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.

Stretching of a Fractal Polymer around a Disc Reveals Kardar-Parisi-Zhang Scaling.

While stretching of a polymer along a flat surface is hardly different from the classical Pincus problem of pulling chain ends in free space, the role of curved geometry in conformational statistics of the stretched chain is an exciting open question. We use scaling analysis and computer simulations to examine stretching of a fractal polymer chain around a disc in 2D (or a cylinder in 3D) of radius R. We reveal that the typical excursions of the polymer away from the surface and curvature-induced correlation length scale as Δ∼R^{ß} and S^{*}∼R^{1/z}, respectively, with the Kardar-Parisi-Zhang (KPZ) growth ß=1/3 and dynamic exponents z=3/2. Although probability distribution of excursions does not belong to KPZ universality class, the KPZ scaling is independent of the fractal dimension of the polymer and, thus, is universal across classical polymer models, e.g., SAW, randomly branching polymers, crumpled unknotted rings. Additionally, our Letter establishes a mapping between stretched polymers in curved geometry and the Balagurov-Vaks model of random walks among traps.

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.

Analysis of English free association network reveals mechanisms of efficient solution of Remote Association Tests.

We study correlations between the structure and properties of a free association network of the English language, and solutions of psycholinguistic Remote Association Tests (RATs). We show that average hardness of individual RATs is largely determined by relative positions of test words (stimuli and response) on the free association network. We argue that the solution of RATs can be interpreted as a first passage search problem on a network whose vertices are words and links are associations between words. We propose different heuristic search algorithms and demonstrate that in "easily-solving" RATs (those that are solved in 15 seconds by more than 64% subjects) the solution is governed by "strong" network links (i.e. strong associations) directly connecting stimuli and response, and thus the efficient strategy consist in activating such strong links. In turn, the most efficient mechanism of solving medium and hard RATs consists of preferentially following sequence of "moderately weak" associations.

Brownian flights over a circle.

The stationary radial distribution, P(ρ), of a random walk with the diffusion coefficient D, which winds at the tangential velocity V around an impenetrable disk of radius R for R≫D/V converges to the distribution involving the Airy function. Typical trajectories are localized in the circular strip [R,R+δR^{1/3}], where δ is a constant which depends on the parameters D and V and is independent of R.

The Histone Deacetylase SIRT6 Restrains Transcription Elongation via Promoter-Proximal Pausing.

Transcriptional regulation in eukaryotes occurs at promoter-proximal regions wherein transcriptionally engaged RNA polymerase II (Pol II) pauses before proceeding toward productive elongation. The role of chromatin in pausing remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 binds to Pol II and prevents the release of the negative elongation factor (NELF), thus stabilizing Pol II promoter-proximal pausing. Genetic depletion of SIRT6 or its chromatin deficiency upon glucose deprivation causes intragenic enrichment of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of cyclin-dependent kinase 9 (CDK9)-that phosphorylates NELF and the carboxyl terminal domain of Pol II-and enrichment of the positive transcription elongation factors MYC, BRD4, PAF1, and the super elongation factors AFF4 and ELL2. These events lead to increased expression of genes involved in metabolism, protein synthesis, and embryonic development. Our results identified SIRT6 as a Pol II promoter-proximal pausing-dedicated histone deacetylase.

Genome-wide RNA pol II initiation and pausing in neural progenitors of the rat.

BACKGROUND: Global RNA sequencing technologies have revealed widespread RNA polymerase II (Pol II) transcription outside of gene promoters. Small 5'-capped RNA sequencing (Start-seq) originally developed for the detection of promoter-proximal Pol II pausing has helped improve annotation of Transcription Start Sites (TSSs) of genes as well as identification of non-genic regulatory elements. However, apart from the most well studied genomes of human and mouse, mammalian transcription has not been profiled with sufficiently high precision. RESULTS: We prepared and sequenced Start-seq libraries from rat (Rattus norgevicus) primary neural progenitor cells. Over 48 million uniquely mappable reads from two independent biological replicates allowed us to define the TSSs of 7365 known genes in the rn6 genome, reannotating 2503 TSSs by more than 5 base pairs, characterize promoter-associated antisense transcription, and profile Pol II pausing. By combining TSS data with polyA-selected RNA sequencing, we also identified thousands of potential new genes producing stable RNA as well as non-genic transcripts representing possible regulatory elements. CONCLUSIONS: Our study has produced the first Start-seq dataset for the rat. Apart from profiling transcription initiation, our data reaffirm the prevalence of Pol II pausing across the rat genome and indicate conservation of pausing mechanisms across metazoan genomes. We suggest that pausing location, at least in mammals, is constrained by a distance from initiation of transcription, whether it occurs at or outside of a gene promoter. Abundant antisense transcription initiation around protein coding genes indicates that Pol II recruited to the vicinity of a promoter is distributed to available start sites of transcription at either DNA strand. Transcriptome profiling of neural progenitors presented here will facilitate further studies of other rat cell types as well as other organisms.

