Transcriptional responses of cancer cells to heat shock-inducing stimuli involve amplification of robust HSF1 binding.

Responses of cells to stimuli are increasingly discovered to involve the binding of sequence-specific transcription factors outside of known target genes. We wanted to determine to what extent the genome-wide binding and function of a transcription factor are shaped by the cell type versus the stimulus. To do so, we induced the Heat Shock Response pathway in two different cancer cell lines with two different stimuli and related the binding of its master regulator HSF1 to nascent RNA and chromatin accessibility. Here, we show that HSF1 binding patterns retain their identity between basal conditions and under different magnitudes of activation, so that common HSF1 binding is globally associated with distinct transcription outcomes. HSF1-induced increase in DNA accessibility was modest in scale, but occurred predominantly at remote genomic sites. Apart from regulating transcription at existing elements including promoters and enhancers, HSF1 binding amplified during responses to stimuli may engage inactive chromatin.

Transcriptome sequencing suggests that pre-mRNA splicing counteracts widespread intronic cleavage and polyadenylation.

Alternative splicing (AS) and alternative polyadenylation (APA) are two crucial steps in the post-transcriptional regulation of eukaryotic gene expression. Protocols capturing and sequencing RNA 3'-ends have uncovered widespread intronic polyadenylation (IPA) in normal and disease conditions, where it is currently attributed to stochastic variations in the pre-mRNA processing. Here, we took advantage of the massive amount of RNA-seq data generated by the Genotype Tissue Expression project (GTEx) to simultaneously identify and match tissue-specific expression of intronic polyadenylation sites with tissue-specific splicing. A combination of computational methods including the analysis of short reads with non-templated adenines revealed that APA events are more abundant in introns than in exons. While the rate of IPA in composite terminal exons and skipped terminal exons expectedly correlates with splicing, we observed a considerable fraction of IPA events that lack AS support and attributed them to spliced polyadenylated introns (SPI). We hypothesize that SPIs represent transient byproducts of a dynamic coupling between APA and AS, in which the spliceosome removes the intron while it is being cleaved and polyadenylated. These findings indicate that cotranscriptional pre-mRNA splicing could serve as a rescue mechanism to suppress premature transcription termination at intronic polyadenylation sites.

Tissue-specific regulation of gene expression via unproductive splicing.

Eukaryotic gene expression is regulated post-transcriptionally by a mechanism called unproductive splicing, in which mRNA is triggered to degrade by the nonsense-mediated decay (NMD) pathway as a result of regulated alternative splicing (AS). Only a few dozen unproductive splicing events (USEs) are currently documented, and many more remain to be identified. Here, we analyzed RNA-seq experiments from the Genotype-Tissue Expression (GTEx) Consortium to identify USEs, in which an increase in the NMD isoform splicing rate is accompanied by tissue-specific down-regulation of the host gene. To characterize RNA-binding proteins (RBPs) that regulate USEs, we superimposed these results with RBP footprinting data and experiments on the response of the transcriptome to the perturbation of expression of a large panel of RBPs. Concordant tissue-specific changes between the expression of RBP and USE splicing rate revealed a high-confidence regulatory network including 27 tissue-specific USEs with strong evidence of RBP binding. Among them, we found previously unknown PTBP1-controlled events in the DCLK2 and IQGAP1 genes, for which we confirmed the regulatory effect using small interfering RNA (siRNA) knockdown experiments in the A549 cell line. In sum, we present a transcriptomic pipeline that allows the identification of tissue-specific USEs, potentially many more than were reported here using stringent filters.

Targeting the Transcriptome Through Globally Acting Components.

