NELF focuses sites of initiation and maintains promoter architecture.

Many factors control the elongation phase of transcription by RNA polymerase II (Pol II), a process that plays an essential role in regulating gene expression. We utilized cells expressing degradation tagged subunits of NELFB, PAF1 and RTF1 to probe the effects of depletion of the factors on nascent transcripts using PRO-Seq and on chromatin architecture using DFF-ChIP. Although NELF is involved in promoter proximal pausing, depletion of NELFB had only a minimal effect on the level of paused transcripts and almost no effect on control of productive elongation. Instead, NELF depletion increased the utilization of downstream transcription start sites and caused a dramatic, genome-wide loss of H3K4me3 marked nucleosomes. Depletion of PAF1 and RTF1 both had major effects on productive transcript elongation in gene bodies and also caused initiation site changes like those seen with NELFB depletion. Our study confirmed that the first nucleosome encountered during initiation and early elongation is highly positioned with respect to the major TSS. In contrast, the positions of H3K4me3 marked nucleosomes in promoter regions are heterogeneous and are influenced by transcription. We propose a model defining NELF function and a general role of the H3K4me3 modification in blocking transcription initiation.

Exhaustivity and Anti-Exhaustivity in the RSA Framework: Testing the Effect of Prior Beliefs.

During communication, the interpretation of utterances is sensitive to a listener's probabilistic prior beliefs. In this paper, we focus on the influence of prior beliefs on so-called exhaustivity interpretations, whereby a sentence such as Mary came is understood to mean that only Mary came. Two theoretical origins for exhaustivity effects have been proposed in the previous literature. On the one hand are perspectives that view these inferences as the result of a purely pragmatic process (as in the classical Gricean view, and more recent Bayesian approaches); on the other hand are proposals that treat them as the result of an encapsulated semantic mechanism (Chierchia, Fox & Spector 2012). We gain traction on adjudicating between these two approaches with new theoretical and experimental evidence, focusing on the behavior of different models for exhaustivity effects, all of which fit under the Rational Speech Act modeling framework (RSA, Frank & Goodman, 2012). Some (but not all!) of these models include an encapsulated semantic mechanism. Theoretically, we demonstrate that many RSA models predict not only exhaustivity, but also anti-exhaustivity, whereby "Mary came" would convey that Mary and someone else came. We evaluate these models against data obtained in a new study which tested the effects of prior beliefs on both production and comprehension, improving on previous empirical work. We find that the models which have the best fit to human behavior include an encapsulated exhaustivity mechanism. We conclude that, on the one hand, in the division of labor between semantics and pragmatics, semantics plays a larger role than is often thought, but, on the other hand, the tradeoff between informativity and cost which characterizes all RSA models does play a central role for genuine pragmatic effects.

The methylome and cell-free DNA: current applications in medicine and pediatric disease.

DNA methylation is an epigenetic mechanism that contributes to cell regulation and development, and different methylation patterns allow for the identification of cell and tissue type. Cell-free DNA (cfDNA) is composed of small circulating fragments of DNA found in plasma and urine. Total cfDNA levels correlate with the presence of inflammation and tissue injury in a variety of disease states. Unfortunately, the utility of cfDNA is limited by its lack of tissue or cell-type specificity. However, methylome analysis of cfDNA allows the identification of the tissue or cell type from which cfDNA originated. Thus, methylation patterns in cfDNA from tissues isolated from direct study may provide windows into health and disease states, thereby serving as a "liquid biopsy". This review will discuss methylation and its role in establishing cellular identity, cfDNA as a biomarker and its pathophysiologic role in the inflammatory process, and the ways cfDNA and methylomics can be jointly applied in medicine. IMPACT: Cell-free DNA (cfDNA) is increasingly being used as a noninvasive diagnostic and disease-monitoring tool in pediatric medicine. However, the lack of specificity of cfDNA limits its utility. Identification of cell type-specific methylation signatures can help overcome the limited specificity of cfDNA. As knowledge of the cfDNA methylome improves, cfDNA will be more broadly applied in medicine, such that clinicians will need to understand the methods and applications of its use.

Dynamic regulation of P-TEFb by 7SK snRNP is integral to the DNA damage response to regulate chemotherapy sensitivity.