Anomalous one-dimensional fluctuations of a simple two-dimensional random walk in a large-deviation regime.

The following question is the subject of our work: could a two-dimensional (2D) random path pushed by some constraints to an improbable "large-deviation regime" possess extreme statistics with one-dimensional (1D) Kardar-Parisi-Zhang (KPZ) fluctuations? The answer is positive, though nonuniversal, since the fluctuations depend on the underlying geometry. We consider in detail two examples of 2D systems for which imposed external constraints force the underlying stationary stochastic process to stay in an atypical regime with anomalous statistics. The first example deals with the fluctuations of a stretched 2D random walk above a semicircle or a triangle. In the second example we consider a 2D biased random walk along a channel with forbidden voids of circular and triangular shapes. In both cases we are interested in the dependence of a typical span 〈d(t)〉∼t^{γ} of the trajectory of t steps above the top of the semicircle or the triangle. We show that γ=1/3, i.e., 〈d(t)〉 shares the KPZ statistics for the semicircle, while γ=0 for the triangle. We propose heuristic derivations of scaling exponents γ for different geometries, justify them by explicit analytic computations, and compare with numeric simulations. For practical purposes, our results demonstrate that the geometry of voids in a channel might have a crucial impact on the width of the boundary layer and, thus, on the heat transfer in the channel.

PARP-1/2 Inhibitor Olaparib Prevents or Partially Reverts EMT Induced by TGF-ß in NMuMG Cells.

Poly- adenosine diphosphate (ADP)-ribose (PAR) is a polymer synthesized as a posttranslational modification by some poly (ADP-ribose) polymerases (PARPs), namely PARP-1, PARP-2, tankyrase-1, and tankyrase-2 (TNKS-1/2). PARP-1 is nuclear and has also been detected in extracellular vesicles. PARP-2 and TNKS-1/2 are distributed in nuclei and cytoplasm. PARP or PAR alterations have been described in tumors, and in particular by influencing the Epithelial- Mesenchymal Transition (EMT), which influences cell migration and drug resistance in cancer cells. Pro-EMT and anti-EMT effects of PARP-1 have been reported while whether PAR changes occur specifically during EMT is currently unknown. The PARP-1/2 inhibitor Olaparib (OLA) is approved by FDA to treat certain patients harboring cancers with impaired homologous recombination. Here, we studied PAR changes and OLA effects on EMT. Total and nuclear PAR increased in EMT while PAR belts were disassembled. OLA prevented EMT, according to: (i) molecular markers evaluated by immuno-cytofluorescence/image quantification, Western blots, and RNA quantitation, (ii) morphological changes expressed as anisotropy, and (iii) migration capacity in the scratch assay. OLA also partially reversed EMT. OLA might work through unconventional mechanisms of action (different from synthetic lethality), even in non-BRCA (breast cancer 1 gene) mutated cancers.

From geometric optics to plants: the eikonal equation for buckling.

Optimal buckling of a tissue, e.g. a plant leaf, growing by means of exponential division of its peripheral cells, is considered in the framework of a conformal approach. It is shown that the boundary profile of a tissue is described by the 2D eikonal equation, which provides the geometric optic approximation for the wavefront propagating in a medium with an inhomogeneous refraction coefficient. A variety of optimal surfaces embedded in 3D is controlled by spatial dependence of the refraction coefficient which, in turn, is dictated by the local growth protocol.