Transcription is a step in gene expression that defines the identity of cells and its dysregulation is associated with diseases. With advancing technologies revealing molecular underpinnings of the cell with ever-higher precision, our ability to view the transcriptomes may have surpassed our knowledge of the principles behind their organization. The human RNA polymerase II (Pol II) machinery comprises thousands of components that, in conjunction with epigenetic and other mechanisms, drive specialized programs of development, differentiation, and responses to the environment. Parts of these programs are repurposed in oncogenic transformation. Targeting of cancers is commonly done by inhibiting general or broadly acting components of the cellular machinery. The critical unanswered question is how globally acting or general factors exert cell type specific effects on transcription. One solution, which is discussed here, may be among the events that take place at genes during early Pol II transcription elongation. This essay turns the spotlight on the well-known phenomenon of promoter-proximal Pol II pausing as a step that separates signals that establish pausing genome-wide from those that release the paused Pol II into the gene. Concepts generated in this rapidly developing field will enhance our understanding of basic principles behind transcriptome organization and hopefully translate into better therapies at the bedside.

Polymorphism of G4 associates: from stacks to wires via interlocks.

We examined the assembly of DNA G-quadruplexes (G4s) into higher-order structures using atomic force microscopy, optical and electrophoretic methods, NMR spectroscopy and molecular modeling. Our results suggest that parallel blunt-ended G4s with single-nucleotide or modified loops may form different types of multimers, ranging from stacks of intramolecular structures and/or interlocked dimers and trimers to wires. Decreasing the annealing rate and increasing salt or oligonucleotide concentrations shifted the equilibrium from intramolecular G4s to higher-order structures. Control antiparallel and hybrid G4s demonstrated no polymorphism or aggregation in our experiments. The modification that mimics abasic sites (1',2'-dideoxyribose residues) in loops enhanced the oligomerization/multimerization of both the 2-tetrad and 3-tetrad G4 motifs. Our results shed light on the rules that govern G4 rearrangements. Gaining control over G4 folding enables the harnessing of the full potential of such structures for guided assembly of supramolecular DNA structures for nanotechnology.

Data on secondary structures and ligand interactions of G-rich oligonucleotides that defy the classical formula for G4 motifs.

The data provided in this article are related to the research article "The expanding repertoire of G4 DNA structures" [1]. Secondary structures of G-rich oligonucleotides (ONs) that represent "imperfect" G-quadruplex (G4) motifs, i.e., contain truncated or interrupted G-runs, were analyzed by optical methods. Presented data on ON structures include circular dichroism (CD) spectra, thermal difference spectra (TDS) and UV -melting curves of the ONs; and rotational relaxation times (RRT) of ethidium bromide (EtBr) complexes with the ONs. TDS, CD spectra and UV-melting curves can be used to characterize the topologies and thermal stabilities of the ON structures. RRTs are roughly proportional to the hydrodynamic volumes of the complexes and thus can be used to distinguish between inter- and intramolecular ON structures. Presented data on ON interactions with small molecules include fluorescence emission spectra of the G4 sensor thioflavin T (ThT) in complexes with the ONs, and CD-melting curves of the ONs in the presence of G4-stabilizing ligands N-methylmesoporphyrin IX (NMM) and pyridostatin (PDS). These data should be useful for comparative analyses of classical G4s and "defective"G4s, such as quadruplexes with vacancies or bulges.

The expanding repertoire of G4 DNA structures.

The definition of DNA and RNA G-quadruplexes (G4s) has recently been broadened to include structures with certain defects: bulges, G-vacancies or mismatches. Despite the striking progress in computational methods for assessing G4 folding propensity, predicting G4s with defects remains problematic, reflecting the enhanced sequential diversity of these motifs. "Imperfect" G4 motifs, i.e., those containing interrupted or truncated G-runs, are typically omitted from genomic analyses. We report here studies of G4s with defects and compare these structures with classical ("perfect") quadruplexes. Thermal stabilities and ligand interactions are also discussed. We exploited a simple in-house computational tool for mining putative G4s with defects in the human genome. The obtained profiles of the genomic distribution of imperfect G4 motifs were analyzed. Collectively, our findings suggest that, similar to classical G4s, imperfect G4s could be considered as potential regulatory elements, pathology biomarkers and therapeutic targets.