Mechanism of gene regulation by STATs and interacting transcriptionfactors

Auf der Basis existierender Publikationen, werden zwei große Gruppen von Genen, nach dem Prinzip ihrer Regulation, voneinander unterschieden. Eine spezielle Kathegorie von Genen wird im Zuge einer ersten/primären Antwort auf einen Stimulus gebildet und kann als PRGs (primary response genes) zusammengefasst werden. Als Unterscheidungskriterium von jenen Genen die zur Gruppe der sekundär exprimierten Gene (SRGs – secondary response genes) gerechnet werden, wird das Vorhandensein der RNA Polymerase II und die Histonmodifikation H3K4me3 am Promoter der betreffenden Gene, bereits vor dem Eintreffen des Stimulus, herangezogen. PRGs zeichnen sich durch einen sehr hohen Anteil, sowohl von RNA Pol II als auch H3K4me3 aus, wohingegen SRGs erst durch einen aktiven Prozeß im Verlauf des Stimulierens, das Binden der RNA Pol II am Promoter des zu regulierenden Gens gewährleisten müßen, einhergehend mit gesteigerter H3K4me3. Dieser zusätzliche Aufwand und das hierfür benötigte Vorhandensein von mehreren aktiven Transkriptionsfaktoren, ist in weiterer Folge, für die späte Expression dieser Gruppe von Genen, im Vergleich zu der Gruppe der PRGs, verantwortlich. Um die Mechanismen der Regulation dieser spät-exprimierten Gene zu analysieren konzentrierten wir uns auf jene Gene, die als Antwort auf Interferone exprimiert werden, und somit unter der Kontrolle der STAT (Signal transducers and activators of transcription) Familie von Transkriptionsfaktoren liegen. Zum Einen behandelten wir Zellen in unseren Untersuchungen mit IFNbeta (Interferon), zum anderen analysierten wir die Effekte einer weiteren Klasse von Interferonen, der Typ I Interferone am Beispiel IFNbeta, im Zuge der Infektion von Makrophagen mit dem Gram+, fakultativ intrazellulärem Bakterium Listeria monocytogenes. Makrophagen produzieren als Antwort auf eine Infektion mit Listerien große Mengen IFNbeta, welches wiederum, im Zusammenwirken mit anderen Signalen, für die Regulation von Genen benötigt wird, die an der Abwehr der Infektion beteiligt sind. Als Paradebeispiel für die große Gruppe der IFNbeta regulierten Gene betrachteten wir die Regulation von Gbp2 (guanylate binding protein). Aufbauend auf bereits bestehende Arbeiten im Labor, welche STAT1 und IRF1 (interferon regulatory factor) als die wichtigsten Transkriptionsfaktoren identifizierten, und darüber hinaus die Bedeutung der Ser 727 Phosphorilierung von STAT1, für die Rekrutierung von HDACs (histone deacetylases) und CPB/p300 (CREB – cyclic AMP response element binding protein – binding protein) an den Gbp2 promoter beschrieben, entdeckten wir die Abhängigkeit der RNA Pol II Rekrutierung von der Anwesenheit von IRF1 am Promoter. Zusätzlich zu IRF1 identifizierten wir IRF7 als noch nicht beschriebenen Schlüßelfaktor für die Regulation von Gbp2, wie auch andere über einen ISRE (interferon stimulated response element) Konsensus regulierte Gene, im Zuge der IFNgamma Antwort. Im Gegensatz zu IRF1, benötigt IRF7 für seine Aktivierung, auch im Kontext der IFNgamma Antwort, die Aktivität der S/T Kinase TBK1 (TANK - TRAF family member associated NFkappaB – nuclear factor of kappa light polypeptide gene enhancer in B-cells - activator - binding kinase). IRF7 Phosphorylierung durch TBK1 zeigte sich als notwendig für die Regulation von Gbp2 und anderen Genen, deren Abwesenheit führte jedoch nicht zu einem Verlust der IRF7 Promoterbindung. SOCS1 Expression zeigte sich im Gegensatz zu GBP2, unabhängig von der IRF7 Phosphorylierung durch TBK1. Die verwandte S/T Kinase IKKepsilon (IkappaB – inhibitor of NFkappaB - kinase) hatte im Gegensatz zu TBK1, einen reprimierenden Effekt auf die GBP2 Expression. Zusammengenommen zeigen diese Daten, dass phosphoryliertes wie unphosphoryliertes IRF7 an der Genregulation von ISRE regulierten Genen, nach Behandlung mit IFNgamma beteiligt ist. Eine weitere Gruppe von Genen innerhalb der SRGs benötigt zusätzlich zu den Faktoren, welche