MicroRNA and mRNA Transcriptome Profiling in Primary Human Astrocytes Infected with Borrelia burgdorferi.

Lyme disease is caused by infection with the bacterium Borrelia burgdorferi (Bb), which is transmitted to humans by deer ticks. The infection manifests usually as a rash and minor systemic symptoms; however, the bacteria can spread to other tissues, causing joint pain, carditis, and neurological symptoms. Lyme neuroborreliosis presents itself in several ways, such as Bell's palsy, meningitis, and encephalitis. The molecular basis for neuroborreliosis is poorly understood. Analysis of the changes in the expression levels of messenger RNAs and non-coding RNAs, including microRNAs, following Bb infection could therefore provide vital information on the pathogenesis and clinical symptoms of neuroborreliosis. To this end, we used cultured primary human astrocytes, key responders to CNS infection and important components of the blood-brain barrier, as a model system to study RNA and microRNA changes in the CNS caused by Bb. Using whole transcriptome RNA-seq, we found significant changes in 38 microRNAs and 275 mRNAs at 24 and 48 hours following Bb infection. Several of the RNA changes affect pathways involved in immune response, development, chromatin assembly (including histones) and cell adhesion. Further, several of the microRNA predicted target mRNAs were also differentially regulated. Overall, our results indicate that exposure to Bb causes significant changes to the transcriptome and microRNA profile of astrocytes, which has implications in the pathogenesis, and hence potential treatment strategies to combat this disease.

RNA polymerase II pausing as a context-dependent reader of the genome.

The RNA polymerase II (Pol II) transcribes all mRNA genes in eukaryotes and is among the most highly regulated enzymes in the cell. The classic model of mRNA gene regulation involves recruitment of the RNA polymerase to gene promoters in response to environmental signals. Higher eukaryotes have an additional ability to generate multiple cell types. This extra level of regulation enables each cell to interpret the same genome by committing to one of the many possible transcription programs and executing it in a precise and robust manner. Whereas multiple mechanisms are implicated in cell type-specific transcriptional regulation, how one genome can give rise to distinct transcriptional programs and what mechanisms activate and maintain the appropriate program in each cell remains unclear. This review focuses on the process of promoter-proximal Pol II pausing during early transcription elongation as a key step in context-dependent interpretation of the metazoan genome. We highlight aspects of promoter-proximal Pol II pausing, including its interplay with epigenetic mechanisms, that may enable cell type-specific regulation, and emphasize some of the pertinent questions that remain unanswered and open for investigation.

On predicting regulatory genes by analysis of functional networks in C. elegans.

BACKGROUND: Connectivity networks, which reflect multiple interactions between genes and proteins, possess not only a descriptive but also a predictive value, as new connections can be extrapolated and tested by means of computational analysis. Integration of different types of connectivity data (such as co-expression and genetic interactions) in one network has proven to benefit 'guilt by association' analysis. However predictive values of connectives of different types, that had their specific functional meaning and topological characteristics were not obvious, and have been addressed in this analysis. METHODS: eQTL data for 3 experimental C.elegans age groups were retrieved from WormQTL. WormNet has been used to obtain pair-wise gene interactions. The Shortest Path Function (SPF) has been adopted for statistical validation of the co-expressed gene clusters and for computational prediction of their potential gene expression regulators from a network context. A new SPF-based algorithm has been applied to genetic interactions sub-networks adjacent to the clusters of co-expressed genes for ranking the most likely gene expression regulators causal to eQTLs. RESULTS: We have demonstrated that known co-expression and genetic interactions between C. elegans genes can be complementary in predicting gene expression regulators. Several algorithms were compared in respect to their predictive potential in different network connectivity contexts. We found that genes associated with eQTLs are highly clustered in a C. elegans co-expression sub-network, and their adjacent genetic interactions provide the optimal functional connectivity environment for application of the new SPF-based algorithm. It was successfully tested in the reverse-prediction analysis on groups of genes with known regulators and applied to co-expressed genes and experimentally observed expression quantitative trait loci (eQTLs). CONCLUSIONS: This analysis demonstrates differences in topology and connectivity of co-expression and genetic interactions sub-networks in WormNet. The modularity of less continuous genetic interaction network does not correspond to modularity of the dense network comprised by gene co-expression interactions. However the genetic interaction network can be used much more efficiently with the SPF method in prediction of potential regulators of gene expression. The developed method can be used for validation of functional significance of suggested eQTLs and a discovery of new regulatory modules.

