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Table S3: List of antibodies used in this study.

Supplementary Figures S1-19 and legends. Figure S1: Overexpression of HIF-1α enhances BTIC characteristics. Figure S2: Characterizations of isolated BTICs by different parameters. Figure S3: The expression of TET1 and TET3 is upregulated by hypoxia. Figure S4: HIF-1α binding to TET1 and TET3 gene promoter in HIF-1α overexpressing cells. Figure S5: Knockdown of TET diminishes hypoxia induced activation of stemness related genes in breast cancer cell lines. Figure S6: Knockdown of TET1/3 represses tumorigenicity of isolated BTICs in vivo. Figure S7: Isotype control for IHC analysis. Figure S8: Gene specific changes in 5hmC level and TET proteins binding in hypoxic, HIF-1α overexpressing cells and isolated BTICs. Figure S9: Starvation is unable to affect the levels of 5hmC on the genome regions of TNFα gene and TNFα gene expression. Figure S10: TET proteins are essential for hypoxia activated TNFα-p38-MAPK signaling. Figure S11: TET proteins are required for hypoxia-induced 5hmC change on the genome regions of the TNFα gene. Figure S12: The enzymatic activity of TET proteins is required for establishing 5hmC signature upon hypoxia. Figure S13: The enzymatic activity of TET proteins is required for activation of TNFα-p38-MAPK signaling upon hypoxia. Figure S14: Protein-protein interaction between TET1 and TET3 in hypoxic cells and isolated BTICs. Figure S15: Co-occupancy of TET1 and TET3 on genome regions of the TNFα gene. Figure S16: Suppression of p38-MAPK signaling reduces BTIC properties. Figure S17: Certain downstream targets of TNFα-p38-MAPK signaling are regulated by hypoxia/TET. Figure S18: The levels of 5hmC and the bindings of TET proteins at pluripotent loci upon hypoxia. Figure S19: The landscape for 5hmC level and TET proteins binding on pluripotent loci upon hypoxia.

Score-based motif enrichment analysis (MEA) is typically applied to regulatory DNA to infer transcription factors (TFs) that may modulate transcription and chromatin state in different conditions. Most MEA methods determine motif enrichment independent of motif position within a sequence, even when those sequences harbor anchor points that motifs and their bound TFs may functionally interact with in a distance-dependent fashion, such as other TF binding motifs, transcription start sites (TSS), sequencing assay cleavage sites, or other biologically meaningful features. We developed motif enrichment positional profiling (MEPP), a novel MEA method that outputs a positional enrichment profile of a given TF’s binding motif relative to key anchor points (e.g. transcription start sites, or other motifs) within the analyzed sequences while accounting for lower-order nucleotide bias. Using transcription initiation and TF binding as test cases, we demonstrate MEPP’s utility in determining the sequence positions where motif presence correlates with measures of biological activity, inferring positional dependencies of binding site function. We demonstrate how MEPP can be applied to interpretation and hypothesis generation from experiments that quantify transcription initiation, chromatin structure, or TF binding measurements. MEPP is available for download from https://github.com/npdeloss/mepp.

The fate of influenza A virus (IAV) infection in the host cell depends on the balance between cellular defence mechanisms and viral evasion strategies. To illuminate the landscape of IAV cellular restriction, we generated and integrated global genetic loss-of-function screens with transcriptomics and proteomics data. Our multi-omics analysis revealed a subset of both IFN-dependent and independent cellular defence mechanisms that inhibit IAV replication. Amongst these, the autophagy regulator TBC1 domain family member 5 (TBC1D5), which binds Rab7 to enable fusion of autophagosomes and lysosomes, was found to control IAV replication in vitro and in vivo and to promote lysosomal targeting of IAV M2 protein. Notably, IAV M2 was observed to abrogate TBC1D5-Rab7 binding through a physical interaction with TBC1D5 via its cytoplasmic tail. Our results provide evidence for the molecular mechanism utilised by IAV M2 protein to escape lysosomal degradation and traffic to the cell membrane, where it supports IAV budding and growth.

