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As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing and/or sequencing capacity, which can also introduce biases 1 – 3 . SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing 4 , 5 . Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We developed and deployed improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detected emerging variants of concern up to 14 days earlier in wastewater samples, and identified multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission. Emerging SARS-CoV-2 variants of concern were detected early and multiple cases of virus spread not captured by clinical genomic surveillance were identified using high-resolution wastewater and clinical sequencing.

Key Points A large number of co-repressor complexes, containing a series of different enzymatic activities, are recruited to DNA by transcription factors. Their function is to mediate chromatin remodelling and modifications of the histone tails, therefore cooperating in establishing and maintaining transcriptional repression. The co-repressors nuclear receptor co-repressor (NCoR, also known as NCOR1) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT, also known as NCOR2) were originally isolated in 1995. Since then our view of the mechanism of action of co-repressors has progressively changed. Here, we describe how models of co-repressor function have evolved from a simple exchange between co-repressors and co-activators to a combinatorial model of co-repressor and co-activator action. Dismissal of co-repressors from regulated promoters is important to allow co-activator recruitment and is achieved through active steps of de-repression that involve post-translational modification of the co-repressors. Gene deletion studies of the NCoR and SMRT co-repressors in mouse models have indicated that they are specifically required for different developmental processes. NCoR is crucial in the development of erythrocytes and thymocytes, whereas SMRT is required for the development of the heart. Transcriptional repression mediated by NCoR and SMRT is crucial in the maintenance of embryonic neural stem cells, and their absence results in differentiation down glial or glial and neuronal pathways, respectively. The classical model of gene activation by a unidirectional switch from co-repressor binding to co-activator binding is changing. This Review discusses emerging themes in the interplay among co-repressor complexes, enzymatic functions and chromatin modifications in controlling gene repression. A crucial aspect of development, homeostasis and prevention of disease is the strict maintenance of patterns of gene repression. Gene repression is largely achieved by the combinatorial action of various enzymatic complexes — known as co-repressor complexes — that are recruited to DNA by transcription factors and often act through enzymatic modification of histone protein tails. Our understanding of how co-repressors act has begun to change over recent years owing to the increased availability of genome-scale data. Here, we consider specific strategies that underlie repression events — for example, those mediated by the nuclear receptor co-repressor (NCoR, also known as NCOR1) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT, also known as NCOR2) co-repressor complexes — and discuss emerging themes in gene repression.

In the tumor microenvironment, local immune dysregulation is driven in part by macrophages and dendritic cells that are polarized to a mixed proinflammatory/immune-suppressive phenotype. The unfolded protein response (UPR) is emerging as the possible origin of these events. Here we report that the inositol-requiring enzyme 1 (IRE1α) branch of the UPR is directly involved in the polarization of macrophages in vitro and in vivo, including the up-regulation of interleukin 6 (IL-6), IL-23, Arginase1, as well as surface expression of CD86 and programmed death ligand 1 (PD-L1). Macrophages in which the IRE1α/X-box binding protein 1 (Xbp1) axis is blocked pharmacologically or deleted genetically have significantly reduced polarization and CD86 and PD-L1 expression, which was induced independent of IFNγ signaling, suggesting a novel mechanism in PD-L1 regulation in macrophages. Mice with IRE1α- but not Xbp1-deficient macrophages showed greater survival than controls when implanted with B16.F10 melanoma cells. Remarkably, we found a significant association between the IRE1α gene signature and CD274 gene expression in tumor-infiltrating macrophages in humans. RNA sequencing (RNASeq) analysis showed that bone marrow-derived macrophages with IRE1α deletion lose the integrity of the gene connectivity characteristic of regulated IRE1α-dependent decay (RIDD) and the ability to activate CD274 gene expression. Thus, the IRE1α/Xbp1 axis drives the polarization of macrophages in the tumor microenvironment initiating a complex immune dysregulation leading to failure of local immune surveillance.