Analysis of paired end Pol II ChIP-seq and short capped RNA-seq in MCF-7 cells.

While a role of promoter-proximal RNA Polymerase II (Pol II) pausing in regulation of eukaryotic gene expression is implied, the mechanisms and dynamics of this process are poorly understood. We performed genome-wide analysis of short capped RNAs (scRNAs) and Pol II chromatin immunoprecipitation sequencing (ChIP-seq) in human breast cancer MCF-7 cells to better understand Pol II pausing (Samarakkody, A., Abbas, A., Scheidegger, A., Warns, J., Nnoli, O., Jokinen, B., Zarns, K., Kubat, B., Dhasarathy, A. and Nechaev, S. (2015) RNA polymerase II pausing can be retained or acquired during activation of genes involved in the epithelial to mesenchymal transition. Nucleic Acids Res43, 3938-3949). The data are available at the NCBI Gene Expression Omnibus under accession number GSE67041. For both ChIP and scRNA samples, we used paired end sequencing on the Illumina MiSeq instrument. For ChIP-seq, the use of paired end sequencing allowed us to avoid ambiguities in center-read definition. For scRNA seq, this allowed us to identify both the 5'-end and the 3'-end in the same run that represent, respectively, the transcription start sites and the locations of Pol II pausing. The sharpening of Pol II ChIP-seqmetagene profiles when aligned against 5'-ends of scRNAs indicates that these RNAs can be used to define the start sites for the majority of mRNA transcription events.

Epigenetic Modulation of Microglial Inflammatory Gene Loci in Helminth-Induced Immune Suppression: Implications for Immune Regulation in Neurocysticercosis.

In neurocysticercosis, parasite-induced immune suppressive effects are thought to play an important role in enabling site-specific inhibition of inflammatory responses to infections. It is axiomatic that microglia-mediated (M1 proinflammatory) response causes central nervous system inflammation; however, the mechanisms by which helminth parasites modulate microglia activation remain poorly understood. Here, we show that microglia display a diminished expression of M1-inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and nitric oxide synthase 2 (NOS2) in murine neurocysticercosis. Microglia also exhibited a lack of myeloid cell maturation marker major histocompatibility complex (MHC)-II in these parasite-infected brains. Treatment of microglia with helminth soluble/secreted factors (HSFs) in vitro did not induce expression of M1-inflammatory signature molecule NOS2 as well as MHC-II in primary microglia. However, HSF treatment completely inhibited lipopolysaccharide-induced increase in expression of MHC-II, NOS2 and nitric oxide production in these cells. As epigenetic modulation of chromatin states that regulates recruitment of RNA polymerase II (Pol-II) is a key regulatory step in determining gene expression and functional outcome, we next evaluated whether HSF induced modulation of these phenomenon in microglia in vitro. Indeed, HSF downregulated Pol-II recruitment to the promoter region of TNF-α, IL-6, NOS2, MHC-II, and transcription factor CIITA (a regulator of MHC-II expression), by itself. Moreover, HSF suppressed the lipopolysaccharide-induced increase in Pol-II recruitment as well. In addition, HSF exposure reduced the positive histone marks H3K4Me3 and H3K9/14Ac at the promoter of TNF-α, IL-6, NOS2, MHC-II, and CIITA. These studies provide a novel mechanistic insight into helminth-mediated immune suppression in microglia via modulation of epigenetic processes.

Bidirectional Transcription Arises from Two Distinct Hubs of Transcription Factor Binding and Active Chromatin.

Anti-sense transcription originating upstream of mammalian protein-coding genes is a well-documented phenomenon, but remarkably little is known about the regulation or function of anti-sense promoters and the non-coding RNAs they generate. Here we define at nucleotide resolution the divergent transcription start sites (TSSs) near mouse mRNA genes. We find that coupled sense and anti-sense TSSs precisely define the boundaries of a nucleosome-depleted region (NDR) that is highly enriched in transcription factor (TF) motifs. Notably, as the distance between sense and anti-sense TSSs increases, so does the size of the NDR, the level of signal-dependent TF binding, and gene activation. We further discover a group of anti-sense TSSs in macrophages with an enhancer-like chromatin signature. Interestingly, this signature identifies divergent promoters that are activated during immune challenge. We propose that anti-sense promoters serve as platforms for TF binding and establishment of active chromatin to further regulate or enhance sense-strand mRNA expression.