über Interferone aktiviert werden, noch die Aktivierung von Transkriptionsfaktoren über andere Signaltransduktionswege, und deren Zusammenspiel um eine effiziente Expression zu erfahren. Ein Beispiel für diese speziell regulierten Gene ist das Nos2 Gen, welches für die Bildung der iNOS (inducible nitric oxide synthase) verantwortlich ist und nach Infektion durch verschiedenste intrazelluläre Pathogene, wie Listerien, gebildet wird. Es benötigt die Aktivierung von NFkappaB im selben Ausmaß wie die IFN abhängige Aktivierung von ISGF3 (interferon stimulated gene factor – ein Komplex aus STAT1, STAT2, und IRF9 welcher im Zuge der Typ I IFN Signaltransduktion aktiviert wird, zum Beispiel IFNbeta). Im Zuge der detailierten Analyse des Mechanismus der iNOS Regulation, konnten wir zeigen, dass die Bindung von NFkappaB an den Nos2 Promoter jener von ISGF3 vorangeht. NFkappaB zeichnet dafür verantwortlich, dass der basale Transkriptionsfaktor TFIIH mit der assoziierten Kinase CDK7 (cyclin dependent kinase), welcher die RNA Pol II am Ser5 des CTD (C-terminal domain) phosphoriliert, an den Promoter rekrutiert, und dort verankert wird. Dies erzeugt einen transkriptionellen Gedächtniszustand, der bestehen bleibt, auch wenn NFkappaB den Promoter wieder verlassen hat. Neben CDK7-TFIIH konnte auch das Rekrutieren von CDK9 p-TEFb (positive transcription elongation factor) als NFkappaB abhängig gezeigt werden. CDK9 aktivität ist notwendig um die NELF (negative elongation factor) abhängigen Blockade der Polymerase am Übergang zur Elongation aufzuheben. Dem Binden von CDK7 an den Promoter folgt die Bindung von TBP (TATA binding protein), und der Polymerase selbst, welche beide auf das Interferonsignal und die daraus resultierende Aktivierung von ISGF3 angewiesen sind. Die so geschaffene RNA Pol II Bindung kann schließlich die iNOS Transkription auslösen. Die beiden Transkriptionsfaktoren kooperieren in der Regulation des Nos2 Gens durch das kombinierte Rekrutieren von verschiedenen Faktoren des PIC (pre initiation complex) und der RNA Pol II selbst. Zusammenfassend zeigen unsere Ergebnisse, dass nicht nur das Rekrutieren der Polymerase selbst ein limitierender Schritt zur Transkription ist, sondern auch das Rekrutieren von Kofaktoren einer strengen Kontrolle unterliegt, und von verschiedenen Transkriptionsfaktoren gesteuert wird. Diese Hypothese findet, anhand unserer Daten, nicht nur Anwendung bei Genen die im Zuge eines einzigen Stimulus exprimiert werden, wie im Fall von IFNgamma aktiviertem STAT1 und IRF1, sondern gilt im selben Maße auch für Gene deren Expression die Zusammenarbeit mehrerer Signaltransduktionswege bedingt, wie am Beispiel der iNOS für NFkappaB und ISGF3 gezeigt. Die getesteten Transkriptionsfaktoren vermitteln die Bindung von Faktoren des PIC und der Polymerase selbst um diese dann zur Expression zu veranlassenAccording to recent studies PRGs (primary response genes) that are expressed rapidly after providing the activating stimulus are characterized by the initial presence of paused polymerases and high levels of H3K4me3 at their promoter. In contrast, SRGs (secondary response genes) are regulated by the active recruitment of RNA Pol II (RNA polymerase II), requiring the interplay of transcription factors and co-factors to induce transcription, and are therefore delayed in their expression, compared to PRGs. To examine the mechanisms driving the expression of SRGs, we focused on the regulation of STAT-regulated genes, stimulated by IFNgamma (Interferon) or IFNbeta. Effects of the latter were studied in macrophages infected with the Gram+, facultative intracellular bacterium Listeria monocytogenes. Infected cells produce IFNbeta to synergize with additional pathways in the upregulation of host-defense genes. To examine the role of STAT1 (signal transducer and activator of transcription) in the regulation of IFNgamma-induced SRGs, we focused on the regulation of the Gbp2 (guanylate binding protein) gene. Previous work in the