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Summary: Helicobacter-induced gastric cancer progresses very slowly, even in animal models, making it difficult to study. Here, we present a protocol to establish an accelerated murine model for Helicobacter-induced gastric cancer. We describe steps for infecting mice with Helicobacter felis, harvesting gastric tissue, assessing disease severity by histopathologic scoring, and performing gene expression studies with RT-qPCR and RNA sequencing. The accelerated model shows rapid progression of the disease, with gastric precancerous lesions developing within 6 months post-infection with Helicobacter.For complete details on the use and execution of this protocol, please refer to Bali et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Many machine learning applications in bioinformatics currently rely on matching gene identities when analyzing input gene signatures and fail to take advantage of preexisting knowledge about gene functions. To further enable comparative analysis of OMICS datasets, including target deconvolution and mechanism of action studies, we develop an approach that represents gene signatures projected onto their biological functions, instead of their identities, similar to how the word2vec technique works in natural language processing. We develop the Functional Representation of Gene Signatures (FRoGS) approach by training a deep learning model and demonstrate that its application to the Broad Institute’s L1000 datasets results in more effective compound-target predictions than models based on gene identities alone. By integrating additional pharmacological activity data sources, FRoGS significantly increases the number of high-quality compound-target predictions relative to existing approaches, many of which are supported by in silico and/or experimental evidence. These results underscore the general utility of FRoGS in machine learning-based bioinformatics applications. Prediction networks pre-equipped with the knowledge of gene functions may help uncover new relationships among gene signatures acquired by large-scale OMICs studies on compounds, cell types, disease models, and patient cohorts.

Summary: Helicobacter pylori (H. pylori) infection is a known cause of many digestive diseases, including gastritis, peptic ulcers, and gastric cancer. However, the underlying mechanisms by which H. pylori infection triggers these disorders are still not clearly understood. Gastric cancer is a slow progressing disease, which makes it difficult to study. We have developed an accelerated disease progression mouse model, which leverages mice deficient in the myeloid differentiation primary response 88 gene (Myd88−/−) infected with Helicobacter felis (H. felis). Using this model and gastric biopsy samples from patients, we report that activation of the Toll/interleukin-1 receptor (TIR)-domain-containing adaptor inducing interferon-β (TRIF)-type I interferon (IFN-I) signaling pathway promotes Helicobacter-induced disease progression toward severe gastric pathology and gastric cancer development. Further, results implicated downstream targets of this pathway in disease pathogenesis. These findings may facilitate stratification of Helicobacter-infected patients and thus enable treatment prioritization of patients.

Hybrid immunity (vaccination + natural infection) to SARS-CoV-2 provides superior protection to re-infection. We performed immune profiling studies during breakthrough infections in mRNA-vaccinated hamsters to evaluate hybrid immunity induction. The mRNA vaccine, BNT162b2, was dosed to induce binding antibody titers against ancestral spike, but inefficient serum virus neutralization of ancestral SARS-CoV-2 or variants of concern (VoCs). Vaccination reduced morbidity and controlled lung virus titers for ancestral virus and Alpha but allowed breakthrough infections in Beta, Delta and Mu-challenged hamsters. Vaccination primed for T cell responses that were boosted by infection. Infection back-boosted neutralizing antibody responses against ancestral virus and VoCs. Hybrid immunity resulted in more cross-reactive sera, reflected by smaller antigenic cartography distances. Transcriptomics post-infection reflects both vaccination status and disease course and suggests a role for interstitial macrophages in vaccine-mediated protection. Therefore, protection by vaccination, even in the absence of high titers of neutralizing antibodies in the serum, correlates with recall of broadly reactive B- and T-cell responses.

Zika virus (ZIKV) infection suppresses the induction of dendritic cell (DC)-derived immunity, but the underlying mechanistical insights are still lacking. Here the authors show, using in vitro systems profiling of DC transcriptome and epigenome, that ZIKV specifically alters SREBP2-related expression of inflammation- and metabolism-related genes to modulate DC functions.

Wiskott-Aldrich syndrome is caused by mutations in WASP, but the underlying mechanisms remain to be explored. Here the authors reveal that WASP deficiency results in aberrant RNA splicing, and that WASP regulates the transcription of splicing factor genes and co-transcriptional RNA splicing via a phase-separation process that involves splicing factors and nascent RNA.