Background: The Breast Cancer Index (BCI) is a gene expression-based signature that consists of two functional biomarker panels, HOXB13/IL17BR (H/I) and Molecular Grade Index (MGI), that interrogate important proliferation and estrogen signaling pathways in breast cancer. The BCI prognostic score reports individualized risk of overall and late distant recurrence and is based on the algorithmic combination of the H/I ratio and MGI, while the predictive component, BCI (H/I), reports a categorical prediction of high versus low likelihood of benefit from extended endocrine therapy.The homeobox transcription factor HOXB13 has previously been shown to reprogram genome-wide binding of the androgen receptor (AR) during prostate cancer tumorigenesis, where it colocalizes with FOXA1 at reprogrammed AR binding sites. The aim of the current study was to characterize the potential role of HOXB13 in estrogen receptor (ER) reprogramming by analyzing changes in the global ER binding pattern induced by transient overexpression of HOXB13 in breast cancer cells. In addition, gene ontology (GO) analysis was performed to delineate the functional role of HOXB13 in modulating estrogen signaling and response to endocrine therapy.Methods: HOXB13 was overexpressed in MCF-7 cells by electroporating HOXB13 mRNA, or eGFP mRNA as control. Cells were harvested at different time points and analyzed by western blot and chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) using antibodies against ER, HOXB13, FOXA1 and H3K27ac. After aligning reads with Bowtie2, peak calling and data integration were performed using HOMER v4.10. Gene ontology (GO) analysis was performed using the Genomic Regions Enrichment of Annotations Tool (GREAT v4.0.4) (http://great.stanford.edu/) to identify annotations enriched among genes near ER genomic binding sites.Results: ChIP-seq analysis revealed substantial binding of HOXB13 to a large number of genomic binding sites compared to the eGFP control. HOXB13 overexpression in ER+ breast cancer cells significantly increased ER binding with a majority of ER binding sites being co-bound by FOXA1 and HOXB13. GO analysis of both proximal and distal genomic regions showed significant enrichment of genes associated with mammary gland development, such as “mammary gland epithelium development”, “mammary gland development”, and “mammary gland epithelial cell differentiation”.Conclusion: Findings from this analysis show that transient HOXB13 overexpression reprograms and expands the ER binding pattern in breast cancer cells. Genomic regions newly bound by ER are enriched for genes involved in mammary gland and epithelial differentiation suggesting that HOXB13 activates ER transcriptional programs that link oncogenic and developmental pathways. These results will be compared to ongoing experiments using an inducible HOXB13 expression system to assess the effects of HOXB13 expression on ER binding and function in response to estradiol and tamoxifen. Citation Format: Kai Treuner, Dominic Schenone, Christopher Benner, Catherine A Schnabel, Sven Heinz. Characterization of HOXB13-induced estrogen receptor reprogramming in breast cancer cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-32.

Before their delivery to and degradation by the 26S proteasome, misfolded transmembrane proteins of the endoplasmic reticulum (ER) and inner-nuclear membrane (INM) must be extracted from lipid bilayers. This extraction process, known as retrotranslocation, requires both quality-control E3 ubiquitin ligases and dislocation factors that diminish the energetic cost of dislodging the transmembrane segments of a protein. Recently, we showed that retrotranslocation of all ER transmembrane proteins requires the Dfm1 rhomboid pseudoprotease. However, we did not investigate whether Dfm1 also mediated retrotranslocation of transmembrane substrates in the INM, which is contiguous with the ER but functionally separated from it by nucleoporins. Here, we show that canonical retrotranslocation occurs during INM-associated degradation (INMAD) but proceeds independently of Dfm1. Despite this independence, ER-associated degradation (ERAD)-M and INMAD cooperate to mitigate proteotoxicity. We show a novel misfolded-transmembrane-protein toxicity that elicits genetic suppression, demonstrating the cell's ability to tolerate a toxic burden of misfolded transmembrane proteins without functional INMAD or ERAD-M. This strikingly contrasted the suppression of the dfm1Δ null, which leads to the resumption of ERAD-M through HRD-complex remodeling. Thus, we conclude that INM retrotranslocation proceeds through a novel, private channel that can be studied by virtue of its role in alleviating membrane-associated proteotoxicity.