lab had shown that Gbp2 belongs to a subgroup of genes that require both STAT1 and IRF1 (interferon regulatory factor) for transcriptional induction upon IFNgamma treatment and that recruitment of HDACs (histone deacetylase) and CBP/p300 (CREB – cyclic AMP response element binding protein – binding protein) to the Gbp2 promoter directly depends on the presence of STAT1 and its phosphorylation on Ser727. We could further demonstrate that IRF1 binding was needed for the recruitment of RNA Pol II. Moreover, IRF7 was involved in the regulation of Gbp2 and other ISRE (interferon stimulated response element)-driven genes after IFNgamma treatment. This finding is novel, since IRF7 was not known to be expressed upon IFNgamma treatment and to participate in the regulation of interferon-induced genes. Contrasting IRF1, IRF7 function in this context relied on the presence and constitutive activity of the S/T kinase TBK1 (TANK - TRAF family member associated NFkappaB – nuclear factor of kappa light polypeptide gene enhancer in B-cells - activator - binding kinase). TBK1-mediated IRF7 phosophorylation was required for GBP2 expression, but did not alter IRF7 binding to the Gbp2 promoter. Expression of the Socs1 gene did require the presence of IRF7, but not its phosphorylation by TBK1. In contrast to TBK1, the related S/T kinase IKKepsilon (IkappaB – inhibitor of NFkappaB - kinase) repressed IRF7 activity to induce GBP2 expression. These findings indicate that phosphorylated, aswell as unphosphorylated IRF7 participates in the regulation of ISRE-driven gene expression after IFNgamma treatment. A distinct subset of SRGs requires the cooperation of signals derived from IFNs as well as additional signals derived from the infecting pathogen to acquire full-fledged transcriptional activity. Paradigmatic for this group is the Nos2 gene, encoding iNOS (inducible nitric oxide synthase), which is highly upregulated in macrophages during infections with various pathogens. In this study we show that the transcription factors NFkappaB and ISGF3 (interferon stimulated gene factor - a complex of STAT1, STAT2, and IRF9 which is formed and activated in response to type I IFNs, like IFNbeta) cooperate in the induction of iNOS and iNOS like genes in macrophages, infected with Listeria monocytogenes. We were able to demonstrate that, NFkappaB preceded ISGF3 at the Nos2 promoter and generated a transcriptional memory effect by depositing basal transcription factor TFIIH with the associated CDK7 (cyclin dependent kinase) kinase for serine 5 phosphorylation of the RNA Pol II - CTD (carboxyterminal domain). Moreover, p-TEFb (positive transcription elongation factor), a complex containing the kinase CDK9, which is required to release the Pol II enzyme from the NELF (negative elongation factor) dependent elongation block, was similarly deposited in an NFkappaB dependent manner. Deposition of CDK7-TFIIH at the proximal Nos2 promoter, was followed by TBP (TATA binding protein) and RNA Pol II binding, which were found to be recruited in an ISGF3 dependent manner. Hence, the two transcription factors cooperate in our infection model by assembling different components of the PIC (pre-initiation complex), and the RNA-Pol II enzyme itself, in order to induce iNOS transcription. Taken together our results indicate that at least for some SRGs not only the recruitment of RNA Pol II is a limiting step to induce gene expression, but also the recruitment of co-factors follows strict rules and results from the division of labor between different transcription factors. According to our findings this hypothesis is valid for transcription factors that are activated within a single pathway, such as STAT1 and IRF1, but also those activated through different signaling pathways, like NFkappaB and ISGF3. Collectively these proteins converge at the promoters of a subset of infection-induced genes to assemble the PIC – Pol II complex, and engage the Pol II enzyme in active transcription