Testicular germ cell tumors and closely related embryonal stem cells are exquisitely sensitive to cisplatin, a feature thought to be linked to their pluripotent state and p53 status. It remains unclear whether and how cellular state is coordinated with p53 to confer cisplatin sensitivity. Here, we report that positive transcription elongation factor b (P-TEFb) determines cell fate upon DNA damage. We find that cisplatin rapidly activates P-TEFb by releasing it from inhibitory 7SK small nuclear ribonucleoprotein complex. P-TEFb directly phosphorylates pluripotency factor estrogen-related receptor beta (ESRRB), and induces its proteasomal degradation to enhance pro-survival glycolysis. On the other hand, P-TEFb is required for the transcription of a substantial portion of p53 target genes, triggering cell death during prolonged cisplatin treatment. These results reveal previously underappreciated roles of P-TEFb to coordinate the DNA damage response. We discuss the implications for using P-TEFb inhibitors to treat cancer and ameliorate cisplatin-induced ototoxicity.

Human Cytomegalovirus Infection Elicits Global Changes in Host Transcription by RNA Polymerases I, II, and III.

How human cytomegalovirus (HCMV) infection impacts the transcription of the host genome remains incompletely understood. Here, we examine the global consequences of infection of primary human foreskin fibroblasts (HFFs) on transcription by RNA polymerase I, II, and III over the course of a lytic infection using PRO-Seq. The expected rapid induction of innate immune response genes is observed with specific subsets of genes exhibiting dissimilar expression kinetics. We find minimal effects on Pol II initiation, but increased rates of the release of paused Pol II into productive elongation are detected by 24 h postinfection and pronounced at late times postinfection. Pol I transcription increases during infection and we provide evidence for a potential Pol I elongation control mechanism. Pol III transcription of tRNA genes is dramatically altered, with many induced and some repressed. All effects are partially dependent on viral genome replication, suggesting a link to viral mRNA levels and/or a viral early-late or late gene product. Changes in tRNA transcription are connected to distinct alterations in the chromatin state around tRNA genes, which were probed with high-resolution DFF-ChIP. Additionally, evidence is provided that the Pol III PIC stably contacts an upstream -1 nucleosome. Finally, we compared and contrasted our HCMV data with results from published experiments with HSV-1, EBV, KSHV, and MHV68. We report disparate effects on Pol II transcription and potentially similar effects on Pol III transcription.

Differences in RNA polymerase II complexes and their interactions with surrounding chromatin on human and cytomegalovirus genomes.

Interactions of the RNA polymerase II (Pol II) preinitiation complex (PIC) and paused early elongation complexes with the first downstream (+1) nucleosome are thought to be functionally important. However, current methods are limited for investigating these relationships, both for cellular chromatin and the human cytomegalovirus (HCMV) genome. Digestion with human DNA fragmentation factor (DFF) before immunoprecipitation (DFF-ChIP) precisely revealed both similarities and major differences in PICs driven by TBP on the host genome in comparison with PICs driven by TBP or the viral-specific, late initiation factor UL87 on the viral genome. Host PICs and paused Pol II complexes are frequently found in contact with the +1 nucleosome and paused Pol II can also be found in a complex involved in the initial invasion of the +1 nucleosome. In contrast, viral transcription complexes have very limited nucleosomal interactions, reflecting a relative lack of chromatinization of transcriptionally active regions of HCMV genomes.

Nuclear export restricts Gdown1 to a mitotic function.

Approximately half of purified mammalian RNA polymerase II (Pol II) is associated with a tightly interacting sub-stoichiometric subunit, Gdown1. Previous studies have established that Gdown1 inhibits transcription initiation through competitive interactions with general transcription factors and blocks the Pol II termination activity of transcription termination factor 2 (TTF2). However, the biological functions of Gdown1 remain poorly understood. Here, we utilized genetic, microscopic, and multi-omics approaches to functionally characterize Gdown1 in three human cell lines. Acute depletion of Gdown1 caused minimal direct effects on transcription. We show that Gdown1 resides predominantly in the cytoplasm of interphase cells, shuttles between the cytoplasm and nucleus, and is regulated by nuclear export. Gdown1 enters the nucleus at the onset of mitosis. Consistently, genetic ablation of Gdown1 is associated with partial de-repression of mitotic transcription, and Gdown1 KO cells present with evidence of aberrant mitoses coupled to p53 pathway activation. Evidence is presented demonstrating that Gdown1 modulates the combined functions of purified productive elongation factors PAF1C, RTF1, SPT6, DSIF and P-TEFb in vitro. Collectively, our findings support a model wherein the Pol II-regulatory function of Gdown1 occurs during mitosis and is required for genome integrity.

Interpreting plural predication: homogeneity and non-maximality.