Mutations in the splicing factor SF3B1 are found in several cancer types and have been associated with various splicing defects. Using transcriptome sequencing data from chronic lymphocytic leukemia, breast cancer and uveal melanoma tumor samples, we show that hundreds of cryptic 3' splice sites (3'SSs) are used in cancers with SF3B1 mutations. We define the necessary sequence context for the observed cryptic 3' SSs and propose that cryptic 3'SS selection is a result of SF3B1 mutations causing a shift in the sterically protected region downstream of the branch point. While most cryptic 3'SSs are present at low frequency (<10%) relative to nearby canonical 3'SSs, we identified ten genes that preferred out-of-frame cryptic 3'SSs. We show that cancers with mutations in the SF3B1 HEAT 5-9 repeats use cryptic 3'SSs downstream of the branch point and provide both a mechanistic model consistent with published experimental data and affected targets that will guide further research into the oncogenic effects of SF3B1 mutation.

Background: A key step in domestication of the grapevine was the transition fromseparate sexes (dioecy) in wild Vitis vinifera ssp. sylvestris (V. sylvestris) to hermaphroditismincultivated Vitis vinifera ssp. sativa (V. vinifera). It is known that V. sylvestris has an XY systemand V. vinifera a modified Y haplotype (Yh) and that the sex locus is small, but it has notpreviously been precisely characterized.Results: We generate a high-quality de novo reference genome for V. sylvestris, onto whichwe map whole-genome re-sequencing data of a cross to locate the sex locus. Assembly ofthe full X, Y, and Yh haplotypes of V. sylvestris and V. vinifera sex locus and examining theirgene content and expression profiles during flower development in wild and cultivatedaccessions show that truncation and deletion of tapetum and pollen development geneson the X haplotype likely causes male sterility, while the upregulation of a Y allele of acytokinin regulator (APRT3) may cause female sterility. The downregulation of this cytokininregulator in the Yh haplotype may be sufficient to trigger reversal to hermaphroditism.Molecular dating of X and Y haplotypes is consistent with the sex locus being as old as theVitis genus, but the mechanism by which recombination was suppressed remainsundetermined.Conclusions: We describe the genomic and evolutionary characterization of the sex locusof cultivated and wild grapevine, providing a coherent model of sex determination in thelatter and for transition from dioecy to hermaphroditism during domestication.

Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Analyzing hundreds of hiPSCs derived from different individuals, we show the proportions of these pluripotent states vary considerably across lines. We discover 13 gene network modules (GNMs) and 13 regulatory network modules (RNMs), which are highly correlated with each other suggesting that the coordinated co-accessibility of regulatory elements in the RNMs likely underlie the coordinated expression of genes in the GNMs. Epigenetic analyses reveal that regulatory networks underlying self-renewal and pluripotency are more complex than previously realized. Genetic analyses identify thousands of regulatory variants that overlapped predicted transcription factor binding sites and are associated with chromatin accessibility in the hiPSCs. We show that the master regulator of pluripotency, the NANOG-OCT4 Complex, and its associated network are significantly enriched for regulatory variants with large effects, suggesting that they play a role in the varying cellular proportions of pluripotency states between hiPSCs. Our work bins tens of thousands of regulatory elements in hiPSCs into discrete regulatory networks, shows that pluripotency and self-renewal processes have a surprising level of regulatory complexity, and suggests that genetic factors may contribute to cell state transitions in human iPSC lines. Stem cells exist in vitro in a spectrum of interconvertible pluripotent states. Here, authors show that pluripotency and self-renewal processes have a high level of regulatory complexity and suggest that genetic factors contribute to cell state transitions in human iPSC lines.