Chromatin accessibility maps of chronic lymphocytic leukemia identify subtypespecific epigenome signatures and associated transcription regulatory networks

Chronic lymphocytic leukemia (CLL) is characterized by substantial clinical heterogeneity, despite relatively few genetic alterations. To provide a basis for studying epigenome deregulation in CLL, we established genome-wide chromatin accessibility maps for 88 CLL samples from 55 patients using the ATAC-seq assay. These data were further complemented by ChIPmentation and RNA-seq profiling in ten samples. Based on this dataset, we devised and applied a bioinformatic method that links chromatin profiles to clinical annotations. Our analysis identified sample-specific variation on top of a shared core of CLL regulatory regions. IGHV mutation status – which distinguishes the two major subtypes of CLL – was accurately predicted by the chromatin profiles, and gene regulatory networks inferred for IGHV-mutated vs. IGHV-unmutated samples identified characteristic regulatory differences between these two disease subtypes. In summary, we found widespread heterogeneity in the CLL chromatin landscape, established a community resource for studying epigenome deregulation in leukemia, and demonstrated the feasibility of chromatin accessibility mapping in cancer cohorts and clinical research

Chromatin mapping and single-cell immune profiling define the temporal dynamics of ibrutinib response in CLL

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib provides effective treatment for patients with chronic lymphocytic leukemia (CLL), despite extensive heterogeneity in this disease. To define the underlining regulatory dynamics, we analyze high-resolution time courses of ibrutinib treatment in patients with CLL, combining immune-phenotyping, single-cell transcriptome profiling, and chromatin mapping. We identify a consistent regulatory program starting with a sharp decrease of NF-kappa B binding in CLL cells, which is followed by reduced activity of lineage-defining transcription factors, erosion of CLL cell identity, and acquisition of a quiescence-like gene signature. We observe patient-to-patient variation in the speed of execution of this program, which we exploit to predict patient-specific dynamics in the response to ibrutinib based on the pre-treatment patient samples. In aggregate, our study describes time-dependent cellular, molecular, and regulatory effects for therapeutic inhibition of B cell receptor signaling in CLL, and it establishes a broadly applicable method for epigenome/transcriptome-based treatment monitoring

Genomic imprinting in mouse blastocysts is predominantly associated with H3K27me3.

In mammalian genomes, differentially methylated regions (DMRs) and histone marks including trimethylation of histone 3 lysine 27 (H3K27me3) at imprinted genes are asymmetrically inherited to control parentally-biased gene expression. However, neither parent-of-origin-specific transcription nor imprints have been comprehensively mapped at the blastocyst stage of preimplantation development. Here, we address this by integrating transcriptomic and epigenomic approaches in mouse preimplantation embryos. We find that seventy-one genes exhibit previously unreported parent-of-origin-specific expression in blastocysts (nBiX: novel blastocyst-imprinted expressed). Uniparental expression of nBiX genes disappears soon after implantation. Micro-whole-genome bisulfite sequencing (µWGBS) of individual uniparental blastocysts detects 859 DMRs. We further find that 16% of nBiX genes are associated with a DMR, whereas most are associated with parentally-biased H3K27me3, suggesting a role for Polycomb-mediated imprinting in blastocysts. nBiX genes are clustered: five clusters contained at least one published imprinted gene, and five clusters exclusively contained nBiX genes. These data suggest that early development undergoes a complex program of stage-specific imprinting involving different tiers of regulation

Non-IgE-reactive allergen peptides deteriorate the skin barrier in house dust mite-sensitized atopic dermatitis patients

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by type 2 cytokine-driven skin inflammation and epithelial barrier dysfunction. The latter is believed to allow the increased penetration of chemicals, toxins, and allergens into the skin. House dust mite allergens, particularly Der p 2, are important triggers in sensitized individuals with AD; the precise actions of these allergens in epithelial biology remain, however, incompletely understood. In this study, we compared the effects of the protein allergen Der p 2 and a mix of non-IgE-reactive Der p 2 peptides on skin cells using patch tests in AD patients and healthy participants. We then analyzed mRNA expression profiles of keratinocytes by single-cell RNA-sequencing. We report that existing barrier deficiencies in the non-lesional skin of AD patients allow deep penetration of Der p 2 and its peptides, leading to local microinflammation. Der p 2 protein specifically upregulated genes involved in the innate immune system, stress, and danger signals in suprabasal KC. Der p 2 peptides further downregulated skin barrier genes, in particular the expression of genes involved in cell–matrix and cell–cell adhesion. Peptides also induced genes involved in hyperproliferation and caused disturbances in keratinocyte differentiation. Furthermore, inflammasome-relevant genes and IL18 were overexpressed, while KRT1 was downregulated. Our data suggest that Der p 2 peptides contribute to AD initiation and exacerbation by augmenting hallmark features of AD, such as skin inflammation, barrier disruption, and hyperplasia of keratinocytes

Life-long epigenetic programming of cortical architecture by maternal ‘Western’ diet during pregnancy