Protein aggregates are a common feature of diseased and aged cells. Membrane proteins comprise a quarter of the proteome, and yet, it is not well understood how aggregation of membrane proteins is regulated and what effects these aggregates can have on cellular health. We have determined in yeast that the derlin Dfm1 has a chaperone-like activity that influences misfolded membrane protein aggregation. We establish that this function of Dfm1 does not require recruitment of the ATPase Cdc48 and it is distinct from Dfm1's previously identified function in dislocating misfolded membrane proteins from the endoplasmic reticulum (ER) to the cytosol for degradation. Additionally, we assess the cellular impacts of misfolded membrane proteins in the absence of Dfm1 and determine that misfolded membrane proteins are toxic to cells in the absence of Dfm1 and cause disruptions to proteasomal and ubiquitin homeostasis.

Substance abuse and addiction represent a significant public health problem that impacts multiple dimensions of society, including healthcare, the economy, and the workforce. In 2021, over 100,000 drug overdose deaths were reported in the US, with an alarming increase in fatalities related to opioids and psychostimulants. Understanding the fundamental gene regulatory mechanisms underlying addiction and related behaviors could facilitate more effective treatments. To explore how repeated drug exposure alters gene regulatory networks in the brain, we combined capped small (cs)RNA-seq, which accurately captures nascent-like initiating transcripts from total RNA, with Hi-C and single nuclei (sn)ATAC-seq. We profiled initiating transcripts in two addiction-related brain regions, the prefrontal cortex (PFC) and the nucleus accumbens (NAc), from rats that were never exposed to drugs or were subjected to prolonged abstinence after oxycodone or cocaine intravenous self-administration (IVSA). Interrogating over 100,000 active transcription start regions (TSRs) revealed that most TSRs had hallmarks of bonafide enhancers and highlighted the KLF/SP1, RFX, and AP1 transcription factors families as central to establishing brain-specific gene regulatory programs. Analysis of rats with addiction-like behaviors versus controls identified addiction-associated repression of transcription at regulatory enhancers recognized by nuclear receptor subfamily 3 group C (NR3C) factors, including glucocorticoid receptors. Cell-type deconvolution analysis using snATAC-seq uncovered a potential role of glial cells in driving the gene regulatory programs associated with addiction-related phenotypes. These findings highlight the power of advanced transcriptomics methods to provide insight into how addiction perturbs gene regulatory programs in the brain.

Abstract Background Motif enrichment analysis (MEA) identifies over-represented transcription factor binding (TF) motifs in the DNA sequence of regulatory regions, enabling researchers to infer which transcription factors can regulate transcriptional response to a stimulus, or identify sequence features found near a target protein in a ChIP-seq experiment. Score-based MEA determines motifs enriched in regions exhibiting extreme differences in regulatory activity, but existing methods do not control for biases in GC content or dinucleotide composition. This lack of control for sequence bias, such as those often found in CpG islands, can obscure the enrichment of biologically relevant motifs. Results We developed Motif Enrichment In Ranked Lists of Peaks (MEIRLOP), a novel MEA method that determines enrichment of TF binding motifs in a list of scored regulatory regions, while controlling for sequence bias. In this study, we compare MEIRLOP against other MEA methods in identifying binding motifs found enriched in differentially active regulatory regions after interferon-beta stimulus, finding that using logistic regression and covariates improves the ability to call enrichment of ISGF3 binding motifs from differential acetylation ChIP-seq data compared to other methods. Our method achieves similar or better performance compared to other methods when quantifying the enrichment of TF binding motifs from ENCODE TF ChIP-seq datasets. We also demonstrate how MEIRLOP is broadly applicable to the analysis of numerous types of NGS assays and experimental designs. Conclusions Our results demonstrate the importance of controlling for sequence bias when accurately identifying enriched DNA sequence motifs using score-based MEA. MEIRLOP is available for download from https://github.com/npdeloss/meirlop under the MIT license.

Abstract Background Mounting evidence suggests several diseases and biological processes target transcription termination to misregulate gene expression. Disruption of transcription termination leads to readthrough transcription past the 3′ end of genes, which can result in novel transcripts, changes in epigenetic states and altered 3D genome structure. Results We developed Automatic Readthrough Transcription Detection (ARTDeco), a tool to detect and analyze multiple features of readthrough transcription from RNA-seq and other next-generation sequencing (NGS) assays that profile transcriptional activity. ARTDeco robustly quantifies the global severity of readthrough phenotypes, and reliably identifies individual genes that fail to terminate (readthrough genes), are aberrantly transcribed due to upstream termination failure (read-in genes), and novel transcripts created as a result of readthrough (downstream of gene or DoG transcripts). We used ARTDeco to characterize readthrough transcription observed during influenza A virus (IAV) infection, validating its specificity and sensitivity by comparing its performance in samples infected with a mutant virus that fails to block transcription termination. We verify ARTDeco’s ability to detect readthrough as well as identify read-in genes from different experimental assays across multiple experimental systems with known defects in transcriptional termination, and show how these results can be leveraged to improve the interpretation of gene expression and downstream analysis. Applying ARTDeco to a gene expression data set from IAV-infected monocytes from different donors, we find strong evidence that read-in gene-associated expression quantitative trait loci (eQTLs) likely regulate genes upstream of read-in genes. This indicates that taking readthrough transcription into account is important for the interpretation of eQTLs in systems where transcription termination is blocked. Conclusions ARTDeco aids researchers investigating readthrough transcription in a variety of systems and contexts.