The contribution of altered mitochondrial Ca2+ handling to metabolic and functional defects in type 2 diabetic (T2D) mouse hearts is not well understood. In this study, we show that the T2D heart is metabolically inflexible and almost exclusively dependent on mitochondrial fatty acid oxidation as a consequence of mitochondrial calcium uniporter complex (MCUC) inhibitory subunit MCUb overexpression. Using a recombinant endonuclease-deficient Cas9-based gene promoter pulldown approach coupled with mass spectrometry, we found that MCUb is upregulated in the T2D heart due to loss of glucose homeostasis regulator nuclear receptor corepressor 2 repression, and chromatin immunoprecipitation assays identified peroxisome proliferator–activated receptor α as a mediator of MCUb gene expression in T2D cardiomyocytes. Upregulation of MCUb limits mitochondrial matrix Ca2+ uptake and impairs mitochondrial energy production via glucose oxidation by depressing pyruvate dehydrogenase complex activity. Gene therapy displacement of endogenous MCUb with a dominant-negative MCUb transgene (MCUbW246R/V251E) in vivo rescued T2D cardiomyocytes from metabolic inflexibility and stimulated cardiac contractile function and adrenergic responsiveness by enhancing phospholamban phosphorylation via protein kinase A. We conclude that MCUb represents one newly discovered molecular effector at the interface of metabolism and cardiac function, and its repression improves the outcome of the chronically stressed diabetic heart.

Histone acetylation levels are reduced during mitosis. To study the mitotic regulation of H3K9ac we used an array of inhibitors targeting specific histone deacetylases. We evaluated the involvement of the targeted enzymes in regulating H3K9ac during all mitotic stages by immunofluorescence and immunoblots. We identified HDAC2, HDAC3 and SIRT1 as modulators of H3K9ac mitotic levels. HDAC2 inhibition increased H3K9ac levels in prophase, whereas HDAC3 or SIRT1 inhibition increased H3K9ac levels in metaphase. Next, we performed ChIP-seq on mitotic-arrested cells following targeted inhibition of these histone deacetylases. We found that both HDAC2 and HDAC3 have a similar impact on H3K9ac, and inhibiting either of these two HDACs substantially increases the levels of this histone acetylation in promoters, enhancers and insulators. Altogether, our results support a model in which H3K9 deacetylation is a stepwise process – at prophase HDAC2 modulates most transcription-associated H3K9ac-marked loci and at metaphase HDAC3 maintains the reduced acetylation, whereas SIRT1 potentially regulates H3K9ac by impacting HAT activity.Summary blurbCombination of immunofluorescence, western blot and ChIP-seq revealed the interplay between HDAC2, HDAC3 and SIRT1 in H3K9 deacetylation during mitosis of mammalian cells.

Summary The emergence of the novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to require at least 12-18 months, and the typical timeline for approval of a novel antiviral therapeutic can exceed 10 years. Thus, repurposing of known drugs could significantly accelerate the deployment of novel therapies for COVID-19. Towards this end, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. We report the identification of 100 molecules that inhibit viral replication, including 21 known drugs that exhibit dose response relationships. Of these, thirteen were found to harbor effective concentrations likely commensurate with achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2–4, and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334. Notably, MDL-28170, ONO 5334, and apilimod were found to antagonize viral replication in human iPSC-derived pneumocyte-like cells, and the PIKfyve inhibitor also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, the known pharmacological and human safety profiles of these compounds will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.