Plural definite descriptions across many languages display two well-known properties. First, they can give rise to so-called non-maximal readings, in the sense that they 'allow for exceptions' (Mary read the books on the reading list, in some contexts, can be judged true even if Mary didn't read all the books on the reading list). Second, while they tend to have a quasi-universal quantificational force in affirmative sentences ('quasi-universal' rather than simply 'universal' due to the possibility of exceptions we have just mentioned), they tend to be interpreted existentially in the scope of negation (a property often referred to as homogeneity, cf. Löbner in Linguist Philos 23:213-308, 2000). Building on previous works (in particular Krifka in Proceedings of SALT VI, Cornell University, pp 136-153, 1996 and Malamud in Semant Pragmat, 5:1-28, 2012), we offer a theory in which sentences containing plural definite expressions trigger a family of possible interpretations, and where general principles of language use account for their interpretation in various contexts and syntactic environments. Our theory solves a number of problems that these previous works encounter, and has broader empirical coverage in that it offers a precise analysis for sentences that display complex interactions between plural definites, quantifiers and bound variables, as well as for cases involving non-distributive predicates. The resulting proposal is briefly compared with an alternative proposal by Kriz (Aspects of homogeneity in the semantics of natural language, University of Vienna, 2015), which has similar coverage but is based on a very different architecture and sometimes makes subtly different predictions.

A unified view of the sequence and functional organization of the human RNA polymerase II promoter.

To better understand human RNA polymerase II (Pol II) promoters in the context of promoter-proximal pausing and local chromatin organization, 5' and 3' ends of nascent capped transcripts and the locations of nearby nucleosomes were accurately identified through sequencing at exceptional depth. High-quality visualization tools revealed a preferred sequence that defines over 177 000 core promoters with strengths varying by >10 000-fold. This sequence signature encompasses and better defines the binding site for TFIID and is surprisingly invariant over a wide range of promoter strength. We identified a sequence motif associated with promoter-proximal pausing and demonstrated that cap methylation only begins once transcripts are about 30 nt long. Mapping also revealed a ∼150 bp periodic downstream sequence element (PDE) following the typical pause location, strongly suggestive of a +1 nucleosome positioning element. A nuclear run-off assay utilizing the unique properties of the DNA fragmentation factor (DFF) coupled with sequencing of DFF protected fragments demonstrated that a +1 nucleosome is present downstream of paused Pol II. Our data more clearly define the human Pol II promoter: a TFIID binding site with built-in downstream information directing ubiquitous promoter-proximal pausing and downstream nucleosome location.

Renal Replacement Therapy in Neonates.

Acute kidney injury (AKI) is a highly prevalent disease entity in the NICU, affecting nearly one-quarter of critically ill neonates by some reports. Though medical management remains the mainstay in the treatment of AKI, renal replacement therapy (RRT) is indicated when conservative measures are unable to maintain electrolytes, fluid balance, toxins, or waste products within a safe margin. Several modalities of RRT exist for use in neonatal populations, including peritoneal dialysis, hemodialysis, and continuous RRT. It is the aim of this review to introduce each of these RRT modalities, as well as to discuss their technical considerations, benefits, indications, contraindications, and complications.

Functional interaction of human Ssu72 with RNA polymerase II complexes.

Phosphorylation of the C-terminal domain (CTD) of the large subunit of human RNA polymerase II (Pol II) is regulated during the transcription cycle by the combined action of specific kinases and phosphatases. Pol II enters into the preinitiation complex (PIC) unphosphorylated, but is quickly phosphorylated by Cdk7 during initiation. How phosphatases alter the pattern and extent of CTD phosphorylation at this early stage of transcription is not clear. We previously demonstrated the functional association of an early-acting, magnesium-independent phosphatase with early elongation complexes. Here we show that Ssu72 is responsible for that activity. We found that the phosphatase enters the transcription cycle during the formation of PICs and that Ssu72 is physically associated with very early elongation complexes. The association of Ssu72 with elongation complexes was stable to extensive washing with up to 200 mM KCl. Interestingly, Ssu72 ceased to function on complexes that contained RNA longer than 28 nt. However, when PICs were washed before initiation, the strict cutoff at 28 nt was lost. This suggests that factor(s) are important for the specific regulation of Ssu72 function during the transition between initiation and pausing. Overall, our results demonstrate when Ssu72 can act on early transcription complexes and suggest that Ssu72 may also function in the PIC prior to initiation.

Induction of calcitonin gene-related peptide expression in rats by cortical spreading depression.

