Figeno: multi-region genomic figures with long-read support.

SUMMARY: The vast amount of publicly available genomic data requires analysis and visualization tools. Here, we present figeno, an application for generating publication-quality FIgures for GENOmics. Figeno particularly focuses on multi-region views across genomic breakpoints and on long reads with base modifications. In addition, we support epigenomic data including ATAC-seq, ChIP-seq or HiC, as well as whole genome sequencing data with copy numbers and structural variants. AVAILABILITY AND IMPLEMENTATION: Figeno is available as a python package with both a command line and graphical user interface. It can be installed via PyPI and the source code is available at https://github.com/CompEpigen/figeno.

EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells.

Introduction: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. Results: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. Conclusion: In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.

DNA methylation biomarkers for noninvasive detection of triple-negative breast cancer using liquid biopsy.

Noninvasive detection of aberrant DNA methylation could provide invaluable biomarkers for earlier detection of triple-negative breast cancer (TNBC) which could help clinicians with easier and more efficient treatment options. We evaluated genome-wide DNA methylation data derived from TNBC and normal breast tissues, peripheral blood of TNBC cases and controls and reference samples of sorted blood and mammary cells. Differentially methylated regions (DMRs) between TNBC and normal breast tissues were stringently selected, verified and externally validated. A machine-learning algorithm was applied to select the top DMRs, which then were evaluated on plasma-derived circulating cell-free DNA (cfDNA) samples of TNBC patients and healthy controls. We identified 23 DMRs accounting for the methylation profile of blood cells and reference mammary cells and then selected six top DMRs for cfDNA analysis. We quantified un-/methylated copies of these DMRs by droplet digital PCR analysis in a plasma test set from TNBC patients and healthy controls and confirmed our findings obtained on tissues. Differential cfDNA methylation was confirmed in an independent validation set of plasma samples. A methylation score combining signatures of the top three DMRs overlapping with the SPAG6, LINC10606 and TBCD/ZNF750 genes had the best capability to discriminate TNBC patients from controls (AUC = 0.78 in the test set and AUC = 0.74 in validation set). Our findings demonstrate the usefulness of cfDNA-based methylation signatures as noninvasive liquid biopsy markers for the diagnosis of TNBC.

A leukemia-protective germline variant mediates chromatin module formation via transcription factor nucleation.

Non-coding variants coordinate transcription factor (TF) binding and chromatin mark enrichment changes over regions spanning >100 kb. These molecularly coordinated regions are named "variable chromatin modules" (VCMs), providing a conceptual framework of how regulatory variation might shape complex traits. To better understand the molecular mechanisms underlying VCM formation, here, we mechanistically dissect a VCM-modulating noncoding variant that is associated with reduced chronic lymphocytic leukemia (CLL) predisposition and disease progression. This common, germline variant constitutes a 5-bp indel that controls the activity of an AXIN2 gene-linked VCM by creating a MEF2 binding site, which, upon binding, activates a super-enhancer-like regulatory element. This triggers a large change in TF binding activity and chromatin state at an enhancer cluster spanning >150 kb, coinciding with subtle, long-range chromatin compaction and robust AXIN2 up-regulation. Our results support a model in which the indel acts as an AXIN2 VCM-activating TF nucleation event, which modulates CLL pathology.

Epigenetic quantification of circulating immune cells in peripheral blood of triple-negative breast cancer patients.