Background & Aims: Colonic stem cells are essential for producing the mucosal lining, which in turn protects stem cells from insult by luminal factors. Discovery of genetic and biochemical events that control stem cell proliferation and differentiation can be leveraged to decipher the causal factors of ulcerative colitis and aid the development of more effective therapy. Methods: We performed in vivo and in vitro studies from control (nuclear receptor corepressor 1 [NCoR1F/F]) and intestinal epithelial cell–specific NCoR1-deficient mice (NCoR1ΔIEC). Mice were challenged with dextran sodium sulfate to induce experimental ulcerative colitis, followed by colitis examination, barrier permeability analysis, cell proliferation immunostaining assays, and RNA sequencing analysis. By using crypt cultures, the organoid-forming efficiency, cell proliferation, apoptosis, and histone acetylation were analyzed after butyrate and/or tumor necrosis factor α treatments. Results: NCoR1ΔIEC mice showed a dramatic increase in disease severity in this colitis model, with suppression of proliferative cells at the crypt base as an early event and a concomitant increase in barrier permeability. Genome expression patterns showed an important role for NCoR1 in colonic stem cell proliferation and secretory cell differentiation. Colonic organoids cultured from NCoR1ΔIEC mice were more sensitive to butyrate-induced cell growth inhibition and apoptosis, which were exaggerated further by tumor necrosis factor α co-treatment, which was accompanied by increased histone acetylation. Conclusions: NCoR1 regulates colonic stem cell proliferation and secretory cell differentiation. When NCoR1 is disrupted, barrier protection is weakened, allowing luminal products such as butyrate to penetrate and synergistically damage the colonic crypt cells. Transcript profiling: RNA sequencing data have been deposited in the GEO database, accession number: GSE136153.

ABSTRACT New or emerging infectious diseases are commonly caused by pathogens that cannot be readily manipulated or studied under common laboratory conditions. These limitations hinder standard experimental approaches and our abilities to define the fundamental molecular mechanisms underlying pathogenesis. The advance of capped small RNA sequencing (csRNA-seq) now enables genome-wide mapping of actively initiated transcripts from genes and other regulatory transcribed start regions (TSRs) such as enhancers at a precise moment from total RNA. As RNA is nonpathogenic and can be readily isolated from inactivated infectious samples, csRNA-seq can detect acute changes in gene regulation within or in response to a pathogen with remarkable sensitivity under common laboratory conditions. Studying valley fever (coccidioidomycosis), an emerging endemic fungal infection that increasingly impacts livestock, pet, and human health, we show how csRNA-seq can unravel transcriptional programs driving pathogenesis. Performing csRNA-seq on RNA isolated from different stages of the valley fever pathogen Coccidioides immitis revealed alternative promoter usage, connected cis-regulatory domains, and a WOPR family transcription factor, which are known regulators of virulence in other fungi, as being critical for pathogenic growth. We further demonstrate that a C. immitis WOPR homologue, CIMG_02671, activates transcription in a WOPR motif-dependent manner. Collectively, these findings provide novel insights into valley fever pathogenesis and provide a proof of principle for csRNA-seq as a powerful means to determine the genes, regulatory mechanisms, and transcription factors that control the pathogenesis of highly infectious agents. IMPORTANCE Infectious pathogens like airborne viruses or fungal spores are difficult to study; they require high-containment facilities, special equipment, and expertise. As such, establishing approaches such as genome editing or other means to identify the factors and mechanisms underlying caused diseases, and, thus, promising drug targets, is costly and time-intensive. These obstacles particularly hinder the analysis of new, emerging, or rare infectious diseases. We recently developed a method termed capped small RNA sequencing (csRNA-seq) that enables capturing acute changes in active gene expression from total RNA. Prior to csRNA-seq, such an analysis was possible only by using living cells or nuclei, in which pathogens are highly infectious. The process of RNA purification, however, inactivates pathogens and thus enables the analysis of gene expression during disease progression under standard laboratory conditions. As a proof of principle, here, we use csRNA-seq to unravel the gene regulatory programs and factors likely critical for the pathogenesis of valley fever, an emerging endemic fungal infection that increasingly impacts livestock, pet, and human health.

