K29-linked free polyubiquitin chains affect ribosome biogenesis and direct ribosomal proteins to the intranuclear quality control compartment.

Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs) Ubp2 and Ubp14, and E3 ligases Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the intranuclear quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with ribosomopathies.

Rif1 and Rif2 shape telomere function and architecture through multivalent Rap1 interactions.

Yeast telomeres comprise irregular TG1₋3 DNA repeats bound by the general transcription factor Rap1. Rif1 and Rif2, along with Rap1, form the telosome, a protective cap that inhibits telomerase, counteracts SIR-mediated transcriptional silencing, and prevents inadvertent recognition of telomeres as DNA double-strand breaks. We provide a molecular, biochemical, and functional dissection of the protein backbone at the core of the yeast telosome. The X-ray structures of Rif1 and Rif2 bound to the Rap1 C-terminal domain and that of the Rif1 C terminus are presented. Both Rif1 and Rif2 have separable and independent Rap1-binding epitopes, allowing Rap1 binding over large distances (42-110 Å). We identify tetramerization (Rif1) and polymerization (Rif2) modules that, in conjunction with the long-range binding, give rise to a higher-order architecture that interlinks Rap1 units. This molecular Velcro relies on Rif1 and Rif2 to recruit and stabilize Rap1 on telomeric arrays and is required for telomere homeostasis in vivo.

Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor.

Pioneer transcription factors (pTFs) bind to target sites within compact chromatin, initiating chromatin remodeling and controlling the recruitment of downstream factors. The mechanisms by which pTFs overcome the chromatin barrier are not well understood. Here, we reveal, using single-molecule fluorescence, how the yeast transcription factor Rap1 invades and remodels chromatin. Using a reconstituted chromatin system replicating yeast promoter architecture, we demonstrate that Rap1 can bind nucleosomal DNA within a chromatin fiber but with shortened dwell times compared to naked DNA. Moreover, we show that Rap1 binding opens chromatin fiber structure by inhibiting inter-nucleosome contacts. Finally, we reveal that Rap1 collaborates with the chromatin remodeler RSC to displace promoter nucleosomes, paving the way for long-lived bound states on newly exposed DNA. Together, our results provide a mechanistic view of how Rap1 gains access and opens chromatin, thereby establishing an active promoter architecture and controlling gene expression.

Fork pausing complex engages topoisomerases at the replisome.

Replication forks temporarily or terminally pause at hundreds of hard-to-replicate regions around the genome. A conserved pair of budding yeast replisome components Tof1-Csm3 (fission yeast Swi1-Swi3 and human TIMELESS-TIPIN) act as a "molecular brake" and promote fork slowdown at proteinaceous replication fork barriers (RFBs), while the accessory helicase Rrm3 assists the replisome in removing protein obstacles. Here we show that the Tof1-Csm3 complex promotes fork pausing independently of Rrm3 helicase by recruiting topoisomerase I (Top1) to the replisome. Topoisomerase II (Top2) partially compensates for the pausing decrease in cells when Top1 is lost from the replisome. The C terminus of Tof1 is specifically required for Top1 recruitment to the replisome and fork pausing but not for DNA replication checkpoint (DRC) activation. We propose that forks pause at proteinaceous RFBs through a "sTOP" mechanism ("slowing down with topoisomerases I-II"), which we show also contributes to protecting cells from topoisomerase-blocking agents.

Sfp1 regulates transcriptional networks driving cell growth and division through multiple promoter-binding modes.

The yeast Sfp1 protein regulates both cell division and growth but how it coordinates these processes is poorly understood. We demonstrate that Sfp1 directly controls genes required for ribosome production and many other growth-promoting processes. Remarkably, the complete set of Sfp1 target genes is revealed only by a combination of ChIP (chromatin immunoprecipitation) and ChEC (chromatin endogenous cleavage) methods, which uncover two promoter binding modes, one requiring a cofactor and the other a DNA-recognition motif. Glucose-regulated Sfp1 binding at cell cycle "START" genes suggests that Sfp1 controls cell size by coordinating expression of genes implicated in mass accumulation and cell division.

