Search

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent efficacy in phase I clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is 1) strictly dependent on pyrimidine nucleotide depletion, 2) independent of canonical antigen presentation pathway transcriptional regulators, and 3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

Pancreatic ductal adenocarcinoma (PDAC) is classified into two key subtypes, classical and basal, with basal PDAC predicting worse survival. Using in vitro drug assays, genetic manipulation experiments, and in vivo drug studies in human patient-derived xenografts (PDXs) of PDAC, we found that basal PDACs were uniquely sensitive to transcriptional inhibition by targeting cyclin-dependent kinase 7 (CDK7) and CDK9, and this sensitivity was recapitulated in the basal subtype of breast cancer. We showed in cell lines, PDXs, and publicly available patient datasets that basal PDAC was characterized by inactivation of the integrated stress response (ISR), which leads to a higher rate of global mRNA translation. Moreover, we identified the histone deacetylase sirtuin 6 (SIRT6) as a critical regulator of a constitutively active ISR. Using expression analysis, polysome sequencing, immunofluorescence, and cycloheximide chase experiments, we found that SIRT6 regulated protein stability by binding activating transcription factor 4 (ATF4) in nuclear speckles and protecting it from proteasomal degradation. In human PDAC cell lines and organoids as well as in murine PDAC genetically engineered mouse models where SIRT6 was deleted or down-regulated, we demonstrated that SIRT6 loss both defined the basal PDAC subtype and led to reduced ATF4 protein stability and a nonfunctional ISR, causing a marked vulnerability to CDK7 and CDK9 inhibitors. Thus, we have uncovered an important mechanism regulating a stress-induced transcriptional program that may be exploited with targeted therapies in particularly aggressive PDAC.

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.

Drawing on Laura Nader’s concept of the vertical slice, this article reviews the hundreds of instances where the work of anthropologists, or anthropologists themselves appear in the leaked US State Department documents known as the “Manning Cables” published by WikiLeaks. The analysis of these documents shows anthropologists engaging with the US government in various ways, including in advisory capacities or bringing cultural or political knowledge from peripheral geographical regions to the core. Ethical, political, and disciplinary dimensions of these interactions are discussed, and Nader’s conception of the vertical slice is used to distinguish political dimensions of these anthropological engagements with state power.

The effects of rapid acute depletion of components of RNA polymerase II (Pol II) general transcription factors (GTFs) that are thought to be critical for formation of preinitiation complexes (PICs) and initiation in vitro were quantified in HAP1 cells using precision nuclear run-on sequencing (PRO-Seq). The average dependencies for each factor across >70 000 promoters varied widely even though levels of depletions were similar. Some of the effects could be attributed to the presence or absence of core promoter elements such as the upstream TBP-specificity motif or downstream G-rich sequences, but some dependencies anti-correlated with such sequences. While depletion of TBP had a large effect on most Pol III promoters only a small fraction of Pol II promoters were similarly affected. TFIIB depletion had the largest general effect on Pol II and also correlated with apparent termination defects downstream of genes. Our results demonstrate that promoter activity is combinatorially influenced by recruitment of TFIID and sequence-specific transcription factors. They also suggest that interaction of the preinitiation complex (PIC) with nucleosomes can affect activity and that recruitment of TFIID containing TBP only plays a positive role at a subset of promoters.

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.

During the 1950s, partially in response to the Soviet Union’s launching of Sputnik, social psychologist Donald Michael launched Project Man in Space, a group of American social scientists organized...

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.

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.

Background The 7SK small nuclear RNA (snRNA) found in most metazoans is a key regulator of P-TEFb which in turn regulates RNA polymerase II elongation. Although its primary sequence varies in protostomes, its secondary structure and function are conserved across evolutionary distant taxa. Results Here, we describe a novel ncRNA sharing many features characteristic of 7SK RNAs, in D. melanogaster. We examined the structure of the corresponding gene and determined the expression profiles of the encoded RNA, called snRNA:7SK:94F, during development. It is probably produced from the transcription of a lncRNA which is processed into a mature snRNA. We also addressed its biological function and we show that, like dm7SK, this alternative 7SK interacts in vivo with the different partners of the P-TEFb complex, i.e. HEXIM, LARP7 and Cyclin T. This novel RNA is widely expressed across tissues. Conclusion We propose that two distinct 7SK genes might contribute to the formation of the 7SK snRNP complex in D. melanogaster.

Beta- and gammaherpesviruses late transcription factors (LTFs) target viral promoters containing a TATT sequence to drive transcription after viral DNA replication has begun. Human cytomegalovirus (HCMV), a betaherpesvirus, uses the UL87 LTF to bind both TATT and host RNA polymerase II (Pol II), whereas the UL79 LTF has been suggested to drive productive elongation. Here we apply integrated functional genomics (dTag system, PRO-Seq, ChIP-Seq, and promoter function assays) to uncover the contribution of diversity in LTF target sequences in determining degree and scope to which LTFs drive viral transcription. We characterize the DNA sequence patterns in LTF-responsive and -unresponsive promoter populations, determine where and when Pol II initiates transcription, identify sites of LTF binding genome-wide, and quantify change in nascent transcripts from individual promoters in relation to core promoter sequences, LTF loss, stage of infection, and viral DNA replication. We find that HCMV UL79 and UL87 LTFs function concordantly to initiate transcription from over half of all active viral promoters in late infection, while not appreciably affecting host transcription. Both LTFs act on and bind to viral early-late and late kinetic-class promoters. Over one-third of these core promoters lack the TATT and instead have a TATAT, TGTT, or YRYT. The TATT and non-TATT motifs are part of a sequence block with a sequence code that correlates with promoter transcription level. LTF occupancy of a TATATA palindrome shared by back-to-back promoters is linked to bidirectional transcription. We conclude that diversity in LTF target sequences shapes the LTF-transformative program that drives the viral early-to-late transcription switch., Author summary Herpesviruses have a group of genes earmarked for expression late in the infection. Beta- and gammaherpesviruses utilize a six-member set of viral late transcription factors to selectively activate these genes by binding to a DNA sequence signature in gene promoters. We made an unexpected discovery that a wider range of differences in sequence signatures configures the late gene expression program for human cytomegalovirus, a beta-herpesvirus of global public health importance. Diversity in signature patterns expands promoter targets and probably pre-sets amount of individual promoter output. A unique palindromic sequence signature is linked to the activation of back-to-back promoters at multiple locations in the viral genome. We deduce that diversity in late transcription factor targets functionally orchestrates the rollout of a productive late-stage infection. This may be a generalizable feature adopted by beta- and gammaherpesvirus subfamilies.

Transcription by RNA polymerase II (Pol II) is controlled during initiation, elongation, and termination by a large variety of transcription factors, the state of chromatin modifications, and environmental conditions. Herein we describe experimental approaches for the examination of Pol II transcription at semi-global and genome-wide scales through analysis of nascent Pol II transcripts. We begin with a description of the nuclear walk-on (NWO) assay, which involves rapid isolation of nuclei in the presence of EDTA, followed by extension of about a quarter of the nascent transcripts with (32)P-CTP. Labeled nascent transcripts are then analyzed by denaturing PAGE and phosphorimaging followed by densitometry analysis to quantify the signal on the gel. A parallel reaction containing α-amanitin to inhibit Pol II reveals transcription due to Pol I and Pol III, which can be subtracted to yield a profile of Pol II transcription. We then describe how to use the NWO as a front end for PRO-Seq and PRO-Cap methods, which permit the genome-wide characterization of Pol II transcription at nucleotide resolution and provide precise information about sites of transcription initiation and pausing. We discuss strategies for optimizing sequencing methods that capture nascent Pol II transcripts, methods of bias reduction, and approaches for normalizing these and other sequencing datasets using spike-in controls.

Renaissance artists represented the Bible as the preeminent monument of classical culture well before humanist scholars began their revolutionary efforts to recover the ancient forms of biblical texts. Once Renaissance humanism and the Reformation turned decisively to biblical philology (and began overturning the authority of the Vulgate Bible and medieval theology), artists supported their creation of innovative conceptualizations of the Bible. Remarkably, the three most influential artists of the Northern Renaissance—Albrecht Dürer, Lucas Cranach the Elder, and Hans Holbein the Younger—made profound contributions to all the major Renaissance and Reformation Bibles in Germany and Switzerland and to the biblical humanist movement generally. The chapter concludes with an introduction to the history of biblical humanism, including the emergence of new authoritative Bibles beginning with Erasmus’s first edition of the New Testament in the original Greek.

During the formative decades of the Reformation, Lucas Cranach filled sacred places with evangelical art distinctively grounded in biblicism. He and his workshop (including Lucas Cranach the Younger) were innovators in all the key areas of art production: the publication of the new Bibles, the invention of biblical imagery for the new theology (motifs including Law and Gospel, Christ blessing the children, Christ and the adulteress, and Crucifixion with the centurion), the reformation of the retable altar (including the first and most influential Protestant altarpieces), and the portrayal of the electors of Saxony as guardians of the new church and the promotion of Luther and Melanchthon as biblical authorities. From the perspective of traditional Christian art, their images often owed as much to a spirit of renovation as to the zeal of revolution. In opposition to iconoclastic Protestants, Cranach consistently demonstrates the vitality of visual biblical art as an evangelizing medium and as a theological discourse.

Lucas Cranach the Elder, a close friend of Martin Luther, not only produced the definitive visual record of the history of the Reformation but also became a major leader in the movement to transform Christianity. From 1518 onward, he designed art to advance the Reformation of the church across Germany and Europe. The Bible stood at the center of his media campaign. Cranach and his workshop designed the first Protestant Bible (1522) as well as subsequent imprints of Luther’s translations. He also developed innovative biblical propaganda (most importantly in the anti-papal Passion of Christ and Antichrist). Frequently in his immense oeuvre (including works designed for both Protestant and Catholic contexts) Cranach anchors the new biblicism in a humanist ideal of the authority of philology. A major accomplishment was his development of the portrait type of the professor of the Bible (preeminently Luther and Philipp Melanchthon) as an icon of the authority of humanist biblical philology for the Reformation.

The new diversity of Bible versions and ensuing sociopolitical upheavals presented challenges with which publishers and artists, such as Hans Holbein, had to contend. Initially receiving commissions from printers in Basel (Johannes Froben, etc.), Holbein designed art for numerous German Bibles, a French humanist translation, the first complete Bible in English (Coverdale Bible, 1535), and a host of Catholic-oriented Bible texts, including the Vulgate and Erasmus’s Bible editions. He also created the first emblem Bible, the Icons of the Old Testament, one of the most influential Bibles of the Renaissance. Holbein focused on the Bible as image and history, not as text or theology, an approach that enabled him to accommodate the heterogeneity of humanist and Reformation Bibles. With few exceptions, Holbein’s designs could be reused in Bibles with different theological agendas, an artistic efficiency that contributed to the stability of the Bible image across a wide humanist and multi-confessional spectrum.

Albrecht Dürer played a significant role in the emergence of the Renaissance Bible, promoting a new perception of its authority at a pivotal moment in the history of Christianity: the beginning of the age of print, followed by the onset of the Protestant Reformation. For artists across Europe, the breakthrough that suddenly raised the woodcut to the status of high art was Dürer’s 1498 publication of illustrated editions of the Book of Revelation, usually called the Apocalypse. During the decade 1494–1504, he developed a methodology for imitating classical art that represented the Bible as part of classical antiquity (Fall of Humanity, 1504). In these efforts, he worked to validate and advance the philological and historical methodologies of biblical humanism, the innovative academic discipline that revolutionized European Christianity and politics. The chapter analyzes Dürer’s Apocalypse, his representations of St. Jerome (author of the Vulgate translation), and his classicizing adaptations of the biblical Passion.

The epilogue traces the impact of the early Protestant Bibles on the explosion of an exceedingly popular new genre: the Bible emblem book, especially those by Bernard Salomon, Virgil Solis, Jost Amman, Tobias Stimmer, and Matthäus Merian. As was the case with Dürer, Cranach, and Holbein, the new Protestant Bible designs were reused and imitated in diverse theological contexts, thereby illustrating the dynamic cultural transfer across language and confessional borders. Solis’s woodcut illustrations, for example, appeared in German, Dutch, Latin, and English Bibles by both Protestant and Catholic translators. Remarkably, Protestant designs informed not only Catholic but also Jewish biblical art (the Amsterdam Haggadah of 1695). Though any concept of textual unity faded with the Reformation, the Bible image emerged as a significant visual source for common cultural knowledge and experience.

High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed in vitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, in vitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.

Though American anthropologists have long engaged in radical political activities, there remains a poorly documented history of American Marxist anthropologists’ engagements with national and global socialist and communist political parties. This article draws on an interview with American anthropologist John Moore, as well as material from Moore’s FBI file, recently released under the Freedom of Information Act, with records from a 1960s Military Intelligence investigation of Moore to document and explore Moore’s involvement in communist and socialist organizations from the 1960s to the 1990s. Moore’s reflections on his political activities highlight a continuity.

During the months and years following the 11 September 2001 terror attacks in the US, it became axiomatic for American policy makers to speak of a need to more directly connect military and intelli...

The epithet “elusive” clings to Hans Holbein the Younger in modern scholarship. Given his contemporary success and renown, not to mention that he worked for powerful patrons (the King of England an...