Hypertriglyceridemia results from accumulation of triglyceride (TG)-rich lipoproteins (TRLs) in the circulation and is associated with increased CVD risk. ApoC-III is an apolipoprotein on TRLs and a prominent negative regulator of TG catabolism. We recently established that in vivo apoC-III predominantly inhibits LDL receptor-mediated and LDL receptor-related protein 1-mediated hepatic TRL clearance and that apoC-III-enriched TRLs are preferentially cleared by syndecan-1 (SDC1). In this study, we determined the impact of apoE, a common ligand for all three receptors, on apoC-III metabolism using apoC-III antisense oligonucleotide (ASO) treatment in mice lacking apoE and functional SDC1 (Apoe−/−Ndst1f/fAlb-Cre+). ApoC-III ASO treatment significantly reduced plasma TG levels in Apoe−/−Ndst1f/fAlb-Cre+ mice without reducing hepatic VLDL production or improving hepatic TRL clearance. Further analysis revealed that apoC-III ASO treatment lowered plasma TGs in Apoe−/−Ndst1f/fAlb-Cre+ mice, which was associated with increased LPL activity in white adipose tissue in the fed state. Finally, clinical data confirmed that ASO-mediated lowering of APOC-III via volanesorsen can reduce plasma TG levels independent of the APOE isoform genotype. Our data indicate that apoE determines the metabolic impact of apoC-III as we establish that apoE is essential to mediate inhibition of TRL clearance by apoC-III and that, in the absence of functional apoE, apoC-III inhibits tissue LPL activity.

With the increasing obtainability of multi-OMICs data comes the need for easy to use data analysis tools. Here, the authors introduce Metascape, a biologist-oriented portal that provides a gene list annotation, enrichment and interactome resource and enables integrated analysis of multi-OMICs datasets.

Abstract Identification of the precise molecular pathways involved in oncogene-induced transformation may help us gain a better understanding of tumor initiation and promotion. Here, we demonstrate that SOX2+ foregut epithelial cells are prone to oncogenic transformation upon mutagenic insults, such as Kras G12D and p53 deletion. GFP-based lineage-tracing experiments indicate that SOX2+ cells are the cells-of-origin of esophagus and stomach hyperplasia. Our observations indicate distinct roles for oncogenic KRAS mutation and P53 deletion. p53 homozygous deletion is required for the acquisition of an invasive potential, and Kras G12D expression, but not p53 deletion, suffices for tumor formation. Global gene expression analysis reveals secreting factors upregulated in the hyperplasia induced by oncogenic KRAS and highlights a crucial role for the CXCR2 pathway in driving hyperplasia. Collectively, the array of genetic models presented here demonstrate that stratified epithelial cells are susceptible to oncogenic insults, which may lead to a better understanding of tumor initiation and aid in the design of new cancer therapeutics.

Transcription factors of the AP-1 family can play diverse roles despite recognizing the same DNA sequence. Here the authors investigate the DNA binding activities of AP-1 members in mouse macrophages and apply a machine learning approach to identify motifs predicted to drive factor-specific binding profiles.

Inflammation is known to affect cancer development, yet the mechanisms by which immune signaling drives transformation remain unclear. Here, the authors provide evidence that chronic TNF-α signaling promotes the enhancer binding and transcriptional interplay between mutant p53 and NFκB.

Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.