Funding: European Research Council (SECRET-CELLS, ERC-2015-AdG-695136; T.H.); Wellcome Trust grant number 094476/Z/10/Z, which funded the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility, University of St. Andrews.The evolution of human diets led to preferences toward polyunsaturated fatty acid (PUFA) content with ‘Western’ diets enriched in ω-6 PUFAs. Mounting evidence points to ω-6 PUFA excess limiting metabolic and cognitive processes that define longevity in humans. When chosen during pregnancy, ω-6 PUFA-enriched ‘Western’ diets can reprogram maternal bodily metabolism with maternal nutrient supply precipitating the body-wide imprinting of molecular and cellular adaptations at the level of long-range intercellular signaling networks in the unborn fetus. Even though unfavorable neurological outcomes are amongst the most common complications of intrauterine ω-6 PUFA excess, cellular underpinnings of life-long modifications to brain architecture remain unknown. Here, we show that nutritional ω-6 PUFA-derived endocannabinoids desensitize CB1 cannabinoid receptors, thus inducing epigenetic repression of transcriptional regulatory networks controlling neuronal differentiation. We found that cortical neurons lose their positional identity and axonal selectivity when mouse fetuses are exposed to excess ω-6 PUFAs in utero. Conversion of ω-6 PUFAs into endocannabinoids disrupted the temporal precision of signaling at neuronal CB1 cannabinoid receptors, chiefly deregulating Stat3-dependent transcriptional cascades otherwise required to execute neuronal differentiation programs. Global proteomics identified the immunoglobulin family of cell adhesion molecules (IgCAMs) as direct substrates, with DNA methylation and chromatin accessibility profiling uncovering epigenetic reprogramming at >1400 sites in neurons after prolonged cannabinoid exposure. We found anxiety and depression-like behavioral traits to manifest in adult offspring, which is consistent with genetic models of reduced IgCAM expression, to suggest causality for cortical wiring defects. Overall, our data uncover a regulatory mechanism whose disruption by maternal food choices could limit an offspring’s brain function for life.PostprintPeer reviewe

Life-long impairment of glucose homeostasis upon prenatal exposure to psychostimulants

Maternal drug abuse during pregnancy is a rapidly escalating societal problem. Psychostimulants, including amphetamine, cocaine, and methamphetamine, are amongst the illicit drugs most commonly consumed by pregnant women. Neuropharmacology concepts posit that psychostimulants affect monoamine signaling in the nervous system by their affinities to neurotransmitter reuptake and vesicular transporters to heighten neurotransmitter availability extracellularly. Exacerbated dopamine signaling is particularly considered as a key determinant of psychostimulant action. Much less is known about possible adverse effects of these drugs on peripheral organs, and if in utero exposure induces lifelong pathologies. Here, we addressed this question by combining human RNA-seq data with cellular and mouse models of neuroendocrine development. We show that episodic maternal exposure to psychostimulants during pregnancy coincident with the intrauterine specification of pancreatic beta cells permanently impairs their ability of insulin production, leading to glucose intolerance in adult female but not male offspring. We link psychostimulant action specifically to serotonin signaling and implicate the sex-specific epigenetic reprogramming of serotonin-related gene regulatory networks upstream from the transcription factor Pet1/Fev as determinants of reduced insulin production.Peer reviewe

Genetic analysis in European ancestry individuals identifies 517 loci associated with liver enzymes

Plasma levels of liver enzymes provide insights into hepatic function and related diseases. Here, the authors perform a genome-wide association study on three liver enzymes, identifying genetic variants associated with their plasma concentration as well as links to metabolic and cardiovascular diseases. Serum concentration of hepatic enzymes are linked to liver dysfunction, metabolic and cardiovascular diseases. We perform genetic analysis on serum levels of alanine transaminase (ALT), alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) using data on 437,438 UK Biobank participants. Replication in 315,572 individuals from European descent from the Million Veteran Program, Rotterdam Study and Lifeline study confirms 517 liver enzyme SNPs. Genetic risk score analysis using the identified SNPs is strongly associated with serum activity of liver enzymes in two independent European descent studies (The Airwave Health Monitoring study and the Northern Finland Birth Cohort 1966). Gene-set enrichment analysis using the identified SNPs highlights involvement in liver development and function, lipid metabolism, insulin resistance, and vascular formation. Mendelian randomization analysis shows association of liver enzyme variants with coronary heart disease and ischemic stroke. Genetic risk score for elevated serum activity of liver enzymes is associated with higher fat percentage of body, trunk, and liver and body mass index. Our study highlights the role of molecular pathways regulated by the liver in metabolic disorders and cardiovascular disease

DNA methylation signature of chronic low-grade inflammation and its role in cardio-respiratory diseases

We performed a multi-ethnic Epigenome Wide Association study on 22,774 individuals to describe the DNA methylation signature of chronic low-grade inflammation as measured by C-Reactive protein (CRP). We find 1,511 independent differentially methylated loci associated with CRP. These CpG sites show correlation structures across chromosomes, and are primarily situated in euchromatin, depleted in CpG islands. These genomic loci are predominantly situated in transcription factor binding sites and genomic enhancer regions. Mendelian randomization analysis suggests altered CpG methylation is a consequence of increased blood CRP levels. Mediation analysis reveals obesity and smoking as important underlying driving factors for changed CpG methylation. Finally, we find that an activated CpG signature significantly increases the risk for cardiometabolic diseases and COPD.publishedVersionPeer reviewe