General Regulatory Factors Control the Fidelity of Transcription by Restricting Non-coding and Ectopic Initiation.

The fidelity of transcription initiation is essential for accurate gene expression, but the determinants of start site selection are not fully understood. Rap1 and other general regulatory factors (GRFs) control the expression of many genes in yeast. We show that depletion of these factors induces widespread ectopic transcription initiation within promoters. This generates many novel non-coding RNAs and transcript isoforms with diverse stability, drastically altering the coding potential of the transcriptome. Ectopic transcription initiation strongly correlates with altered nucleosome positioning. We provide evidence that Rap1 can suppress ectopic initiation by a "place-holder" mechanism whereby it physically occludes inappropriate sites for pre-initiation complex formation. These results reveal an essential role for GRFs in the fidelity of transcription initiation and in the suppression of pervasive transcription, profoundly redefining current models for their function. They have important implications for the mechanism of transcription initiation and the control of gene expression.

Sequence-Directed Action of RSC Remodeler and General Regulatory Factors Modulates +1 Nucleosome Position to Facilitate Transcription.

Accessible chromatin is important for RNA polymerase II recruitment and transcription initiation at eukaryotic promoters. We investigated the mechanistic links between promoter DNA sequence, nucleosome positioning, and transcription. Our results indicate that positioning of the transcription start site-associated +1 nucleosome in yeast is critical for efficient TBP binding and is driven by two key factors, the essential chromatin remodeler RSC and a small set of ubiquitous general regulatory factors (GRFs). Our findings indicate that the strength and directionality of RSC action on promoter nucleosomes depends on the arrangement and proximity of two specific DNA motifs. This, together with the effect on nucleosome position observed in double depletion experiments, suggests that, despite their widespread co-localization, RSC and GRFs predominantly act through independent signals to generate accessible chromatin. Our results provide mechanistic insight into how the promoter DNA sequence instructs trans-acting factors to control nucleosome architecture and stimulate transcription initiation.

Distinct patterns of histone acetyltransferase and Mediator deployment at yeast protein-coding genes.

The transcriptional coactivators Mediator and two histone acetyltransferase (HAT) complexes, NuA4 and SAGA, play global roles in transcriptional activation. Here we explore the relative contributions of these factors to RNA polymerase II association at specific genes and gene classes by rapid nuclear depletion of key complex subunits. We show that the NuA4 HAT Esa1 differentially affects certain groups of genes, whereas the SAGA HAT Gcn5 has a weaker but more uniform effect. Relative dependence on Esa1 and Tra1, a shared component of NuA4 and SAGA, distinguishes two large groups of coregulated growth-promoting genes. In contrast, we show that the activity of Mediator is particularly important at a separate, small set of highly transcribed TATA-box-containing genes. Our analysis indicates that at least three distinct combinations of coactivator deployment are used to generate moderate or high transcription levels and suggests that each may be associated with distinct forms of regulation.

A Reply to "MNase-Sensitive Complexes in Yeast: Nucleosomes and Non-histone Barriers," by Chereji et al.

In this issue of Molecular Cell, Chereji et al. (2017) present new data on MNase-sensitive particles previously identified upstream of transcription start sites at many promoters in budding yeast, and they argue, based upon negative histone-ChIP results, that they are non-nucleosomal signals generated by transcription factors (TFs). We show instead, based upon functional experiments where the relevant TFs are rapidly depleted, that this explanation does not hold, and we argue instead that histone ChIP and chemical cleavage assays have a limited capacity to capture these highly dynamic, MNase-sensitive "fragile" nucleosomes.

A Molecular Titration System Coordinates Ribosomal Protein Gene Transcription with Ribosomal RNA Synthesis.