Background Ménière's disease (MD) is a disorder of the inner ear that causes episodic bouts of severe dizziness, roaring tinnitus, and fluctuating hearing loss. To date, no targeted therapy exists. As such, we have undertaken a large whole genome sequencing study on carefully phenotyped unilateral MD patients with the goal of gene/pathway discovery and a move towards targeted intervention. This study was a retrospective review of patients with a history of Ménière's disease. Genomic DNA, acquired from saliva samples, was purified and subjected to whole genome sequencing. Results Stringent variant calling, performed on 511 samples passing quality checks, followed by gene-based filtering by recurrence and proximity in molecular interaction networks, led to 481 high priority MD genes. These high priority genes, including MPHOSPH8, MYO18A, TRIOBP, OTOGL, TNC, and MYO6, were previously implicated in hearing loss, balance, and cochlear function, and were significantly enriched in common variant studies of hearing loss. Validation in an independent MD cohort confirmed 82 recurrent genes. Pathway analysis pointed to cell–cell adhesion, extracellular matrix, and cellular energy maintenance as key mediators of MD. Furthermore, the MD-prioritized genes were highly expressed in human inner ear hair cells and dark/vestibular cells, and were differentially expressed in a mouse model of hearing loss. Conclusion By enabling the development of model systems that may lead to targeted therapies and MD screening panels, the genes and variants identified in this study will inform diagnosis and treatment of MD.

The SMRT transcriptional co-repressor is shown to have a crucial role in forebrain development and in maintaining neural stem cells, and it represses expression of a histone H3 trimethyl K27 demethylase, which can activate components of the neurogenic programme. A series of transcription factors critical for maintenance of the neural stem cell state have been identified 1 , 2 , 3 , but the role of functionally important corepressors 4 , 5 , 6 , 7 in maintenance of the neural stem cell state and early neurogenesis remains unclear. Previous studies have characterized the expression of both SMRT (also known as NCoR2 , nuclear receptor co-repressor 2) and NCoR in a variety of developmental systems 8 ; however, the specific role of the SMRT corepressor in neurogenesis is still to be determined. Here we report a critical role for SMRT in forebrain development and in maintenance of the neural stem cell state. Analysis of a series of markers in SMRT -gene-deleted mice revealed the functional requirement of SMRT in the actions of both retinoic-acid-dependent and Notch-dependent forebrain development. In isolated cortical progenitor cells, SMRT was critical for preventing retinoic-acid-receptor-dependent induction of differentiation along a neuronal pathway in the absence of any ligand. Our data reveal that SMRT represses expression of the jumonji-domain containing gene JMJD3 , a direct retinoic-acid-receptor target that functions as a histone H3 trimethyl K27 demethylase and which is capable of activating specific components of the neurogenic program.

Throughout the COVID-19 pandemic, massive sequencing and data sharing efforts enabled the real-time surveillance of novel SARS-CoV-2 strains throughout the world, the results of which provided public health officials with actionable information to prevent the spread of the virus. However, with great sequencing comes great computation, and while cloud computing platforms bring high-performance computing directly into the hands of all who seek it, optimal design and configuration of a cloud compute cluster requires significant system administration expertise. We developed ViReflow, a user-friendly viral consensus sequence reconstruction pipeline enabling rapid analysis of viral sequence datasets leveraging Amazon Web Services (AWS) cloud compute resources and the Reflow system. ViReflow was developed specifically in response to the COVID-19 pandemic, but it is general to any viral pathogen. Importantly, when utilized with sufficient compute resources, ViReflow can trim, map, call variants, and call consensus sequences from amplicon sequence data from 1000 SARS-CoV-2 samples at 1000X depth in < 10 min, with no user intervention. ViReflow’s simplicity, flexibility, and scalability make it an ideal tool for viral molecular epidemiological efforts.

As metagenomic studies move to increasing numbers of samples, communities like the human gut may benefit more from the assembly of abundant microbes in many samples, rather than the exhaustive assembly of fewer samples. We term this approach leaderboard metagenome sequencing. To explore protocol optimization for leaderboard metagenomics in real samples, we introduce a benchmark of library prep and sequencing using internal references generated by synthetic long-read technology, allowing us to evaluate high-throughput library preparation methods against gold-standard reference genomes derived from the samples themselves. We introduce a low-cost protocol for high-throughput library preparation and sequencing.