RNA polymerase II pausing can be retained or acquired during activation of genes involved in the epithelial to mesenchymal transition.

Promoter-proximal RNA polymerase II (Pol II) pausing is implicated in the regulation of gene transcription. However, the mechanisms of pausing including its dynamics during transcriptional responses remain to be fully understood. We performed global analysis of short capped RNAs and Pol II Chromatin Immunoprecipitation sequencing in MCF-7 breast cancer cells to map Pol II pausing across the genome, and used permanganate footprinting to specifically follow pausing during transcriptional activation of several genes involved in the epithelial to mesenchymal transition (EMT). We find that the gene for EMT master regulator Snail (SNAI1), but not Slug (SNAI2), shows evidence of Pol II pausing before activation. Transcriptional activation of the paused SNAI1 gene is accompanied by a further increase in Pol II pausing signal, whereas activation of non-paused SNAI2 gene results in the acquisition of a typical pausing signature. The increase in pausing signal reflects increased transcription initiation without changes in Pol II pausing. Activation of the heat shock HSP70 gene involves pausing release that speeds up Pol II turnover, but does not change pausing location. We suggest that Pol II pausing is retained during transcriptional activation and can further undergo regulated release in a signal-specific manner.

Effects of topological constraints on globular polymers.

Topological constraints can affect both equilibrium and dynamic properties of polymer systems and can play a role in the organization of chromosomes. Despite many theoretical studies, the effects of topological constraints on the equilibrium state of a single compact polymer have not been systematically studied. Here we use simulations to address this longstanding problem. We find that sufficiently long unknotted polymers differ from knotted ones in the spatial and topological states of their subchains. The unknotted globule has subchains that are mostly unknotted and form asymptotically compact RG(s)∼s1/3 crumples. However, crumples display a high fractal dimension of the surface db=2.8, forming excessive contacts and interpenetrating each other. We conclude that this topologically constrained equilibrium state resembles a conjectured crumpled globule [Grosberg et al., Journal de Physique, 1988, 49, 2095], but differs from its idealized hierarchy of self-similar, isolated and compact crumples.

A statistical model of intra-chromosome contact maps.

A statistical model describing a fine structure of the intra-chromosome maps obtained by a genome-wide chromosome conformation capture method (Hi-C) is proposed. The model combines hierarchical chain folding with a quenched heteropolymer structure of primary chromatin sequences. It is conjectured that the observed Hi-C maps are statistical averages over many different ways of hierarchical genome folding. It is shown that the existence of a quenched primary structure coupled with hierarchical folding induces a full range of features observed in experimental Hi-C maps: hierarchical elements, chess-board intermittency and large-scale compartmentalization.

Searching the Human Genome for Snail and Slug With DNA@Home.

DNA@Home is a volunteer computing project that aims to use Gibbs Sampling for the identification and location of DNA control signals on full genome-scale datasets. A fault tolerant and asynchronous implementation of Gibbs sampling using the Berkeley Open Infrastructure for Network Computing (BOINC) was used to identify the location of binding sites of the SNAI1 (Snail) and SNAI2 (Slug) transcription factors across the human genome. Genes regulated by Slug but not Snail, and genes regulated by Snail but not Slug provided two datasets with known motifs. These datasets contained up to 994 DNA sequences which to our knowledge is largest scale use of Gibbs sampling for discovery of binding sites. 1000 parallel sampling walks were used to search for the presence of 1, 2 or 3 possible motifs using small, medium, and full size sets of these sequences. These runs were performed over a period of two months using over 1500 volunteered computing hosts and generated over 2.2 Terabytes of sampling data. High performance computing resources were used for post processing. This paper presents intra and inter walk analyses used to determine walk convergence. The results were validated against current biological knowledge of the Snail and Slug promoter regions and present avenues for further biological study.