Cell growth potential is determined by the rate of ribosome biogenesis, a complex process that requires massive and coordinated transcriptional output. In the yeast Saccharomyces cerevisiae, ribosome biogenesis is highly regulated at the transcriptional level. Although evidence for a system that coordinates ribosomal RNA (rRNA) and ribosomal protein gene (RPG) transcription has been described, the molecular mechanisms remain poorly understood. Here we show that an interaction between the RPG transcriptional activator Ifh1 and the rRNA processing factor Utp22 serves to coordinate RPG transcription with that of rRNA. We demonstrate that Ifh1 is rapidly released from RPG promoters by a Utp22-independent mechanism following growth inhibition, but that its long-term dissociation requires Utp22. We present evidence that RNA polymerase I activity inhibits the ability of Utp22 to titrate Ifh1 from RPG promoters and propose that a dynamic Ifh1-Utp22 interaction fine-tunes RPG expression to coordinate RPG and rRNA transcription.

Nucleosome Stability Distinguishes Two Different Promoter Types at All Protein-Coding Genes in Yeast.

Previous studies indicate that eukaryotic promoters display a stereotypical chromatin landscape characterized by a well-positioned +1 nucleosome near the transcription start site and an upstream -1 nucleosome that together demarcate a nucleosome-free (or -depleted) region. Here we present evidence that there are two distinct types of promoters distinguished by the resistance of the -1 nucleosome to micrococcal nuclease digestion. These different architectures are characterized by two sequence motifs that are broadly deployed at one set of promoters where a nuclease-sensitive ("fragile") nucleosome forms, but concentrated in a narrower, nucleosome-free region at all other promoters. The RSC nucleosome remodeler acts through the motifs to establish stable +1 and -1 nucleosome positions, while binding of a small set of general regulatory (pioneer) factors at fragile nucleosome promoters plays a key role in their destabilization. We propose that the fragile nucleosome promoter architecture is adapted for regulation of highly expressed, growth-related genes.

Developmental changes in audiotactile event perception.

The fission and fusion illusions provide measures of multisensory integration. The sound-induced tap fission illusion occurs when a tap is paired with two distractor sounds, resulting in the perception of two taps; the sound-induced tap fusion illusion occurs when two taps are paired with a single sound, resulting in the perception of a single tap. Using these illusions, we measured integration in three groups of children (9-, 11-, and 13-year-olds) and compared them with a group of adults. Based on accuracy, we derived a measure of magnitude of illusion and used a signal detection analysis to estimate perceptual discriminability and decisional criterion. All age groups showed a significant fission illusion, whereas only the three groups of children showed a significant fusion illusion. When compared with adults, the 9-year-olds showed larger fission and fusion illusions (i.e., reduced discriminability and greater bias), whereas the 11-year-olds were adult-like for fission but showed some differences for fusion: significantly worse discriminability and marginally greater magnitude and criterion. The 13-year-olds were adult-like on all measures. Based on the pattern of data, we speculate that the developmental trajectories for fission and fusion differ. We discuss these developmental results in the context of three non-mutually exclusive theoretical frameworks: sensory dominance, maximum likelihood estimation, and causal inference.

Lung and extrathoracic cancer incidence among underground uranium miners exposed to radon progeny in the Príbram region of the Czech Republic: a case-cohort study.

OBJECTIVES: Radon is carcinogenic, but more studies are needed to understand relationships with lung cancer and extrathoracic cancers at low exposures. There are few studies evaluating associations with cancer incidence or assessing the modifying effects of smoking. METHODS: We conducted a case-cohort study with 16 434 underground uranium miners in the Czech Republic with cancer incidence follow-up 1977-1996. Associations between radon exposure and lung cancer, and extrathoracic cancer, were estimated with linear excess relative rate (ERR) models. We examined potential modifying effects of smoking, time since exposure and exposure rate. RESULTS: Under a simple ERR model, assuming a 5-year exposure lag, the estimated ERR of lung cancer per 100 working level months (WLM) was 0.54 (95% CI 0.33 to 0.83) and the estimated ERR of extrathoracic cancer per 100 WLM was 0.07 (95% CI -0.17 to 0.72). Most lung cancer cases were observed among smokers (82%), and the estimated ERR of lung cancer per 100 WLM was larger among smokers (ERR/100 WLM=1.35; 95% CI 0.84 to 2.15) than among never smokers (ERR/100 WLM=0.12; 95% CI -0.05 to 0.49). Among smokers, the estimated ERR of lung cancer per 100 WLM decreased with time since exposure from 3.07 (95% CI -0.04 to 10.32) in the period 5-14 years after exposure to 1.05 (95% CI 0.49 to 1.87) in the period 25+ years after exposure. CONCLUSIONS: We observed positive associations between cumulative radon exposure and lung cancer, consistent with prior studies. We observed a positive association between cumulative radon exposure and extrathoracic cancers, although the estimates were small. There was evidence that the association between radon and lung cancer was modified by smoking in a multiplicative or super-multiplicative fashion.