BACKGROUND: A shift in the proportions of blood immune cells is a hallmark of cancer development. Here, we investigated whether methylation-derived immune cell type ratios and methylation-derived neutrophil-to-lymphocyte ratios (mdNLRs) are associated with triple-negative breast cancer (TNBC). METHODS: Leukocyte subtype-specific unmethylated/methylated CpG sites were selected, and methylation levels at these sites were used as proxies for immune cell type proportions and mdNLR estimation in 231 TNBC cases and 231 age-matched controls. Data were validated using the Houseman deconvolution method. Additionally, the natural killer (NK) cell ratio was measured in a prospective sample set of 146 TNBC cases and 146 age-matched controls. RESULTS: The mdNLRs were higher in TNBC cases compared with controls and associated with TNBC (odds ratio (OR) range (2.66-4.29), all Padj. < 1e-04). A higher neutrophil ratio and lower ratios of NK cells, CD4 + T cells, CD8 + T cells, monocytes, and B cells were associated with TNBC. The strongest association was observed with decreased NK cell ratio (OR range (1.28-1.42), all Padj. < 1e-04). The NK cell ratio was also significantly lower in pre-diagnostic samples of TNBC cases compared with controls (P = 0.019). CONCLUSION: This immunomethylomic study shows that a shift in the ratios/proportions of leukocyte subtypes is associated with TNBC, with decreased NK cell showing the strongest association. These findings improve our knowledge of the role of the immune system in TNBC and point to the possibility of using NK cell level as a non-invasive molecular marker for TNBC risk assessment, early detection, and prevention.

Random forest-based modelling to detect biomarkers for prostate cancer progression.

BACKGROUND: The clinical course of prostate cancer (PCa) is highly variable, demanding an individualized approach to therapy. Overtreatment of indolent PCa cases, which likely do not progress to aggressive stages, may be associated with severe side effects and considerable costs. These could be avoided by utilizing robust prognostic markers to guide treatment decisions. RESULTS: We present a random forest-based classification model to predict aggressive behaviour of prostate cancer. DNA methylation changes between PCa cases with good or poor prognosis (discovery cohort with n = 70) were used as input. DNA was extracted from formalin-fixed tumour tissue, and genome-wide DNA methylation differences between both groups were assessed using Illumina HumanMethylation450 arrays. For the random forest-based modelling, the discovery cohort was randomly split into a training (80%) and a test set (20%). Our methylation-based classifier demonstrated excellent performance in discriminating prognosis subgroups in the test set (Kaplan-Meier survival analyses with log-rank p value < 0.0001). The area under the receiver operating characteristic curve (AUC) for the sensitivity analysis was 95%. Using the ICGC cohort of early- and late-onset prostate cancer (n = 222) and the TCGA PRAD cohort (n = 477) for external validation, AUCs for sensitivity analyses were 77.1% and 68.7%, respectively. Cancer progression-related DNA hypomethylation was frequently located in 'partially methylated domains' (PMDs)-large-scale genomic areas with progressive loss of DNA methylation linked to mitotic cell division. We selected several candidate genes with differential methylation in gene promoter regions for additional validation at the protein expression level by immunohistochemistry in > 12,000 tissue micro-arrayed PCa cases. Loss of ZIC2 protein expression was associated with poor prognosis and correlated with significantly shorter time to biochemical recurrence. The prognostic value of ZIC2 proved to be independent from established clinicopathological variables including Gleason grade, tumour stage, nodal stage and prostate-specific-antigen. CONCLUSIONS: Our results highlight the prognostic relevance of methylation loss in PMD regions, as well as of several candidate genes not previously associated with PCa progression. Our robust and externally validated PCa classification model either directly or via protein expression analyses of the identified top-ranked candidate genes will support the clinical management of prostate cancer.

Acute Exercise Increases the Expression of KIR2DS4 by Promoter Demethylation in NK Cells.

Positive effects of exercise on cancer prevention and progression have been proposed to be mediated by stimulating natural killer (NK) cells. Because NK cell receptors are regulated by epigenetic modifications, we investigated whether acute aerobic exercise and training change promoter DNA methylation and gene expression of the activating KIR2DS4 and the inhibiting KIR3DL1 gene. Sixteen healthy women (50-60 years) performed a graded exercise test (GXT) and were randomized into either a passive control group or an intervention group performing a four-week endurance exercise intervention. Blood samples (pre-, post-GXT and post-training) were used for isolation of DNA/RNA of NK cells to assess DNA promoter methylation by targeted deep-amplicon sequencing and gene expression by qRT-PCR. Potential changes in NK cell subsets were determined by flow cytometry. Acute and chronic exercise did not provoke significant alterations of NK cell proportions. Promoter methylation decreased and gene expression increased for KIR2DS4 after acute exercise. A high gene expression correlated with a low methylation of CpGs that were altered by acute exercise. Chronic exercise resulted in a minor decrease of DNA methylation and did not alter gene expression. Acute exercise provokes epigenetic modifications, affecting the balance between the activating KIR2DS4 and the inhibiting KIR3DL1, with potential benefits on NK cell function.