Hosts mount prudently tuned responses to viral infection in an attempt to block nearly every step of the replication cycle. Viruses must adapt to replicate in this hostile antiviral cellular state. Interferon stimulation or pathogen challenge robustly induces expression of IFIT (interferon-induced proteins with tetratricopeptide repeats) proteins. IFITs are a family of proteins that bind RNA and play antiviral roles during infection. Thus, we were surprised to identify the IFIT family as top candidate proviral host factors for influenza A virus (IAV) in a genome-wide CRISPR–Cas9 knockout screen. We validated the proviral activity of IFIT2 by showing that IFIT2-deficient cells support lower levels of IAV replication and exhibit defects in viral gene expression. The molecular functions of IFIT2, let alone how they are used by influenza virus, are unknown. Using CLIP-seq, we showed that IFIT2 binds directly to viral and cellular mRNAs in AU-rich regions largely in the 3’UTR, with a preference for a subset of interferon-stimulated mRNAs. IFIT2 also associates with actively translating ribosomes in infected cells to facilitate the translation of viral messages. IFIT2-responsive elements from an IAV mRNA were sufficient to confer translational enhancement to exogenous transcripts in cis. Conversely, mutation of these elements or the use of an IFIT2 RNA-binding mutant ablated stimulation of viral gene expression. Together, these data link the RNA-binding capability of IFIT2 to changes in translational efficiency of target viral mRNAs and the stimulation of viral replication. They establish a model for the normal function of IFIT2 as an antiviral protein affecting the post-transcriptional fate of cellular mRNAs and explain how influenza virus repurposes IFIT2 to support viral replication. Our work highlights a new node for the regulation of translation during interferon responses and highlights how canonical antiviral responses may be repurposed to support viral replication.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus linked to devastating neurologic diseases. Immune responses to flaviviruses may be pathogenic or protective. Our understanding of human immune responses to ZIKV in vivo remains limited. Therefore, we performed a longitudinal molecular and phenotypic characterization of innate and adaptive immune responses during an acute ZIKV infection. We found that innate immune transcriptional and genomic responses were both cell type- and time-dependent. While interferon stimulated gene induction was common to all innate immune cells, the upregulation of important inflammatory cytokine genes was primarily limited to monocyte subsets. Additionally, genomic analysis revealed substantial chromatin remodeling at sites containing cell-type specific transcription factor binding motifs that may explain the observed changes in gene expression. In this dengue virus-experienced individual, adaptive immune responses were rapidly mobilized with T cell transcriptional activity and ZIKV neutralizing antibody responses peaking 6 days after the onset of symptoms. Collectively this study characterizes the development and resolution of an in vivo human immune response to acute ZIKV infection in an individual with pre-existing flavivirus immunity.

ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, is frequently mutated in cancer. Deficiency in its homolog ARID1B is synthetically lethal with ARID1A mutation. However, the functional relationship between these homologs has not been explored. Here, we use ATAC-seq, genome-wide histone modification mapping, and expression analysis to examine colorectal cancer cells lacking one or both ARID proteins. We find that ARID1A has a dominant role in maintaining chromatin accessibility at enhancers, while the contribution of ARID1B is evident only in the context of ARID1A mutation. Changes in accessibility are predictive of changes in expression and correlate with loss of H3K4me and H3K27ac marks, nucleosome spacing, and transcription factor binding, particularly at growth pathway genes including MET. We find that ARID1B knockdown in ARID1A mutant ovarian cancer cells causes similar loss of enhancer architecture, suggesting that this is a conserved function underlying the synthetic lethality between ARID1A and ARID1B.

Genome-wide maps of DNase I hypersensitive sites (DHSs) reveal that most human promoters contain perpetually active cis-regulatory elements between -150 bp and +50 bp (-150/+50 bp) relative to the transcription start site (TSS). Transcription factors (TFs) recruit cofactors (chromatin remodelers, histone/protein-modifying enzymes, and scaffold proteins) to these elements in order to organize the local chromatin structure and coordinate the balance of post-translational modifications nearby, contributing to the overall regulation of transcription. However, the rules of TF-mediated cofactor recruitment to the -150/+50 bp promoter regions remain poorly understood. Here, we provide evidence for a general model in which a series of cis-regulatory elements (here termed 'cardinal' motifs) prefer acting individually, rather than in fixed combinations, within the -150/+50 bp regions to recruit TFs that dictate cofactor signatures distinctive of specific promoter subsets. Subsequently, human promoters can be subclassified based on the presence of cardinal elements and their associated cofactor signatures. In this study, furthermore, we have focused on promoters containing the nuclear respiratory factor 1 (NRF1) motif as the cardinal cis-regulatory element and have identified the pervasive association of NRF1 with the cofactor lysine-specific demethylase 1 (LSD1/KDM1A). This signature might be distinctive of promoters regulating nuclear-encoded mitochondrial and other particular genes in at least some cells. Together, we propose that decoding a signature-based, expanded model of control at proximal promoter regions should lead to a better understanding of coordinated regulation of gene transcription.

Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Comprehensive genome-wide analysis of the epigenetic and transcription status of the TLR4-induced transcriptional program in macrophages suggests that Toll-like receptor 4 (TLR4)-dependent activation of nearly all immediate/early- (I/E) and late-response genes results from a sequential process in which signal-independent factors initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by encoding a distinct set of signal-dependent transcription factor elements, including TATA boxes, which lead to preferential binding of TBP and basal enrichment for RNA polymerase II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO) sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases the overall rates of both transcriptional initiation and the efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. Collectively, these findings reveal broadly utilized mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses.