Identifikation neuer Bindungspartner des c-MYC/MIZ-1–Netzwerks und Charakterisierung der Regulation der Transkription durch die c-MYC–Kofaktoren H2A.Z und MED24

Das Proto-Onkogen c-MYC kodiert für einen Transkriptionsfaktor, der die Expression von Zielgenen entweder aktiviert oder reprimiert. Die Repression wird bei Zielgenen wie P15INK4B und P21CIP1 durch Bindung an den ansonsten aktivierend wirkenden Transkriptionsfaktor MIZ-1 vermittelt, wodurch dessen aktivierende Wirkung gehemmt wird. Nach chromatographischer Anreicherung von MIZ-1 konnten durch massenspektrometrische Analyse die putativen Bindungspartner Mi2-beta/CHD4 (Chromodomäne–Helikase–DNA-bindendes Protein 4) und Insulinrezeptorsubstrat 4 (IRS4) identifiziert und durch Koimmunpräzipitationen Interaktionen von diesen mit c-MYC gezeigt werden. Neben Mi2-/CHD4 wurden auch andere Komponenten des NuRD (nucleosome remodeling and histone deacetylation)-Komplexes, der als Repressor der Transkription beschrieben wurde, in den analysierten Fraktionen nachgewiesen. Dies deutet auf einen möglichen Mechanismus der Repression durch c-MYC und MIZ-1 hin. Für ein anderes Mitglied der Insulinrezeptorsubstratfamilie, IRS1, wurde eine Funktion als Transkriptionsfaktor gezeigt, was eine transkriptionsregulierende Wechselwirkung von IRS4 mit dem c-MYC/MIZ-1–Netzwerk vermuten läßt. H2A.Z ist eine hochkonservierte Variante des kanonischen Histons H2A, welche bei Säugetieren essentiell ist und eine Rolle bei der Regulation der Transkription spielt. In Drosophila melanogaster wird die zu H2A.Z orthologe Histonvariante H2Av durch die ATPase Domino des Tip60 (Tat-interagierendes Protein, 60 kDa)-Komplexes ausgetauscht; c-MYC kann in Säugetierzellen den Tip60-Komplex rekrutieren. Hier konnte in c-MYC–induzierbaren 3T3-immortalisierten Mausfibroblasten gezeigt werden, daß während der Genaktivierung durch c-MYC an den Promotoren von Zielgenen H2A.Z zunächst nahe der Transkriptionsstartstelle rekrutiert und daraufhin wieder entfernt wird. Dieser Vorgang korrelierte mit einem deutlichen Anstieg der Expression des c-MYC–Zielgens ccnd2 (Zyklin D2). Eine shRNA-vermittelte Herunterregulation der Expression von H2A.Z in c-MYC–induzierbaren humanen Osteosarkomzellen bewirkte sowohl eine verminderte die Rolle eines Vermittlers zwischen Transkriptionsfaktoren und der RNA-Polymerase II. c-MYC interagiert in menschlichen Zellinien direkt mit der Mediator-Untereinheit MED24/TRAP100. Durch shRNA-vermittelte Herunterregulation der Expression von med24 und cdk8 (welches für eine weitere, fakultative Mediator-Untereinheit kodiert) in c-MYC–induzierbaren immortalisierten Mausfibroblasten wurde hier gezeigt, daß die Interaktion von c-MYC mit Med24 sowie das Vorhandensein von Cdk8 für die Aktivierung und auch die Repression nahezu aller c-MYC–Zielgene notwendig ist. Außerdem bewirkte die verminderte Expression von med24 und cdk8 das Ansteigen des Expressionsniveaus einiger, jedoch nicht aller untersuchten aktivierten c-MYC–Zielgene. Desweiteren konnte in c-MYC–induzierbaren humanen Osteosarkomzellen am CCND2-Promotor eine c-MYC–abhängige Rekrutierung der Mediator-Untereinheiten MED1 und CDK8 gezeigt werden, was auf eine Beteiligung des Mediator-Komplexes inklusive seines fakultativen CDK8-Moduls an der Aktivierung des Promotors Aktivierung als auch eine verminderte Repression von hindeutet. c-MYC–Zielgenen. Der hochkonservierte Mediator-Komplex spiel

Chromatin Binding of c-REL and p65 Is Not Limiting for Macrophage IL12B Transcription During Immediate Suppression by Ovarian Carcinoma Ascites

Tumors frequently exploit homeostatic mechanisms that suppress expression of IL-12, a central mediator of inflammatory and anti-tumor responses. The p40 subunit of the IL-12 heterodimer, encoded by IL12B, is limiting for these functions. Ovarian carcinoma patients frequently produce ascites which exerts immunosuppression by means of soluble factors. The NFκB pathway is necessary for transcription of IL12B, which is not expressed in macrophages freshly isolated from ascites. This raises the possibility that ascites prevents IL12B expression by perturbing NFκB binding to chromatin. Here, we show that ascites-mediated suppression of IL12B induction by LPS plus IFNγ in primary human macrophages is rapid, and that suppression can be reversible after ascites withdrawal. Nuclear translocation of the NFκB transcription factors c-REL and p65 was strongly reduced by ascites. Surprisingly, however, their binding to the IL12B locus and to CXCL10, a second NFκB target gene, was unaltered, and the induction of CXCL10 transcription was not suppressed by ascites. These findings indicate that, despite its reduced nuclear translocation, NFκB function is not generally impaired by ascites, suggesting that ascites-borne signals target additional pathways to suppress IL12B induction. Consistent with these data, IL-10, a clinically relevant constituent of ascites and negative regulator of NFκB translocation, only partially recapitulated IL12B suppression by ascites. Finally, restoration of a defective IL-12 response by appropriate culture conditions was observed only in macrophages from a subset of donors, which may have important implications for the understanding of patient-specific immune responses

Reverse crosstalk of TGFβ and PPARβ/δ signaling identified by transcriptional profiling

Previous work has provided strong evidence for a role of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) and transforming growth factor-β (TGFβ) in inflammation and tumor stroma function, raising the possibility that both signaling pathways are interconnected. We have addressed this hypothesis by microarray analyses of human diploid fibroblasts induced to myofibroblastic differentiation, which revealed a substantial, mostly reverse crosstalk of both pathways and identified distinct classes of genes. A major class encompasses classical PPAR target genes, including ANGPTL4, CPT1A, ADRP and PDK4. These genes are repressed by TGFβ, which is counteracted by PPARβ/δ activation. This is mediated, at least in part, by the TGFβ-induced recruitment of the corepressor SMRT to PPAR response elements, and its release by PPARβ/δ ligands, indicating that TGFβ and PPARβ/δ signals are integrated by chromatin-associated complexes. A second class represents TGFβ-induced genes that are downregulated by PPARβ/δ agonists, exemplified by CD274 and IL6, which is consistent with the anti-inflammatory properties of PPARβ/δ ligands. Finally, cooperative regulation by both ligands was observed for a minor group of genes, including several regulators of cell proliferation. These observations indicate that PPARβ/δ is able to influence the expression of distinct sets of both TGFβ-repressed and TGFβ-activated genes in both directions

Genomewide Analyses Define Different Modes of Transcriptional Regulation by Peroxisome Proliferator-Activated Receptor-β/δ (PPARβ/δ)

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors with essential functions in lipid, glucose and energy homeostasis, cell differentiation, inflammation and metabolic disorders, and represent important drug targets. PPARs heterodimerize with retinoid X receptors (RXRs) and can form transcriptional activator or repressor complexes at specific DNA elements (PPREs). It is believed that the decision between repression and activation is generally governed by a ligand-mediated switch. We have performed genomewide analyses of agonist-treated and PPARβ/δ-depleted human myofibroblasts to test this hypothesis and to identify global principles of PPARβ/δ-mediated gene regulation. Chromatin immunoprecipitation sequencing (ChIP-Seq) of PPARβ/δ, H3K4me3 and RNA polymerase II enrichment sites combined with transcriptional profiling enabled the definition of 112 bona fide PPARβ/δ target genes showing either of three distinct types of transcriptional response: (I) ligand-independent repression by PPARβ/δ; (II) ligand-induced activation and/or derepression by PPARβ/δ; and (III) ligand-independent activation by PPARβ/δ. These data identify PPRE-mediated repression as a major mechanism of transcriptional regulation by PPARβ/δ, but, unexpectedly, also show that only a subset of repressed genes are activated by a ligand-mediated switch. Our results also suggest that the type of transcriptional response by a given target gene is connected to the structure of its associated PPRE(s) and the biological function of its encoded protein. These observations have important implications for understanding the regulatory PPAR network and PPARβ/δ ligand-based drugs

Software

Python script for Pi-seq GU

Pi-seq: A low-cost customizable multichannel syringe pump for microfluidics

An open source, multichannel, zero-backflow microfluidics device based on syringe pumps controlled by a Raspberry Pi computer. It uses both readily available and 3D-printed parts as well as a custom PCB and is easily serviceable. We demonstrate a use case for single-cell RNA sequencing, however the Pi-seq is fully customizable for various applications

Droplet generation movie

5 s of droplet generatio