Mechanisms coordinating ribosomal protein gene transcription in response to stress.

While expression of ribosomal protein genes (RPGs) in the budding yeast has been extensively studied, a longstanding enigma persists regarding their co-regulation under fluctuating growth conditions. Most RPG promoters display one of two distinct arrangements of a core set of transcription factors (TFs) and are further differentiated by the presence or absence of the HMGB protein Hmo1. However, a third group of promoters appears not to be bound by any of these proteins, raising the question of how the whole suite of genes is co-regulated. We demonstrate here that all RPGs are regulated by two distinct, but complementary mechanisms driven by the TFs Ifh1 and Sfp1, both of which are required for maximal expression in optimal conditions and coordinated downregulation upon stress. At the majority of RPG promoters, Ifh1-dependent regulation predominates, whereas Sfp1 plays the major role at all other genes. We also uncovered an unexpected protein homeostasis-dependent binding property of Hmo1 at RPG promoters. Finally, we show that the Ifh1 paralog Crf1, previously described as a transcriptional repressor, can act as a constitutive RPG activator. Our study provides a more complete picture of RPG regulation and may serve as a paradigm for unravelling RPG regulation in multicellular eukaryotes.

Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.

In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and -1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these "fragile" nucleosomes play an important role in regulating RPG transcriptional output.

Transcriptional control of ribosome biogenesis in yeast: links to growth and stress signals.

Ribosome biogenesis requires prodigious transcriptional output in rapidly growing yeast cells and is highly regulated in response to both growth and stress signals. This minireview focuses on recent developments in our understanding of this regulatory process, with an emphasis on the 138 ribosomal protein genes (RPGs) themselves and a group of >200 ribosome biogenesis (RiBi) genes whose products contribute to assembly but are not part of the ribosome. Expression of most RPGs depends upon Rap1, a pioneer transcription factor (TF) required for the binding of a pair of RPG-specific TFs called Fhl1 and Ifh1. RPG expression is correlated with Ifh1 promoter binding, whereas Rap1 and Fhl1 remain promoter-associated upon stress-induced down regulation. A TF called Sfp1 has also been implicated in RPG regulation, though recent work reveals that its primary function is in activation of RiBi and other growth-related genes. Sfp1 plays an important regulatory role at a small number of RPGs where Rap1-Fhl1-Ifh1 action is subsidiary or non-existent. In addition, nearly half of all RPGs are bound by Hmo1, which either stabilizes or re-configures Fhl1-Ifh1 binding. Recent studies identified the proline rotamase Fpr1, known primarily for its role in rapamycin-mediated inhibition of the TORC1 kinase, as an additional TF at RPG promoters. Fpr1 also affects Fhl1-Ifh1 binding, either independently or in cooperation with Hmo1. Finally, a major recent development was the discovery of a protein homeostasis mechanism driven by unassembled ribosomal proteins, referred to as the Ribosome Assembly Stress Response (RASTR), that controls RPG transcription through the reversible condensation of Ifh1.

Radon and cancer mortality among underground uranium miners in the Príbram region of the Czech Republic.

