RNA polymerases are highly regulated molecular machines. We present a method (global run-on sequencing, GRO-seq) that maps the position, amount, and orientation of transcriptionally engaged RNA ...polymerases genome-wide. In this method, nuclear run-on RNA molecules are subjected to large-scale parallel sequencing and mapped to the genome. We show that peaks of promoter-proximal polymerase reside on ~30% of human genes, transcription extends beyond pre-messenger RNA 3' cleavage, and antisense transcription is prevalent. Additionally, most promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene. This divergent polymerase is associated with active genes but does not elongate effectively beyond the promoter. These results imply that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription.
We report the immediate effects of estrogen signaling on the transcriptome of breast cancer cells using global run-on and sequencing (GRO-seq). The data were analyzed using a new bioinformatic ...approach that allowed us to identify transcripts directly from the GRO-seq data. We found that estrogen signaling directly regulates a strikingly large fraction of the transcriptome in a rapid, robust, and unexpectedly transient manner. In addition to protein-coding genes, estrogen regulates the distribution and activity of all three RNA polymerases and virtually every class of noncoding RNA that has been described to date. We also identified a large number of previously undetected estrogen-regulated intergenic transcripts, many of which are found proximal to estrogen receptor binding sites. Collectively, our results provide the most comprehensive measurement of the primary and immediate estrogen effects to date and a resource for understanding rapid signal-dependent transcription in other systems.
Display omitted
► Global run-on sequencing shows immediate transcriptional effects of estrogen signaling ► Estrogen regulates 25% of the breast cancer transcriptome rapidly and transiently ► Estrogen regulates the production of all classes of coding and noncoding transcripts ► Estrogen regulates the production of many as yet unannotated intergenic transcripts
Recent genome-wide studies in metazoans have shown that RNA polymerase II (Pol II) accumulates to high densities on many promoters at a rate-limited step in transcription. However, the status of this ...Pol II remains an area of debate. Here, we compare quantitative outputs of a global run-on sequencing assay and chromatin immunoprecipitation sequencing assays and demonstrate that the majority of the Pol II on Drosophila promoters is transcriptionally engaged; very little exists in a preinitiation or arrested complex. These promoter-proximal polymerases are inhibited from further elongation by detergent-sensitive factors, and knockdown of negative elongation factor, NELF, reduces their levels. These results not only solidify the notion that pausing occurs at most promoters, but demonstrate that it is the major rate-limiting step in early transcription at these promoters. Finally, the divergent elongation complexes seen at mammalian promoters are far less prevalent in Drosophila, and this specificity in orientation correlates with directional core promoter elements, which are abundant in Drosophila.
Display omitted
► Unlike mammals, Drosophila promoters lack an upstream divergent peak of Pol II ► Promoter Pol II is functionally distinct from actively elongating Pol II ► Transcription at Drosophila enhancers can be bidirectional and involves pausing ► Most Pol II detected at promoters is paused, not uninitiated or arrested
The ability to regulate RNA transcription is critical for organismal development and survival. Lis and colleagues use several measurements to quantify different forms of RNA polymerase II (Pol II) throughout the Drosophila genome. They find that the major form of Pol II at promoters and enhancers is transcriptionally engaged and paused rather than in a preinitiation complex or arrested. This cements pausing as a major rate-limiting step that is likely to be co-opted by various signaling pathways to regulate transcription.