Background & Aims Chronic alcohol consumption is a leading risk factor for the development of hepatocellular carcinoma (HCC), which is associated with a marked increase in hepatic expression of pro-inflammatory IL-17A and its receptor IL-17RA. Methods Genetic deletion and pharmacological blocking were used to characterize the role of IL-17A/IL-17RA signaling in the pathogenesis of HCC in mouse models and human specimens. Results We demonstrate that the global deletion of the Il-17ra gene suppressed HCC in alcohol-fed diethylnitrosamine-challenged Il-17ra–/– and major urinary protein-urokinase-type plasminogen activator/Il-17ra–/– mice compared with wild-type mice. When the cell-specific role of IL-17RA signaling was examined, the development of HCC was decreased in both alcohol-fed Il-17raΔMΦ and Il-17raΔHep mice devoid of IL-17RA in myeloid cells and hepatocytes, but not in Il-17raΔHSC mice (deficient in IL-17RA in hepatic stellate cells). Deletion of Il-17ra in myeloid cells ameliorated tumorigenesis via suppression of pro-tumorigenic/inflammatory and pro-fibrogenic responses in alcohol-fed Il-17raΔMΦ mice. Remarkably, despite a normal inflammatory response, alcohol-fed Il-17raΔHep mice developed the fewest tumors (compared with Il-17raΔMΦ mice), with reduced steatosis and fibrosis. Steatotic IL-17RA-deficient hepatocytes downregulated the expression of Cxcl1 and other chemokines, exhibited a striking defect in tumor necrosis factor (TNF)/TNF receptor 1-dependent caspase-2-SREBP1/2-DHCR7-mediated cholesterol synthesis, and upregulated the production of antioxidant vitamin D3. The pharmacological blocking of IL-17A/Th-17 cells using anti-IL-12/IL-23 antibodies suppressed the progression of HCC (by 70%) in alcohol-fed mice, indicating that targeting IL-17 signaling might provide novel strategies for the treatment of alcohol-induced HCC. Conclusions Overall, IL-17A is a tumor-promoting cytokine, which critically regulates alcohol-induced hepatic steatosis, inflammation, fibrosis, and HCC. Lay summary IL-17A is a tumor-promoting cytokine, which critically regulates inflammatory responses in macrophages (Kupffer cells and bone-marrow-derived monocytes) and cholesterol synthesis in steatotic hepatocytes in an experimental model of alcohol-induced HCC. Therefore, IL-17A may be a potential therapeutic target for patients with alcohol-induced HCC.

Developmental progression depends on temporally defined changes in gene expression mediated by transient exposure of lineage intermediates to signals in the progenitor niche. To determine whether cell-intrinsic epigenetic mechanisms contribute to signal-induced transcriptional responses, here we manipulate the signalling environment and activity of the histone demethylase LSD1 during differentiation of hESC-gut tube intermediates into pancreatic endocrine cells. We identify a transient requirement for LSD1 in endocrine cell differentiation spanning a short time-window early in pancreas development, a phenotype we reproduced in mice. Examination of enhancer and transcriptome landscapes revealed that LSD1 silences transiently active retinoic acid (RA)-induced enhancers and their target genes. Furthermore, prolonged RA exposure phenocopies LSD1 inhibition, suggesting that LSD1 regulates endocrine cell differentiation by limiting the duration of RA signalling. Our findings identify LSD1-mediated enhancer silencing as a cell-intrinsic epigenetic feedback mechanism by which the duration of the transcriptional response to a developmental signal is limited., How epigenetic regulation affects pancreatic development is unclear. Here, the authors show that the histone demethylase LSD1 regulates the epigenetic state of developmental enhancers during pancreatic specification and controls how these enhancers respond to extracellular signals, namely retinoic acid.