Molecular Evolution of Early-Onset Prostate Cancer Identifies Molecular Risk Markers and Clinical Trajectories.

Identifying the earliest somatic changes in prostate cancer can give important insights into tumor evolution and aids in stratifying high- from low-risk disease. We integrated whole genome, transcriptome and methylome analysis of early-onset prostate cancers (diagnosis ≤55 years). Characterization across 292 prostate cancer genomes revealed age-related genomic alterations and a clock-like enzymatic-driven mutational process contributing to the earliest mutations in prostate cancer patients. Our integrative analysis identified four molecular subgroups, including a particularly aggressive subgroup with recurrent duplications associated with increased expression of ESRP1, which we validate in 12,000 tissue microarray tumors. Finally, we combined the patterns of molecular co-occurrence and risk-based subgroup information to deconvolve the molecular and clinical trajectories of prostate cancer from single patient samples.

A systems biology network analysis of nutri(epi)genomic changes in endothelial cells exposed to epicatechin metabolites.

Although vasculo-protective effects of flavan-3-ols are widely accepted today, their impact on endothelial cell functions and molecular mechanisms of action involved is not completely understood. The aim of this study was to characterize the potential endothelium-protective effects of circulating epicatechin metabolites and to define underlying mechanisms of action by an integrated systems biology approach. Reduced leukocyte rolling over vascular endothelium was observed following epicatechin supplementation in a mouse model of inflammation. Integrative pathway analysis of transcriptome, miRNome and epigenome profiles of endothelial cells exposed to epicatechin metabolites revealed that by acting at these different levels of regulation, metabolites affect cellular pathways involved in endothelial permeability and interaction with immune cells. In-vitro experiments on endothelial cells confirmed that epicatechin metabolites reduce monocyte adhesion and their transendothelial migration. Altogether, our in-vivo and in-vitro results support the outcome of a systems biology based network analysis which suggests that epicatechin metabolites mediate their vasculoprotective effects through dynamic regulation of endothelial cell monocyte adhesion and permeability. This study illustrates complex and multimodal mechanisms of action by which epicatechin modulate endothelial cell integrity.

Impact of dietary gut microbial metabolites on the epigenome.

Within the past decade, epigenetic mechanisms and their modulation by natural products have gained increasing interest. Dietary bioactive compounds from various sources, including green tea, soya, fruit and berries, cruciferous vegetables, whole grain foods, fish and others, have been shown to target enzymes involved in epigenetic gene regulation, including DNA methyltransferases, histone acetyltransferases, deacetylases and demethylases in vitro and in cell culture. Also, many dietary agents were shown to alter miRNA expression. In vivo studies in animal models and humans are still limited. Recent research has indicated that the gut microbiota and gut microbial metabolites might be important mediators of diet-epigenome interactions. Inter-individual differences in the gut microbiome might affect release, metabolism and bioavailability of dietary agents and explain variability in response to intervention in human studies. Only a few microbial metabolites, including folate, phenolic acids, S-(-)equol, urolithins, isothiocyanates, and short- and long-chain fatty acids have been tested with respect to their potential to influence epigenetic mechanisms. Considering that a complex mixture of intermediary and microbial metabolites is present in human circulation, a more systematic interdisciplinary investigation of nutri-epigenetic activities and their impact on human health is called for.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Modulation of Adipocyte Differentiation and Proadipogenic Gene Expression by Sulforaphane, Genistein, and Docosahexaenoic Acid as a First Step to Counteract Obesity.

Obesity is characterized by excess body fat accumulation due to an increase in the size and number of differentiated mature adipocytes. Adipocyte differentiation is regulated by genetic and environmental factors, and its inhibition could represent a strategy for obesity prevention and treatment. The current study was designed with two aims: (i) to evaluate the changes in the expression of adipogenic markers (C/EBPα, PPARγ variant 1 and variant 2, and GLUT4) in 3T3-L1 murine preadipocytes at four stages of the differentiation process and (ii) to compare the effectiveness of sulforaphane, genistein, and docosahexaenoic acid in reducing lipid accumulation and modulating C/EBPα, PPARγ1, PPARγ2, and GLUT4 mRNA expression in mature adipocytes. All bioactive compounds were shown to suppress adipocyte differentiation, although with different effectiveness. These results set the stage for further studies considering natural food constituents as important agents in preventing or treating obesity.

Intratumor heterogeneity in epigenetic patterns.

Analogous to life on earth, tumor cells evolve through space and time and adapt to different micro-environmental conditions. As a result, tumors are composed of millions of genetically diversified cells at the time of diagnosis. Profiling these variants contributes to understanding tumors' clonal origins and might help to better understand response to therapy. However, even genetically homogenous cell populations show remarkable diversity in their response to different environmental stimuli, suggesting that genetic heterogeneity does not explain the full spectrum of tumor plasticity. Understanding epigenetic diversity across cancer cells provides important additional information about the functional state of subclones and therefore allows better understanding of tumor evolution and resistance to current therapies.

Erratum: DNMT and HDAC inhibitors induce cryptic transcription start sites encoded in long terminal repeats.

This corrects the article DOI: 10.1038/ng.3889.

Identification of differentially methylated BRCA1 and CRISP2 DNA regions as blood surrogate markers for cardiovascular disease.

Genome-wide Illumina InfiniumMethylation 450 K DNA methylation analysis was performed on blood samples from clinical atherosclerosis patients (n = 8) and healthy donors (n = 8) in the LVAD study (NCT02174133, NCT01799005). Multiple differentially methylated regions (DMR) could be identified in atherosclerosis patients, related to epigenetic control of cell adhesion, chemotaxis, cytoskeletal reorganisations, cell proliferation, cell death, estrogen receptor pathways and phagocytic immune responses. Furthermore, a subset of 34 DMRs related to impaired oxidative stress, DNA repair, and inflammatory pathways could be replicated in an independent cohort study of donor-matched healthy and atherosclerotic human aorta tissue (n = 15) and human carotid plaque samples (n = 19). Upon integrated network analysis, BRCA1 and CRISP2 DMRs were identified as most central disease-associated DNA methylation biomarkers. Differentially methylated BRCA1 and CRISP2 regions were verified by MassARRAY Epityper and pyrosequencing assays and could be further replicated in blood, aorta tissue and carotid plaque material of atherosclerosis patients. Moreover, methylation changes at BRCA1 and CRISP2 specific CpG sites were consistently associated with subclinical atherosclerosis measures (coronary calcium score and carotid intima media thickness) in an independent sample cohort of middle-aged men with subclinical cardiovascular disease in the Aragon Workers' Health Study (n = 24). Altogether, BRCA1 and CRISP2 DMRs hold promise as novel blood surrogate markers for early risk stratification and CVD prevention.

DNMT and HDAC inhibitors induce cryptic transcription start sites encoded in long terminal repeats.

Several mechanisms of action have been proposed for DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi), primarily based on candidate-gene approaches. However, less is known about their genome-wide transcriptional and epigenomic consequences. By mapping global transcription start site (TSS) and chromatin dynamics, we observed the cryptic transcription of thousands of treatment-induced non-annotated TSSs (TINATs) following DNMTi and HDACi treatment. The resulting transcripts frequently splice into protein-coding exons and encode truncated or chimeric ORFs translated into products with predicted abnormal or immunogenic functions. TINAT transcription after DNMTi treatment coincided with DNA hypomethylation and gain of classical promoter histone marks, while HDACi specifically induced a subset of TINATs in association with H2AK9ac, H3K14ac, and H3K23ac. Despite this mechanistic difference, both inhibitors convergently induced transcription from identical sites, as we found TINATs to be encoded in solitary long terminal repeats of the ERV9/LTR12 family, which are epigenetically repressed in virtually all normal cells.

Epigenetic silencing of triple negative breast cancer hallmarks by Withaferin A.

Triple negative breast cancer (TNBC) is characterized by poor prognosis and a DNA hypomethylation profile. Withaferin A (WA) is a plant derived steroidal lactone which holds promise as a therapeutic agent for treatment of breast cancer (BC). We determined genome-wide DNA methylation changes in weakly-metastatic and aggressive, metastatic BC cell lines, following 72h treatment to a sub-cytotoxic concentration of WA. In contrast to the DNA demethylating agent 5-aza-2'-deoxycytidine (DAC), WA treatment of MDA-MB-231 cells rather tackles an epigenetic cancer network through gene-specific DNA hypermethylation of tumor promoting genes including ADAM metallopeptidase domain 8 (ADAM8), urokinase-type plasminogen activator (PLAU), tumor necrosis factor (ligand) superfamily, member 12 (TNFSF12), and genes related to detoxification (glutathione S-transferase mu 1, GSTM1), or mitochondrial metabolism (malic enzyme 3, ME3). Gene expression and pathway enrichment analysis further reveals epigenetic suppression of multiple cancer hallmarks associated with cell cycle regulation, cell death, cancer cell metabolism, cell motility and metastasis. Remarkably, DNA hypermethylation of corresponding CpG sites in PLAU, ADAM8, TNSF12, GSTM1 and ME3 genes correlates with receptor tyrosine-protein kinase erbB-2 amplification (HER2)/estrogen receptor (ESR)/progesterone receptor (PR) status in primary BC tumors. Moreover, upon comparing differentially methylated WA responsive target genes with DNA methylation changes in different clinical subtypes of breast cancer patients in the cancer genome atlas (TCGA), we found that WA silences HER2/PR/ESR-dependent gene expression programs to suppress aggressive TNBC characteristics in favor of luminal BC hallmarks, with an improved therapeutic sensitivity. In this respect, WA may represent a novel and attractive phyto-pharmaceutical for TNBC treatment.

Suppression of indoleamine-2,3-dioxygenase 1 expression by promoter hypermethylation in ER-positive breast cancer.

Kynurenine formation by tryptophan-catabolic indoleamine-2,3-dioxygenase 1 (IDO1) plays a key role in tumor immune evasion and inhibition of IDO1 is efficacious in preclinical models of breast cancer. As the response of breast cancer to immune checkpoint inhibitors may be limited, a better understanding of the expression of additional targetable immunomodulatory pathways is of importance. We therefore investigated the regulation of IDO1 expression in different breast cancer subtypes. We identified estrogen receptor α (ER) as a negative regulator of IDO1 expression. Serum kynurenine levels as well as tumoral IDO1 expression were lower in patients with ER-positive than ER-negative tumors and an inverse relationship between IDO1 and estrogen receptor mRNA was observed across 14 breast cancer data sets. Analysis of whole genome bisulfite sequencing, 450k, MassARRAY and pyrosequencing data revealed that the IDO1 promoter is hypermethylated in ER-positive compared with ER-negative breast cancer. Reduced induction of IDO1 was also observed in human ER-positive breast cancer cell lines. IDO1 induction was enhanced upon DNA demethylation in ER-positive but not in ER-negative cells and methylation of an IDO1 promoter construct reduced IDO1 expression, suggesting that enhanced methylation of the IDO1 promoter suppresses IDO1 in ER-positive breast cancer. The association of ER overexpression with epigenetic downregulation of IDO1 appears to be a particular feature of breast cancer as IDO1 was not suppressed by IDO1 promoter hypermethylation in the presence of high ER expression in cervical or endometrial cancer.

[Isoflavone-containing dietary supplements]. / Isoflavonhaltige Nahrungsergänzungsmittel.