OBJECTIVE: The neuropeptide calcitonin gene-related peptide (CGRP) has now been established as a key player in migraine. However, the mechanisms underlying the reported elevation of CGRP in the serum and cerebrospinal fluid of some migraineurs are not known. A candidate mechanism is cortical spreading depression (CSD), which is associated with migraine with aura and traumatic brain injury. The aim of this study was to investigate whether CGRP gene expression may be induced by experimental CSD in the rat cerebral cortex. METHODS: CSD was induced by topical application of KCl and monitored using electrophysiological methods. Quantitative PCR and ELISA were used to measure CGRP mRNA and peptide levels in discrete ipsilateral and contralateral cortical regions of the rat brain 24 hours following CSD events and compared with sham treatments. RESULTS: The data show that multiple, but not single, CSD events significantly increase CGRP mRNA levels at 24 hours post-CSD in the ipsilateral rat cerebral cortex. Increased CGRP was observed in the ipsilateral frontal, motor, somatosensory, and visual cortices, but not the cingulate cortex, or contralateral cortices. CSD also induced CGRP peptide expression in the ipsilateral, but not contralateral, cortex. CONCLUSIONS: Repeated CSD provides a mechanism for prolonged elevation of CGRP in the cerebral cortex, which may contribute to migraine and post-traumatic headache.

Revealing abstract semantic mechanisms through priming: The distributive/collective contrast.

Sentences such as The bags are light allow both collective (they are light together) and distributive interpretations (each bag is light). We report the results of two experiments showing that this collective/distributive contrast gives rise to priming effects. These findings suggest that collective and distributive readings involve different interpretative mechanisms, which are at play during real comprehension and can be targeted by priming, independently of the specific verification strategy associated with each interpretation.

Oxidative stress rapidly stabilizes promoter-proximal paused Pol II across the human genome.

Oxidative stress has pervasive effects on cells but how they respond transcriptionally upon the initial insult is incompletely understood. We developed a nuclear walk-on assay that semi-globally quantifies nascent transcripts in promoter-proximal paused RNA polymerase II (Pol II). Using this assay in conjunction with ChIP-Seq, in vitro transcription, and a chromatin retention assay, we show that within a minute, hydrogen peroxide causes accumulation of Pol II near promoters and enhancers that can best be explained by a rapid decrease in termination. Some of the accumulated polymerases slowly move or 'creep' downstream. This second effect is correlated with and probably results from loss of NELF association and function. Notably, both effects were independent of DNA damage and ADP-ribosylation. Our results demonstrate the unexpected speed at which a global transcriptional response can occur. The findings provide strong support for the residence time of paused Pol II elongation complexes being much shorter than estimated from previous studies.

Bladder Neck Contracture Following Radical Retropubic versus Robotic-Assisted Laparoscopic Prostatectomy.

INTRODUCTION: Radical retropubic prostatectomy (RRP) and robotic-assisted laparoscopic prostatectomy (RALP) are co-standard surgical therapies for localized prostatic adenocarcinoma. These surgical modalities offer similar outcomes; however, lower rate of bladder neck contracture (BNC) is amongst the touted benefits of RALP. The differences between approaches are largely elucidated through multiple-surgeon comparisons, which can be biased by differential experience and practice patterns. We aimed to eliminate inter-surgeon bias through this single-surgeon comparison of BNC rates following RRP and RALP. MATERIALS AND METHODS: We retrospectively reviewed all RRPs and RALPs performed by one surgeon over 4 years. We compared clinical characteristics, intraoperative and postoperative outcomes. RESULTS: RRP patients had more advanced cancer and a higher biochemical recurrence rate. No significant differences were noted between groups in rates of anastomotic leakage, BNC, or 12-month postoperative pad-free continence. CONCLUSION: RRP offers similar outcomes to RALP with regard to postoperative urinary extravasation, urinary continence, and BNC.

Priming methods in semantics and pragmatics.

Structural priming is a powerful method to inform linguistic theories. We argue that this method extends nicely beyond syntax to theories of meaning. Priming, however, should still be seen as only one of the tools available for linguistic data collection. Specifically, because priming can occur at different, potentially conflicting levels, it cannot detect every aspect of linguistic representations.

Molecular mechanisms underlying genotype-dependent responses to dietary restriction.

Dietary restriction (DR) increases lifespan and attenuates age-related phenotypes in many organisms; however, the effect of DR on longevity of individuals in genetically heterogeneous populations is not well characterized. Here, we describe a large-scale effort to define molecular mechanisms that underlie genotype-specific responses to DR. The effect of DR on lifespan was determined for 166 single gene deletion strains in Saccharomyces cerevisiae. Resulting changes in mean lifespan ranged from a reduction of 79% to an increase of 103%. Vacuolar pH homeostasis, superoxide dismutase activity, and mitochondrial proteostasis were found to be strong determinants of the response to DR. Proteomic analysis of cells deficient in prohibitins revealed induction of a mitochondrial unfolded protein response (mtUPR), which has not previously been described in yeast. Mitochondrial proteotoxic stress in prohibitin mutants was suppressed by DR via reduced cytoplasmic mRNA translation. A similar relationship between prohibitins, the mtUPR, and longevity was also observed in Caenorhabditis elegans. These observations define conserved molecular processes that underlie genotype-dependent effects of DR that may be important modulators of DR in higher organisms.