtracking and its effects in human cells, we used HEK293 TREX cells in which we overexpressed, under the control of a dox-promoter, a dominant negative form of TFIIS (TFIIS mut), an elongation factor necessary for stimulating RNAPII intrinsic cleavage activity. TFIISmut cells were maintained in the presence of Dox to ensure over-expression for 48 hours prior to harvest..
Publication Title
Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Homo sapiens
- 13 Downloadable Samples
- Illumina HumanHT-12 V4.0 expression beadchip
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
UV Irradiation Induces a Non-coding RNA that Functionally Opposes the Protein Encoded by the Same Gene.
Sample Metadata Fields
Cell line, Treatment, Time
View Samples- Homo sapiens
- 1 Downloadable Sample
- Illumina HumanHT-12 V4.0 expression beadchip
Description
The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ~25 kilobases is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter transcript isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The protein-coding ASCC3 isoform counteracts the function of the non-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and noncoding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage
Publication Title
UV Irradiation Induces a Non-coding RNA that Functionally Opposes the Protein Encoded by the Same Gene.
Sample Metadata Fields
Cell line, Treatment, Time
View Samples- Homo sapiens
- 12 Downloadable Samples
- Illumina HumanHT-12 V4.0 expression beadchip
Description
The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ~25 kilobases is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter transcript isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The protein-coding ASCC3 isoform counteracts the function of the non-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and noncoding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage
Publication Title
UV Irradiation Induces a Non-coding RNA that Functionally Opposes the Protein Encoded by the Same Gene.
Sample Metadata Fields
Cell line, Treatment, Time
View Samples
SRP094802- Homo sapiens
- 6 Downloadable Samples
- Illumina HiSeq 2000
Description
The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ~25 kilobases is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter transcript isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The protein-coding ASCC3 isoform counteracts the function of the non-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and noncoding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage. Overall design: Cells were treated with DRB (100 µM, 3.5 hrs), followed by UVC irradiation (15 J/m2) or left untreated. Cells were washed with PBS to remove DRB immediately after UV irradiation and incubated for 10, 25 or 40 minutes, followed by cell lysis and nuclei isolation. Nuclei were processed for GRO-Seq.
Publication Title
UV Irradiation Induces a Non-coding RNA that Functionally Opposes the Protein Encoded by the Same Gene.
Sample Metadata Fields
Cell line, Treatment, Subject, Time
View Samples- Homo sapiens
- 16 Downloadable Samples
- Affymetrix Human Exon 1.0 ST Array [transcript (gene) version (huex10st)
Description
Glucocorticoid excess is linked to central obesity, adipose tissue insulin resistance and type 2 diabetes mellitus. The aim of our study was to investigate the effects of dexamethasone on gene expression in human subcutaneous and omental adipose tissue, in order to identify potential novel mechanisms and biomarkers for glucocorticoid-induced insulin resistance in adipose tissue. Dexamethasone changed the expression of 527 genes in both subcutaneous and omental adipose tissue. FKBP5 and CNR1 were the most responsive genes in both depots (~7-fold increase). Dexamethasone increased FKBP5 gene and protein expression in a dose-dependent manner in both depots, but FKBP5 protein levels were 10-fold higher in omental than subcutaneous adipose tissue. FKBP5 gene expression in subcutaneous adipose tissue was positively correlated with serum insulin, HOMA-IR and subcutaneous adipocyte diameter, while fold change in gene expression by dexamethasone was negatively correlated with clinical markers of insulin resistance, i.e. HbA1c, BMI, HOMA-IR and serum insulin. Only one gene, SERTM1, clearly differed in response to dexamethasone between the two depots. Dexamethasone at high concentrations, influences gene expression in both subcutaneous and omental adipose tissue in a similar pattern and promotes gene expression of FKBP5, a gene that may be implicated in glucocorticoid-induced insulin resistance.
Publication Title
FKBP5 expression in human adipose tissue increases following dexamethasone exposure and is associated with insulin resistance.
Sample Metadata Fields
Sex, Age, Specimen part
View Samples- Mus musculus
- 24 Downloadable Samples
- Affymetrix Mouse Gene 1.0 ST Array (mogene10st)
Description
BACKGROUND: The transcript levels of many genes exhibit significant variation in tissue samples from inbred laboratory mice. A microarray experiment was designed to separate transcript abundance variation across samples from adipose, heart, kidney, and liver tissues of C57BL/6J mice into within-mouse and between-mouse components. Within-mouse variance captures variation due to heterogeneity of gene expression within tissues, RNA-extraction, and array processing. Between-mouse variance reflects differences in transcript levels between these genetically identical mice. Many biological sources can contribute to heterogeneous transcript levels within a tissue sample including inherent stochasticity of biochemical processes such as intrinsic and extrinsic noise within cells and differences in cell-type composition which can result from heterogeneity of stem and progenitor cell populations. Differences in global signaling patterns between individuals and micro-environmental influences such as interactions with pathogens and cage mates can also contribute to variation, but are likely to contribute more to the between-mouse variance component.
Publication Title
Stochastic variation of transcript abundance in C57BL/6J mice.
Sample Metadata Fields
Sex, Age, Specimen part
View Samples