To understand the genetic mechanisms driving variant and IGHV4-34-expressing hairy-cell leukemias, we performed whole-exome sequencing of leukemia samples from ten affected individuals, including six ...with matched normal samples. We identified activating mutations in the MAP2K1 gene (encoding MEK1) in 5 of these 10 samples and in 10 of 21 samples in a validation set (overall frequency of 15/31), suggesting potential new strategies for treating individuals with these diseases.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Quantitative computational models play an increasingly important role in modern biology. Such models typically involve many free parameters, and assigning their values is often a substantial obstacle ...to model development. Directly measuring in vivo biochemical parameters is difficult, and collectively fitting them to other experimental data often yields large parameter uncertainties. Nevertheless, in earlier work we showed in a growth-factor-signaling model that collective fitting could yield well-constrained predictions, even when it left individual parameters very poorly constrained. We also showed that the model had a "sloppy" spectrum of parameter sensitivities, with eigenvalues roughly evenly distributed over many decades. Here we use a collection of models from the literature to test whether such sloppy spectra are common in systems biology. Strikingly, we find that every model we examine has a sloppy spectrum of sensitivities. We also test several consequences of this sloppiness for building predictive models. In particular, sloppiness suggests that collective fits to even large amounts of ideal time-series data will often leave many parameters poorly constrained. Tests over our model collection are consistent with this suggestion. This difficulty with collective fits may seem to argue for direct parameter measurements, but sloppiness also implies that such measurements must be formidably precise and complete to usefully constrain many model predictions. We confirm this implication in our growth-factor-signaling model. Our results suggest that sloppy sensitivity spectra are universal in systems biology models. The prevalence of sloppiness highlights the power of collective fits and suggests that modelers should focus on predictions rather than on parameters.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
After priming, naïve CD8
T lymphocytes establish specific heritable transcription programs that define progression to long-lasting memory cells or to short-lived effector cells. Although lineage ...specification is critical for protection, it remains unclear how chromatin dynamics contributes to the control of gene expression programs. We explored the role of gene silencing by the histone methyltransferase Suv39h1. In murine CD8
T cells activated after
infection, Suv39h1-dependent trimethylation of histone H3 lysine 9 controls the expression of a set of stem cell-related memory genes. Single-cell RNA sequencing revealed a defect in silencing of stem/memory genes selectively in
-defective T cell effectors. As a result,
-defective CD8
T cells show sustained survival and increased long-term memory reprogramming capacity. Thus, Suv39h1 plays a critical role in marking chromatin to silence stem/memory genes during CD8
T effector terminal differentiation.
Gastrointestinal stromal tumors (GIST) harbor driver mutations of signal transduction kinases such as KIT, or, alternatively, manifest loss-of-function defects in the mitochondrial succinate ...dehydrogenase (SDH) complex, a component of the Krebs cycle and electron transport chain. We have uncovered a striking divergence between the DNA methylation profiles of SDH-deficient GIST (n = 24) versus KIT tyrosine kinase pathway-mutated GIST (n = 39). Infinium 450K methylation array analysis of formalin-fixed paraffin-embedded tissues disclosed an order of magnitude greater genomic hypermethylation relative to SDH-deficient GIST versus the KIT-mutant group (84.9 K vs. 8.4 K targets). Epigenomic divergence was further found among SDH-mutant paraganglioma/pheochromocytoma (n = 29), a developmentally distinct SDH-deficient tumor system. Comparison of SDH-mutant GIST with isocitrate dehydrogenase-mutant glioma, another Krebs cycle-defective tumor type, revealed comparable measures of global hypo- and hypermethylation. These data expose a vital connection between succinate metabolism and genomic DNA methylation during tumorigenesis, and generally implicate the mitochondrial Krebs cycle in nuclear epigenomic maintenance.
Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary ...transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ∼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, "bivalent" ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5' proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation.
Disruption of splicing patterns due to mutations of genes coding splicing factors in tumors represents a potential source of tumor neoantigens, which would be both public (shared between patients) ...and tumor-specific (not expressed in normal tissues). In this study, we show that mutations of the splicing factor
in uveal melanoma generate such immunogenic neoantigens. Memory CD8
T cells specific for these neoantigens are preferentially found in 20% of patients with uveal melanoma bearing
-mutated tumors. Single-cell analyses of neoepitope-specific T cells from the blood identified large clonal T-cell expansions, with distinct effector transcription patterns. Some of these expanded T-cell receptors are also present in the corresponding tumors. CD8
T-cell clones specific for the neoepitopes specifically recognize and kill
-mutated tumor cells, supporting the use of this new family of neoantigens as therapeutic targets. SIGNIFICANCE: Mutations of the splicing factor
in uveal melanoma generate shared neoantigens that are uniquely expressed by tumor cells, leading to recognition and killing by specific CD8 T cells. Mutations in splicing factors can be sources of new therapeutic strategies applicable to diverse tumors.
.
Switch/sucrose nonfermentable–deficient cancers may respond to immune checkpoint blockade. Combinations with epidrugs or radiation therapies can offer new specific therapeutic vulnerabilities.
PDF