Isoflavones (IFs) from soy and other legumes have weak estrogenic properties. Isolated IFs are available as dietary supplements and advertised to alleviate symptoms of menopause. The present chapter provides an overview of the occurrence, the chemical structure of IFs and their metabolites, the market situation and reviews the current evidence on the efficacy and safety of IF-containing dietary supplements.The biological effectiveness of IFs is attributable to the activation of the estrogen receptor (ER). Studies on the influence of IFs on endogenous estrogen levels in women show inconsistent results. So far, the European Food Safety Authority (EFSA) has rejected all submitted health claims for IFs due to insufficient scientific evidence for any of the postulated health effects. Based on the results of their recent risk assessment, the EFSA concluded that the available human studies did not support the hypothesis of adverse effects of isolated IFs on the human mammary gland, uterus or thyroid in healthy postmenopausal women. However, the assessment does not allow a general statement on the safety of IF-containing dietary supplements. Studies in animal models are often not comparable with the complex interactions in humans due to differences in the metabolism of IFs, in the developmental stage at time of consumption and in the temporarily restricted uptake of IFs during certain stages of life. CONCLUSION: So far, for none of the advertised functions is unequivocal scientific evidence available. On the basis of available data, potential unwanted side effects cannot be fully excluded. This holds particularly true for women with undiagnosed diseases, especially for those with undetected precancerous lesions in the mammary gland.

Synthesis of Resveratrol Derivatives and In Vitro Screening for Potential Cancer Chemopreventive Activities.

New resveratrol (trans-3,4',5-trihydroxystilbene) analogs were synthesized and screened for their in vitro cancer chemopreventive potential using various bioassays relevant for the prevention of carcinogenesis in humans: two assays to detect modulators of carcinogen metabolism (Cyp1A inhibition; determination of NAD(P)H/quinone reductase (QR) activity), three assays to identify radical scavenging and antioxidant properties (DPPH, ORAC, superoxide anion radicals in differentiated HL-60 cells), four assays to determine anti-inflammatory and anti-hormonal effects (iNOS, Cox-1 and aromatase inhibition, anti-estrogenic potential). 3,4',5-Tri-O-methyl resveratrol 1a was about sevenfold more active than resveratrol in inhibiting Cyp1A activity, it was a potent inducer of QR activity, and it showed pure anti-estrogenic activity (whereas resveratrol is a known mixed estrogen (ant)agonist with both estrogenic and anti-estrogenic properties). Dual estrogen ant-/agonist activity was restored in the mono-O-benzyl-substituted derivatives 4b (4'-O-benzyl resveratrol) and 5b (3-O-benzyl resveratrol). With respect to aromatase inhibition (Cyp19), which provided the highest number of actives, the benzyl-substituted series was more potent than the methyl-substituted derivatives of resveratrol, and 3-O-benzyl resveratrol 5b was about eightfold more active than resveratrol. Overall, 3,4',5-tri-O-pivaloyl resveratrol oxide 7c was identified as a potent inducer of phase 2 enzymes concomitant with inhibition of LPS-mediated iNOS induction.

A click chemistry approach identifies target proteins of xanthohumol.

SCOPE: Many phytochemicals with beneficial pharmacological properties contain electrophilic sites, e.g. α,ß-unsaturated carbonyl (enone) groups. There is increasing evidence that many biological effects of electrophilic compounds depend on covalent conjugation to reactive protein thiols. For example, the reaction of electrophiles with cysteinyl residues of the sensor protein Keap1 activates the cell-protective Nrf2 response. Thus it is of interest to identify more generally the proteins to which small molecule electrophiles bind covalently. METHODS AND RESULTS: Here we use a Click chemistry approach to identify target proteins of the chemopreventive phytochemical xanthohumol (XN), an enone-containing chalcone from hops (Humulus lupulus L.). Using an alkynylated analog of XN (XN-alkyne), we purified covalent protein-electrophile conjugates from cell lysates. We confirm the previously described conjugation of XN to Keap1. One of the newly identified candidate target proteins is glucose-6-phosphate dehydrogenase (G6PDH). We confirm that XN attenuates intracellular G6PDH activity at low micromolar concentrations. CONCLUSION: We find support for the notion that XN modulates multiple pathways and processes by covalent modification of proteins with reactive cysteines.