Alcoholic liver disease (ALD) is a leading cause of cirrhosis in the United States, which is characterized by extensive deposition of extracellular matrix proteins and formation of a fibrous scar. Hepatic stellate cells (HSCs) are the major source of collagen type 1 producing myofibroblasts in ALD fibrosis. However, the mechanism of alcohol‐induced activation of human and mouse HSCs is not fully understood. We compared the gene‐expression profiles of primary cultured human HSCs (hHSCs) isolated from patients with ALD (n = 3) or without underlying liver disease (n = 4) using RNA‐sequencing analysis. Furthermore, the gene‐expression profile of ALD hHSCs was compared with that of alcohol‐activated mHSCs (isolated from intragastric alcohol‐fed mice) or CCl4‐activated mouse HSCs (mHSCs). Comparative transcriptome analysis revealed that ALD hHSCs, in addition to alcohol‐activated and CCl4‐activated mHSCs, share the expression of common HSC activation (Col1a1 [collagen type I alpha 1 chain], Acta1 [actin alpha 1, skeletal muscle], PAI1 [plasminogen activator inhibitor‐1], TIMP1 [tissue inhibitor of metalloproteinase 1], and LOXL2 [lysyl oxidase homolog 2]), indicating that a common mechanism underlies the activation of human and mouse HSCs. Furthermore, alcohol‐activated mHSCs most closely recapitulate the gene‐expression profile of ALD hHSCs. We identified the genes that are similarly and uniquely up‐regulated in primary cultured alcohol‐activated hHSCs and freshly isolated mHSCs, which include CSF1R (macrophage colony‐stimulating factor 1 receptor), PLEK (pleckstrin), LAPTM5 (lysosmal‐associated transmembrane protein 5), CD74 (class I transactivator, the invariant chain), CD53, MMP9 (matrix metallopeptidase 9), CD14, CTSS (cathepsin S), TYROBP (TYRO protein tyrosine kinase‐binding protein), and ITGB2 (integrin beta‐2), and other genes (compared with CCl4‐activated mHSCs). Conclusion: We identified genes in alcohol‐activated mHSCs from intragastric alcohol‐fed mice that are largely consistent with the gene‐expression profile of primary cultured hHSCs from patients with ALD. These genes are unique to alcohol‐induced HSC activation in two species, and therefore may become targets or readout for antifibrotic therapy in experimental models of ALD., We identified genes in alcohol‐activated mHSCs from IG alcohol‐fed mice that are largely consistent with the gene‐expression profile of primary cultured hHSCs from patients with ALD. These genes are unique to alcohol‐induced HSC activation in two species, and therefore may become targets or readout for antifibrotic therapy in experimental models of ALD.

SUMMARYAccumulation of misfolded proteins is a known source of cellular stress and can be detrimental to cellular health. While protein aggregation is a known hallmark of many diseases, the mechanisms by which protein aggregates cause toxicity and the molecular machines that prevent this toxicity are not completely understood. Here, we show that the accumulated misfolded membrane proteins form endoplasmic reticulum (ER) localized aggregates, impacting ubiquitin and proteasome homeostasis. Additionally, we have identified a chaperone ability of the yeast rhomboid pseudoprotease Dfm1 to influence solubilization of misfolded membrane proteins and prevent toxicity from misfolded membrane proteins. 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 to the cytosol for degradation.

Ly6Chi inflammatory monocytes show high IFN responses, and contribute to both protective and pathogenic functions following influenza virus infection. In order to understand the significance of IFN responses in this subset, we examined monocytes during infection with a lethal H5N1 virus that induces high levels of IFN and a low-pathogenicity H1N1 virus that induces low levels of IFN. We show that H5N1 infection results in early recruitment of high numbers of Ly6Chi monocytes and induction of chemokines and Ifnb1. Using unbiased transcriptomic and proteomic approaches, we also find that monocytes are significantly enriched during H5N1 infection and are associated with chemokine and IFN signatures in mice, and with severity of symptoms after influenza virus infection in humans. Recruited Ly6Chi monocytes subsequently become infected in the lung, produce IFN-β, and mature into FasL+ monocyte-derived cells (FasL+MCs) expressing dendritic cell markers. Both Ccr2-/- and Faslgld mice are protected from lethal infection, indicating that monocytes contribute to pathogenesis. Global loss of type I and type III IFN signaling in Stat2-/- mice results in loss of monocyte recruitment, likely reflecting a requirement for IFN-dependent chemokine induction. Here we show that IFN is not directly required for monocyte recruitment on an IFN-sufficient background, but is required for maturation to FasL+MCs. Loss of IFN signaling skews to a Ly6Clo phenotype associated with tissue repair, suggesting that IFN signaling in monocytes is a critical determinant of influenza virus pathogenesis.

