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Large-scale whole-genome sequencing studies have enabled analysis of noncoding rare-variant (RV) associations with complex human diseases and traits. Variant-set analysis is a powerful approach to study RV association. However, existing methods have limited ability in analyzing the noncoding genome. We propose a computationally efficient and robust noncoding RV association detection framework, STAARpipeline, to automatically annotate a whole-genome sequencing study and perform flexible noncoding RV association analysis, including gene-centric analysis and fixed window-based and dynamic window-based non-gene-centric analysis by incorporating variant functional annotations. In gene-centric analysis, STAARpipeline uses STAAR to group noncoding variants based on functional categories of genes and incorporate multiple functional annotations. In non-gene-centric analysis, STAARpipeline uses SCANG-STAAR to incorporate dynamic window sizes and multiple functional annotations. We apply STAARpipeline to identify noncoding RV sets associated with four lipid traits in 21,015 discovery samples from the Trans-Omics for Precision Medicine (TOPMed) program and replicate several of them in an additional 9,123 TOPMed samples. We also analyze five non-lipid TOPMed traits.

Proteolytic cleavage of cell surface proteins triggers critical processes including cell-cell interactions, receptor activation, and shedding of signaling proteins. Consequently, dysregulated extracellular proteases contribute to malignant cell phenotypes including most cancers. To understand these effects, methods are needed that identify proteolyzed membrane proteins within diverse cellular contexts. Herein we report a proteomic approach, called cell surface N-terminomics, to broadly identify precise cleavage sites (neo-N-termini) on the surface of living cells. First, we functionalized the engineered peptide ligase, called stabiligase, with an N-terminal nucleophile that enables covalent attachment to naturally occurring glycans. Upon the addition of a biotinylated peptide ester, glycan-tethered stabiligase efficiently tags extracellular neo-N-termini for proteomic analysis. To demonstrate the versatility of this approach, we identified and characterized 1532 extracellular neo-N-termini across a panel of different cell types including primary immune cells. The vast majority of cleavages were not identified by previous proteomic studies. Lastly, we demonstrated that single oncogenes, KRAS(G12V) and HER2, induce extracellular proteolytic remodeling of proteins involved in cancerous cell growth, invasion, and migration. Cell surface N-terminomics is a generalizable platform that can reveal proteolyzed, neoepitopes to target using immunotherapies.

BackgroundThe availability of whole-genome sequencing data in large studies has enabled the assessment of coding and noncoding variants across the allele frequency spectrum for their associations with blood pressure.MethodsWe conducted a multiancestry whole-genome sequencing analysis of blood pressure among 51 456 Trans-Omics for Precision Medicine and Centers for Common Disease Genomics program participants (stage-1). Stage-2 analyses leveraged array data from UK Biobank (N=383 145), Million Veteran Program (N=318 891), and Reasons for Geographic and Racial Differences in Stroke (N=10 643) participants, along with whole-exome sequencing data from UK Biobank (N=199 631) participants.ResultsTwo blood pressure signals achieved genome-wide significance in meta-analyses of stage-1 and stage-2 single variant findings (P

Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the antitumor immune response. T cell surface receptors and surface proteins that influence interactions and function in the TME are proven targets for cancer immunotherapy. However, how the entire surface proteome remodels in primary human T cells in response to specific suppressive factors in the TME remains to be broadly and systematically characterized. Here, using a reductionist cell culture approach with primary human T cells and stable isotopic labeling with amino acids in cell culture-based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8+ surface proteome (surfaceome). Surprisingly, coculturing primary CD8+ T cells with Tregs only modestly affected the CD8+ surfaceome but did partially reverse activation-induced surfaceomic changes. In contrast, hypoxia drastically altered the CD8+ surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4+ T cell surfaceome similarly responded to hypoxia, revealing a common hypoxia-induced surface receptor program. Our surfaceomics findings suggest that hypoxic environments create a challenge for T cell activation. These studies provide global insight into how Tregs and hypoxia remodel the T cell surfaceome and we believe represent a valuable resource to inform future therapeutic efforts to enhance T cell function.

Genetic studies on telomere length are important for understanding age-related diseases. Prior GWAS for leukocyte TL have been limited to European and Asian populations. Here, we report the first sequencing-based association study for TL across ancestrally-diverse individuals (European, African, Asian and Hispanic/Latino) from the NHLBI Trans-Omics for Precision Medicine (TOPMed) program. We used whole genome sequencing (WGS) of whole blood for variant genotype calling and the bioinformatic estimation of telomere length in n=109,122 individuals. We identified 59 sentinel variants (p-value

The heightened cardiovascular disease (CVD) risk observed among omnivores is thought to be linked, in part, to gut microbiota-dependent generation of trimethylamine-N-oxide (TMAO) from L-carnitine, a nutrient abundant in red meat. Gut microbial transformation of L-carnitine into trimethylamine (TMA), the precursor of TMAO, occurs via the intermediate γ-butyrobetaine (γBB). However, the interrelationship of γBB, red meat ingestion and CVD risks, as well as the gut microbial genes responsible for the transformation of γBB to TMA, are unclear. In the present study, we show that plasma γBB levels in individuals from a clinical cohort (n = 2,918) are strongly associated with incident CVD event risks. Culture of human faecal samples and microbial transplantation studies in gnotobiotic mice with defined synthetic communities showed that the introduction of Emergencia timonensis, a human gut microbe that can metabolize γBB into TMA, is sufficient to complete the carnitine → γBB → TMA transformation, elevate TMAO levels and enhance thrombosis potential in recipients after arterial injury. RNA-sequencing analyses of E. timonensis identified a six-gene cluster, herein named the γBB utilization (gbu) gene cluster, which is upregulated in response to γBB. Combinatorial cloning and functional studies identified four genes (gbuA, gbuB, gbuC and gbuE) that are necessary and sufficient to recapitulate the conversion of γBB to TMA when coexpressed in Escherichia coli. Finally, reanalysis of samples (n = 113) from a clinical, randomized diet, intervention study showed that the abundance of faecal gbuA correlates with plasma TMAO and a red meat-rich diet. Our findings reveal a microbial gene cluster that is critical to dietary carnitine → γBB → TMA → TMAO transformation in hosts and contributes to CVD risk.

The genetic determinants of fasting glucose (FG) and fasting insulin (FI) have been studied mostly through genome arrays, resulting in over 100 associated variants. We extended this work with high-coverage whole genome sequencing analyses from fifteen cohorts in NHLBI's Trans-Omics for Precision Medicine (TOPMed) program. Over 23,000 non-diabetic individuals from five race-ethnicities/populations (African, Asian, European, Hispanic and Samoan) were included. Eight variants were significantly associated with FG or FI across previously identified regions MTNR1B, G6PC2, GCK, GCKR and FOXA2. We additionally characterize suggestive associations with FG or FI near previously identified SLC30A8, TCF7L2, and ADCY5 regions as well as APOB, PTPRT, and ROBO1. Functional annotation resources including the Diabetes Epigenome Atlas were compiled for each signal (chromatin states, annotation principal components, and others) to elucidate variant-to-function hypotheses. We provide a catalog of nucleotide-resolution genomic variation spanning intergenic and intronic regions creating a foundation for future sequencing-based investigations of glycemic traits.

Intratumoral heterogeneity is a common feature of many myeloid leukemias and a significant reason for treatment failure and relapse. Thus, identifying the cells responsible for residual disease and leukemia re-growth is critical to better understanding how they are regulated. Here, we show that a knock-in reporter mouse for the stem cell gene Musashi 2 (Msi2) allows identification of leukemia stem cells in aggressive myeloid malignancies, and provides a strategy for defining their core dependencies. Specifically, we carry out a high throughput screen using Msi2-reporter blast crisis chronic myeloid leukemia (bcCML) and identify several adhesion molecules that are preferentially expressed in therapy resistant bcCML cells and play a key role in bcCML. In particular, we focus on syndecan-1, whose deletion triggers defects in bcCML growth and propagation and markedly improves survival of transplanted mice. Further, live imaging reveals that the spatiotemporal dynamics of leukemia cells are critically dependent on syndecan signaling, as loss of this signal impairs their localization, migration and dissemination to distant sites. Finally, at a molecular level, syndecan loss directly impairs integrin β7 function, suggesting that syndecan exerts its influence, at least in part, by coordinating integrin activity in bcCML. These data present a platform for delineating the biological underpinnings of leukemia stem cell function, and highlight the Sdc1-Itgβ7 signaling axis as a key regulatory control point for bcCML growth and dissemination.

Mono- or dual-checkpoint inhibitors for immunotherapy have changed the paradigm of cancer care; however, only a minority of patients responds to such treatment. Combining small molecule immuno-stimulators can improve treatment efficacy, but they are restricted by poor pharmacokinetics. In this study, TLR7 agonists conjugated onto silica nanoparticles showed extended drug localization after intratumoral injection. The nanoparticle-based TLR7 agonist increased immune stimulation by activating the TLR7 signaling pathway. When treating CT26 colon cancer, nanoparticle conjugated TLR7 agonists increased T cell infiltration into the tumors by > 4× and upregulated expression of the interferon γ gene compared to its unconjugated counterpart by ~2×. Toxicity assays established that the conjugated TLR7 agonist is a safe agent at the effective dose. When combined with checkpoint inhibitors that target programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), a 10-100× increase in immune cell migration was observed; furthermore, 100 mm3 tumors were treated and a 60% remission rate was observed including remission at contralateral non-injected tumors. The data show that nanoparticle based TLR7 agonists are safe and can potentiate the effectiveness of checkpoint inhibitors in immunotherapy resistant tumor models and promote a long-term specific memory immune function.

Like many other crops, the cultivated peanut (Arachis hypogaea L.) is of hybrid origin and has a polyploid genome that contains essentially complete sets of chromosomes from two ancestral species. Here we report the genome sequence of peanut and show that after its polyploid origin, the genome has evolved through mobile-element activity, deletions and by the flow of genetic information between corresponding ancestral chromosomes (that is, homeologous recombination). Uniformity of patterns of homeologous recombination at the ends of chromosomes favors a single origin for cultivated peanut and its wild counterpart A. monticola. However, through much of the genome, homeologous recombination has created diversity. Using new polyploid hybrids made from the ancestral species, we show how this can generate phenotypic changes such as spontaneous changes in the color of the flowers. We suggest that diversity generated by these genetic mechanisms helped to favor the domestication of the polyploid A. hypogaea over other diploid Arachis species cultivated by humans.

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

Vernal pool clam shrimp (Eulimnadia texana) are a promising model system due to their ease of lab culture, short generation time, modest sized genome, a somewhat rare stable androdioecious sex determination system, and a requirement to reproduce via desiccated diapaused eggs. We generated a highly contiguous genome assembly using 46× of PacBio long read data and 216× of Illumina short reads, and annotated using Illumina RNAseq obtained from adult males or hermaphrodites. Of the 120 Mb genome 85% is contained in the largest eight contigs, the smallest of which is 4.6 Mb. The assembly contains 98% of transcripts predicted via RNAseq. This assembly is qualitatively different from scaffolded Illumina assemblies: It is produced from long reads that contain sequence data along their entire length, and is thus gap free. The contiguity of the assembly allows us to order the HOX genes within the genome, identifying two loci that contain HOX gene orthologs, and which approximately maintain the order observed in other arthropods. We identified a partial duplication of the Antennapedia complex adjacent to the few genes homologous to the Bithorax locus. Because the sex chromosome of an androdioecious species is of special interest, we used existing allozyme and microsatellite markers to identify the E. texana sex chromosome, and find that it comprises nearly half of the genome of this species. Linkage patterns indicate that recombination is extremely rare and perhaps absent in hermaphrodites, and as a result the location of the sex determining locus will be difficult to refine using recombination mapping.

The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16% to

Cysteine can be specifically functionalized by a myriad of acid-base conjugation strategies for applications ranging from probing protein function to antibody-drug conjugates and proteomics. In contrast, selective ligation to the other sulfur-containing amino acid, methionine, has been precluded by its intrinsically weaker nucleophilicity. Here, we report a strategy for chemoselective methionine bioconjugation through redox reactivity, using oxaziridine-based reagents to achieve highly selective, rapid, and robust methionine labeling under a range of biocompatible reaction conditions. We highlight the broad utility of this conjugation method to enable precise addition of payloads to proteins, synthesis of antibody-drug conjugates, and identification of hyperreactive methionine residues in whole proteomes.