BACKGROUND: This study aims to estimate the association between radon and site-specific cancer mortality among a large contemporary cohort of male uranium miners. METHODS: Annual occupational radon exposure was estimated based on a worker's duration of underground mining in a year and estimates of potential alpha energy of radon progeny in their location of work. Cancer mortality over the period 1977-1992 was ascertained for a cohort of 16 434 male underground uranium miners employed in the Czech Republic between 1946 and 1992. Poisson regression was used to estimate relationships between cumulative radiation exposure (in working level months [WLM]) and site-specific cancer mortality. RESULTS: Radon is positively associated with lung cancer mortality (excess relative rate [ERR] per 100 WLM = 0.2; 95% confidence interval [CI]: 0.10, 0.37). The best fit of the dose-response relationship between radon and lung cancer mortality was linear and estimates of radon-lung cancer associations varied by windows of time-since-exposure. Positive associations between radon and several types of cancer other than lung cancer were identified, notably chronic lymphocytic leukemia (CLL) (ERR/100 WLM = 0.24; 95% CI: [not determined [ND], 5.10]) and extrathoracic cancer (ERR/100 WLM = 0.12; 95% CI: [ND, 0.69]). We observed no associations between radon and stomach cancer, nor between radon and several hematopoietic cancer subtypes. CONCLUSIONS: This study confirms the established radon-lung cancer association and suggests that radon may also be associated with other types of cancer mortality. Further investigations of extrathoracic and CLL cancer, with the aim of obtaining more precise estimates, are warranted to understand associations between radon and cancers other than lung.

Today's Leadership Lesson: Mind the Wildlife and Prepare for Tomorrow's Disruption.

As the world reacts with unprecedented efforts to contain the COVID-19 pandemic, the role of organizational leaders is to embark on a parallel track to keep mission-critical initiatives moving forward. One track includes preparing their organizations for the next "novel" virus. After all, organizations do not hire leaders to maintain the status quo; they are hired to drive the future. As much as death and taxes are inevitable, it is equally predictable that all organizations will sooner or later confront a black swan event. History teaches us that while the order of magnitude may vary, management crises are not entirely novel. This article explores a series of early risk mitigation strategies to prevent the next COVID-19 and prepare leadership to face this inevitable challenge.

Divergence and Convergence of Royalty Determinations between Compulsory Licensing under the TRIPS Agreement and Ongoing Royalties as an Equitable Remedy.

Patent rights are recognized as a property asset with an attendant right to exclude. However, recent policy developments highlight that the right to exclude is not inviolable. This paper explores two rapidly evolving exceptions to patent exclusivity, both of which take the form of compulsory licenses. First, under the international Agreement on Trade-Related Aspects of Intellectual Property Rights ("TRIPS"), national governments can compel patent owners to out-license technology in service of greater good. These egalitarian compulsory licenses improve access to technology but undermine patent value. Second, compulsory licenses are increasingly relied upon as an equitable remedy in U.S. patent litigation. Typically referred to as "ongoing royalties," these court-mandated compulsory licenses are a modern alternative to injunctions against adjudged infringers. TRIPS compulsory licenses and ongoing royalties arise under independent legal frameworks, but necessarily invoke parallel economic considerations. While the wisdom of each has been discussed at length by others, this paper explores principles of royalty determination employed in each context. Considering both frameworks, an analysis of where each succeeds and fails is provided, together with an exploration of optimized royalty frameworks.

ChECing out Rif1 action in freely cycling cells.

In buddying yeast, like all eukaryotes examined so far, DNA replication is under temporal control, such that some origins fire early and some late during S phase. This replication timing program is established in G1 phase, where chromatin states are thought to prevent binding of key-limiting initiation factors at late-firing origins. Although many factors are involved in replication initiation, a new player, Rif1, has recently entered the scene, with a spate of papers revealing a global role for the protein in the control of replication initiation timing from yeasts to humans. Since budding yeast Rif1 was known to bind only to telomeric and silent mating loci regions, it remained controversial whether Rif1 acts directly at replication origins or instead influences origin activity indirectly. In this perspective, we discuss our recent finding that Rif1 binds directly to the replication origins that it controls. In this study, we also found that Rif1's regulatory activity at origins is best revealed by an assay (sort-seq) that measures replication in unperturbed, freely cycling cultures, as opposed to commonly used protocols in which cells are first blocked in the G1 phase of the cell cycle by mating pheromone, then released into a synchronous S phase. Finally, we discuss how the sequestration of Rif1 at telomeres, through an interaction with the arrays of Rap1 molecules bound there, plays an important role in limiting Rif1's action primarily to telomere-proximal replication origins.