Chinese hamster ovary (CHO) cells are widely used for producing biopharmaceuticals, and engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the published Chinese hamster genome sequence were computationally predicted and are frequently inaccurate. Here, we use nascent transcription start site sequencing methods to revise TSS annotations for 15 308 Chinese hamster genes and 3034 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. We further capture tens of thousands of putative transcribed enhancer regions with this method. Our revised TSSs improves upon the RefSeq annotation by revealing core sequence features of gene regulation such as the TATA box and the Initiator and, as exemplified by targeting the glycosyltransferase gene Mgat3, facilitate activating silent genes by CRISPRa. Together, we envision our revised annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

The contribution of transcription factors (TFs) and gene regulatory programs in the immune response to COVID-19 and their relationship to disease outcome is not fully understood. Analysis of genome-wide changes in transcription at both promoter-proximal and distal cis-regulatory DNA elements, collectively termed the ’active cistrome,’ offers an unbiased assessment of TF activity identifying key pathways regulated in homeostasis or disease. Here, we profiled the active cistrome from peripheral leukocytes of critically ill COVID-19 patients to identify major regulatory programs and their dynamics during SARS-CoV-2 associated acute respiratory distress syndrome (ARDS). We identified TF motifs that track the severity of COVID- 19 lung injury, disease resolution, and outcome. We used unbiased clustering to reveal distinct cistrome subsets delineating the regulation of pathways, cell types, and the combinatorial activity of TFs. We found critical roles for regulatory networks driven by stimulus and lineage determining TFs, showing that STAT and E2F/MYB regulatory programs targeting myeloid cells are activated in patients with poor disease outcomes and associated with single nucleotide genetic variants implicated in COVID-19 susceptibility. Integration with single-cell RNA-seq found that STAT and E2F/MYB activation converged in specific neutrophils subset found in patients with severe disease. Collectively we demonstrate that cistrome analysis facilitates insight into disease mechanisms and provides an unbiased approach to evaluate global changes in transcription factor activity and stratify patient disease severity.

Coccidioidomycosis (Valley Fever) is an emerging endemic fungal infection with a rising incidence and an expanding geographic range. It is caused by Coccidiodes, which are thermally dimorphic fungi that grow as mycelia in soil but transition in the lung to form pathogenic spherules. The regulatory mechanisms underlying this transition are not understood. Exploiting capped small (cs)RNA-seq, which identifies actively initiated stable and unstable transcripts and thereby detects acute changes in gene regulation with remarkable sensitivity, here we report the changes in architectural organization and key sequence features underlying phase transition of this highly pathogenic fungus. Spherule transition was accompanied by large-scale transcriptional reprogramming, functional changes in transcript isoforms, and a massive increase in promoter-distal transcription of ncRNAs. Analysis of spherule-activated regulatory elements revealed a motif predicted to recruit a WOPR family transcription factor, which are known regulators of virulence in other fungi. We identify CIMG_02671 as a C. immitis WOPR homologue and show that it activates transcription in a WOPR motif-dependent manner, suggesting it is an important regulator of pathogenic phase transition. Collectively, this also highlights csRNA-seq as a powerful means to identify transcriptional mechanisms that control pathogenesis.

A critical component in the interpretation of systems-level studies is the inference of enriched biological pathways and protein complexes contained within OMICs datasets. Successful analysis requires the integration of a broad set of current biological databases and the application of a robust analytical pipeline to produce readily interpretable results. Metascape is a web-based portal designed to provide a comprehensive gene list annotation and analysis resource for experimental biologists. In terms of design features, Metascape combines functional enrichment, interactome analysis, gene annotation, and membership search to leverage over 40 independent knowledgebases within one integrated portal. Additionally, it facilitates comparative analyses of datasets across multiple independent and orthogonal experiments. Metascape provides a significantly simplified user experience through a one-click Express Analysis interface to generate interpretable outputs. Taken together, Metascape is an effective and efficient tool for experimental biologists to comprehensively analyze and interpret OMICs-based studies in the big data era., 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.

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 KrasG12D 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 KrasG12D 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. Electronic supplementary material The online version of this article (10.1007/s13238-019-0630-3) contains supplementary material, which is available to authorized users.