Pan-cancer analysis of post-translational modifications reveals shared patterns of protein regulation.

Post-translational modifications (PTMs) play key roles in regulating cell signaling and physiology in both normal and cancer cells. Advances in mass spectrometry enable high-throughput, accurate, and sensitive measurement of PTM levels to better understand their role, prevalence, and crosstalk. Here, we analyze the largest collection of proteogenomics data from 1,110 patients with PTM profiles across 11 cancer types (10 from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium [CPTAC]). Our study reveals pan-cancer patterns of changes in protein acetylation and phosphorylation involved in hallmark cancer processes. These patterns revealed subsets of tumors, from different cancer types, including those with dysregulated DNA repair driven by phosphorylation, altered metabolic regulation associated with immune response driven by acetylation, affected kinase specificity by crosstalk between acetylation and phosphorylation, and modified histone regulation. Overall, this resource highlights the rich biology governed by PTMs and exposes potential new therapeutic avenues.

Pan-cancer proteogenomics connects oncogenic drivers to functional states.

Cancer driver events refer to key genetic aberrations that drive oncogenesis; however, their exact molecular mechanisms remain insufficiently understood. Here, our multi-omics pan-cancer analysis uncovers insights into the impacts of cancer drivers by identifying their significant cis-effects and distal trans-effects quantified at the RNA, protein, and phosphoprotein levels. Salient observations include the association of point mutations and copy-number alterations with the rewiring of protein interaction networks, and notably, most cancer genes converge toward similar molecular states denoted by sequence-based kinase activity profiles. A correlation between predicted neoantigen burden and measured T cell infiltration suggests potential vulnerabilities for immunotherapies. Patterns of cancer hallmarks vary by polygenic protein abundance ranging from uniform to heterogeneous. Overall, our work demonstrates the value of comprehensive proteogenomics in understanding the functional states of oncogenic drivers and their links to cancer development, surpassing the limitations of studying individual cancer types.

Eukaryotic Base Excision Repair: New Approaches Shine Light on Mechanism.

Genomic DNA is susceptible to endogenous and environmental stresses that modify DNA structure and its coding potential. Correspondingly, cells have evolved intricate DNA repair systems to deter changes to their genetic material. Base excision DNA repair involves a number of enzymes and protein cofactors that hasten repair of damaged DNA bases. Recent advances have identified macromolecular complexes that assemble at the DNA lesion and mediate repair. The repair of base lesions generally requires five enzymatic activities: glycosylase, endonuclease, lyase, polymerase, and ligase. The protein cofactors and mechanisms for coordinating the sequential enzymatic steps of repair are being revealed through a range of experimental approaches. We discuss the enzymes and protein cofactors involved in eukaryotic base excision repair, emphasizing the challenge of integrating findings from multiple methodologies. The results provide an opportunity to assimilate biochemical findings with cell-based assays to uncover new insights into this deceptively complex repair pathway.

Pathogenesis and host control of gammaherpesviruses: lessons from the mouse.

Gammaherpesviruses are lymphotropic viruses that are associated with the development of lymphoproliferative diseases, lymphomas, as well as other nonlymphoid cancers. Most known gammaherpesviruses establish latency in B lymphocytes. Research on Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68/γHV68/MHV4) has revealed a complex relationship between virus latency and the stage of B cell differentiation. Available data support a model in which gammaherpesvirus infection drives B cell proliferation and differentiation. In general, the characterized gammaherpesviruses exhibit a very narrow host tropism, which has severely limited studies on the human gammaherpesviruses EBV and Kaposi's sarcoma-associated herpesvirus. As such, there has been significant interest in developing animal models in which the pathogenesis of gammaherpesviruses can be characterized. MHV68 represents a unique model to define the effects of chronic viral infection on the antiviral immune response.

Integrated Proteogenomic Characterization of Human High-Grade Serous Ovarian Cancer.

To provide a detailed analysis of the molecular components and underlying mechanisms associated with ovarian cancer, we performed a comprehensive mass-spectrometry-based proteomic characterization of 174 ovarian tumors previously analyzed by The Cancer Genome Atlas (TCGA), of which 169 were high-grade serous carcinomas (HGSCs). Integrating our proteomic measurements with the genomic data yielded a number of insights into disease, such as how different copy-number alternations influence the proteome, the proteins associated with chromosomal instability, the sets of signaling pathways that diverse genome rearrangements converge on, and the ones most associated with short overall survival. Specific protein acetylations associated with homologous recombination deficiency suggest a potential means for stratifying patients for therapy. In addition to providing a valuable resource, these findings provide a view of how the somatic genome drives the cancer proteome and associations between protein and post-translational modification levels and clinical outcomes in HGSC. VIDEO ABSTRACT.

Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System.

CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease helicase for target degradation. Here, we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways dictated by the presence or absence of a protospacer-adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short single-stranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent on the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes and support a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.

TnpB homologues exapted from transposons are RNA-guided transcription factors.

Transposon-encoded tnpB and iscB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination1-4. These widespread gene families were repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12 (refs. 5,6). We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR-Cas adaptive immunity. Here, using phylogenetics, structural predictions, comparative genomics and functional assays, we uncover multiple independent genesis events of programmable transcription factors, which we name TnpB-like nuclease-dead repressors (TldRs). These proteins use naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPR interference technologies invented by humans7. Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility8, phage susceptibility9, and host immunity10. Collectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of transposon-encoded genes, and reveals the evolutionary trajectory of diverse RNA-guided transcription factors.

Evolutionary and mechanistic diversity of Type I-F CRISPR-associated transposons.

Canonical CRISPR-Cas systems utilize RNA-guided nucleases for targeted cleavage of foreign nucleic acids, whereas some nuclease-deficient CRISPR-Cas complexes have been repurposed to direct the insertion of Tn7-like transposons. Here, we established a bioinformatic and experimental pipeline to comprehensively explore the diversity of Type I-F CRISPR-associated transposons. We report DNA integration for 20 systems and identify a highly active subset that exhibits complete orthogonality in transposon DNA mobilization. We reveal the modular nature of CRISPR-associated transposons by exploring the horizontal acquisition of targeting modules and by characterizing a system that encodes both a programmable, RNA-dependent pathway, and a fixed, RNA-independent pathway. Finally, we analyzed transposon-encoded cargo genes and found the striking presence of anti-phage defense systems, suggesting a role in transmitting innate immunity between bacteria. Collectively, this study substantially advances our biological understanding of CRISPR-associated transposon function and expands the suite of RNA-guided transposases for programmable, large-scale genome engineering.

Transposon-encoded nucleases use guide RNAs to promote their selfish spread.

Insertion sequences are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance1. IS200- and IS605-family transposons undergo 'peel-and-paste' transposition catalysed by a TnpA transposase2, but they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively3,4. Recent studies have demonstrated that TnpB and IscB function as RNA-guided DNA endonucleases5,6, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB and IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related insertion sequences from Geobacillus stearothermophilus that encode several TnpB and IscB orthologues, and showed that a single TnpA transposase was broadly active for transposon mobilization. The donor joints formed upon religation of transposon-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB and IscB nucleases, and co-expression of TnpB and TnpA led to substantially greater transposon retention relative to conditions in which TnpA was expressed alone. Notably, TnpA and TnpB also stimulated recombination frequencies, surpassing rates observed with TnpB alone. Collectively, this study reveals that RNA-guided DNA cleavage arose as a primal biochemical activity to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defence.

Rewiring cancer drivers to activate apoptosis.

Genes that drive the proliferation, survival, invasion and metastasis of malignant cells have been identified for many human cancers1-4. Independent studies have identified cell death pathways that eliminate cells for the good of the organism5,6. The coexistence of cell death pathways with driver mutations suggests that the cancer driver could be rewired to activate cell death using chemical inducers of proximity (CIPs). Here we describe a new class of molecules called transcriptional/epigenetic CIPs (TCIPs) that recruit the endogenous cancer driver, or a downstream transcription factor, to the promoters of cell death genes, thereby activating their expression. We focused on diffuse large B cell lymphoma, in which the transcription factor B cell lymphoma 6 (BCL6) is deregulated7. BCL6 binds to the promoters of cell death genes and epigenetically suppresses their expression8. We produced TCIPs by covalently linking small molecules that bind BCL6 to those that bind to transcriptional activators that contribute to the oncogenic program, such as BRD4. The most potent molecule, TCIP1, increases binding of BRD4 by 50% over genomic BCL6-binding sites to produce transcriptional elongation at pro-apoptotic target genes within 15 min, while reducing binding of BRD4 over enhancers by only 10%, reflecting a gain-of-function mechanism. TCIP1 kills diffuse large B cell lymphoma cell lines, including chemotherapy-resistant, TP53-mutant lines, at EC50 of 1-10 nM in 72 h and exhibits cell-specific and tissue-specific effects, capturing the combinatorial specificity inherent to transcription. The TCIP concept also has therapeutic applications in regulating the expression of genes for regenerative medicine and developmental disorders.

A mouse following in the footsteps of human prehistory.

One of the strongest signals of positive selection in humans surrounds the V370A variant of Ectodysplasin A receptor (EDAR). However, its phenotypic consequences and impetus for selection are not well understood. Kamberov et al. nail down when it originated and, using transgenic mice, delineate its phenotypic impacts.

Observing a DNA polymerase choose right from wrong.

DNA polymerase (pol) ß is a model polymerase involved in gap-filling DNA synthesis utilizing two metals to facilitate nucleotidyl transfer. Previous structural studies have trapped catalytic intermediates by utilizing substrate analogs (dideoxy-terminated primer or nonhydrolysable incoming nucleotide). To identify additional intermediates during catalysis, we now employ natural substrates (correct and incorrect nucleotides) and follow product formation in real time with 15 different crystal structures. We are able to observe molecular adjustments at the active site that hasten correct nucleotide insertion and deter incorrect insertion not appreciated previously. A third metal binding site is transiently formed during correct, but not incorrect, nucleotide insertion. Additionally, long incubations indicate that pyrophosphate more easily dissociates after incorrect, compared to correct, nucleotide insertion. This appears to be coupled to subdomain repositioning that is required for catalytic activation/deactivation. The structures provide insights into a fundamental chemical reaction that impacts polymerase fidelity and genome stability.

The somatic genomic landscape of glioblastoma.

We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.

Selective TnsC recruitment enhances the fidelity of RNA-guided transposition.

Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD1, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs2,3. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins4. How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous-hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.

Regulation of immune responses to food by commensal microbes.

The increasing prevalence of immune-mediated non-communicable chronic diseases, such as food allergies, has prompted a deeper investigation into the role of the gut microbiome in modulating immune responses. Here, we explore the complex interactions between commensal microbes and the host immune system, highlighting the critical role of gut bacteria in maintaining immune homeostasis. We examine how modern lifestyle practices and environmental factors have disrupted co-evolved host-microbe interactions and discuss how changes in microbiome composition impact epithelial barrier function, responses to food allergens, and susceptibility to allergic diseases. Finally, we examine the potential of bioengineered microbiome-based therapies, and live biotherapeutic products, for reestablishing immune homeostasis to prevent or treat food allergies.

Integrating phylogenies into single-cell RNA sequencing analysis allows comparisons across species, genes, and cells.

Comparisons of single-cell RNA sequencing (scRNA-seq) data across species can reveal links between cellular gene expression and the evolution of cell functions, features, and phenotypes. These comparisons evoke evolutionary histories, as depicted by phylogenetic trees, that define relationships between species, genes, and cells. This Essay considers each of these in turn, laying out challenges and solutions derived from a phylogenetic comparative approach and relating these solutions to previously proposed methods for the pairwise alignment of cellular dimensional maps. This Essay contends that species trees, gene trees, cell phylogenies, and cell lineages can all be reconciled as descriptions of the same concept-the tree of cellular life. By integrating phylogenetic approaches into scRNA-seq analyses, challenges for building informed comparisons across species can be overcome, and hypotheses about gene and cell evolution can be robustly tested.

Single-fly genome assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1 Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Developmental and biophysical determinants of grass leaf size worldwide.

One of the most notable ecological trends-described more than 2,300  years ago by Theophrastus-is the association of small leaves with dry and cold climates, which has recently been recognized for eudicotyledonous plants at a global scale1-3. For eudicotyledons, this pattern has been attributed to the fact that small leaves have a thinner boundary layer that helps to avoid extreme leaf temperatures4 and their leaf development results in vein traits that improve water transport under cold or dry climates5,6. However, the global distribution of leaf size and its adaptive basis have not been tested in the grasses, which represent a diverse lineage that is distinct in leaf morphology and that contributes 33% of terrestrial primary productivity (including the bulk of crop production)7. Here we demonstrate that grasses have shorter and narrower leaves under colder and drier climates worldwide. We show that small grass leaves have thermal advantages and vein development that contrast with those of eudicotyledons, but that also explain the abundance of small leaves in cold and dry climates. The worldwide distribution of leaf size in grasses exemplifies how biophysical and developmental processes result in convergence across major lineages in adaptation to climate globally, and highlights the importance of leaf size and venation architecture for grass performance in past, present and future ecosystems.

Excess natural-cause mortality in US counties and its association with reported COVID-19 deaths.

In the United States, estimates of excess deaths attributable to the COVID-19 pandemic have consistently surpassed reported COVID-19 death counts. Excess deaths reported to non-COVID-19 natural causes may represent unrecognized COVID-19 deaths, deaths caused by pandemic health care interruptions, and/or deaths from the pandemic's socioeconomic impacts. The geographic and temporal distribution of these deaths may help to evaluate which explanation is most plausible. We developed a Bayesian hierarchical model to produce monthly estimates of excess natural-cause mortality for US counties over the first 30 mo of the pandemic. From March 2020 through August 2022, 1,194,610 excess natural-cause deaths occurred nationally [90% PI (Posterior Interval): 1,046,000 to 1,340,204]. A total of 162,886 of these excess natural-cause deaths (90% PI: 14,276 to 308,480) were not reported to COVID-19. Overall, 15.8 excess deaths were reported to non-COVID-19 natural causes for every 100 reported COVID-19 deaths. This number was greater in nonmetropolitan counties (36.0 deaths), the West (Rocky Mountain states: 31.6 deaths; Pacific states: 25.5 deaths), and the South (East South Central states: 26.0 deaths; South Atlantic states: 25.0 deaths; West South Central states: 24.2 deaths). In contrast, reported COVID-19 death counts surpassed estimates of excess natural-cause deaths in metropolitan counties in the New England and Middle Atlantic states. Increases in reported COVID-19 deaths correlated temporally with increases in excess deaths reported to non-COVID-19 natural causes in the same and/or prior month. This suggests that many excess deaths reported to non-COVID-19 natural causes during the first 30 mo of the pandemic in the United States were unrecognized COVID-19 deaths.

Antigenic cartography using variant-specific hamster sera reveals substantial antigenic variation among Omicron subvariants.

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) has developed substantial antigenic variability. As the majority of the population now has pre-existing immunity due to infection or vaccination, the use of experimentally generated animal immune sera can be valuable for measuring antigenic differences between virus variants. Here, we immunized Syrian hamsters by two successive infections with one of nine SARS-CoV-2 variants. Their sera were titrated against 16 SARS-CoV-2 variants, and the resulting titers were visualized using antigenic cartography. The antigenic map shows a condensed cluster containing all pre-Omicron variants (D614G, Alpha, Delta, Beta, Mu, and an engineered B.1+E484K variant) and considerably more diversity among a selected panel of Omicron subvariants (BA.1, BA.2, BA.4/BA.5, the BA.5 descendants BF.7 and BQ.1.18, the BA.2.75 descendant BN.1.3.1, the BA.2-derived recombinants XBB.2 and EG.5.1, and the BA.2.86 descendant JN.1). Some Omicron subvariants were as antigenically distinct from each other as the wildtype is from the Omicron BA.1 variant. Compared to titers measured in human sera, titers in hamster sera are of higher magnitude, show less fold change, and result in a more compact antigenic map topology. The results highlight the potential of sera from hamsters for the continued antigenic characterization of SARS-CoV-2.