PPAR TGFβ Kaddatz, Kerstin Kaddatz Kerstin ths Dr. Müller-Brüsselbach Sabine Müller-Brüsselbach, Sabine (Dr.) Der „peroxisome proliferator activated receptor β/δ“ (PPARβ/δ) ist ein Liganden-induzierbarer Transkriptionsfaktor, der neben einer essentiellen Rolle im Lipidmetabolismus und der Energiehomöostase auch Funktionen bei der Regulation der Zelldifferenzierung, Proliferation und Apoptose besitzt. Im ersten Teil der vorliegenden Arbeit konnten anhand von Expressionsanalysen und ChIP-Sequenzierung drei Klassen von Zielgenen in humanen Myofibroblasten identifiziert werden. Gene der ersten Klasse werden durch PPARβ/δ reprimiert und durch Agonisten schnell und stark aktiviert. Die zweite Klasse von Genen zeigt keine Repression durch PPARβ/δ. Die Induktion erfolgt durch Agonisten deutlich schwächer und langsamer und die Expression wird stark durch Antagonisten reprimiert. Die dritte Klasse enthält Gene, deren Expression direkt mit dem PPARβ/δ-Niveau korreliert, wobei die Regulation liganden-unabhängig ist. Desweiteren erfolgt die Bindung von PPARβ/δ im Gegensatz zur Klasse I und II ohne nachweisbare „PPAR response elements“ (PPREs). Zusammenfassend erlauben diese Daten somit die Definition unterschiedlicher Klassen von PPARβ/δ-Zielgenen, die sich in den Mechanismen ihrer Regulation unterscheiden. PPARβ/δ spielt nicht nur eine Schlüsselrolle in der Regulation metabolischer Signalwege sondern moduliert zudem inflammatorische Prozesse und besitzt eine essentielle Funktion im Tumorstroma, was auf eine funktionelle Interaktion von PPARβ/δ und Zytokin-Signalwegen hinweist. Im zweiten Teil der Arbeit konnte mittels genomweiter Expressionsanalyse gezeigt werden, dass PPARβ/δ- und „transforming growth factor β“ (TGFβ)-Signalwege in humanen Myofibroblasten funktionell miteinander agieren. Eine Anzahl von Genen werden kooperativ durch TGFβ und PPARβ/δ aktiviert. Für das Modellgen „angiopoeitin-like 4“ (ANGPTL4) konnten zwei Enhancer Regionen identifiziert werden, die für die synergistische Aktivierung verantwortlich sind. Ein TGFβ-induzierbarer, stromaufwärts vom Transkriptionsstart (TS) gelegener Enhancer (ca. -8,5 kb relativ zum TS) wird durch einen Mechanismus reguliert, der SMAD3, ETS1, RUNX2 und AP-1 Transkriptionsfaktoren einbezieht, welche mit mehreren benachbarten Bindestellen interagieren. Ein zweiter Enhancer (PPAR-E), der aus drei nebeneinander liegenden PPREs besteht, befindet sich im Intron 3 des ANGPTL4-Gens (ca. +3,5 kb relativ zum TS). Der PPAR-E wird durch alle drei PPAR-Subtypen stark aktiviert, wobei ein neuartiges PPRE Motiv eine zentrale Rolle einnimmt. Obwohl der PPAR-E nicht durch TGFβ reguliert wird, interagiert diese Region mit SMAD3, ETS1, RUNX2 und AP-1 in vivo, was eine mögliche mechanistische Erklärung für den beobachteten Synergismus liefert. 2010-10-26 https://doi.org/10.17192/z2010.0567 Molekularbiologie und Tumorforschung Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg TGFβ ppn:230043135 German 2010 Komplexität der Transkriptionsregulation durch PPARβ/δ PPAR application/pdf Peroxisome-proliferator activated receptor β/δ (PPARβ/δ) is a ligand-inducible transcription factor that plays an essential role in lipid metabolism and energy homoeostasis and has been connected to different cellular processes like differentiation, proliferation and apoptosis. In the first part of the thesis, three different classes of target genes were identified in human myofibroblasts by expression profiling and genome-wide chromatin immunoprecipitation analysis (ChIP-sequencing). Class I genes are repressed by PPARβ/δ and show strong and rapid induction by specific agonists. Class II genes exhibit no PPARβ/δ-mediated repression. Their induction by agonists is comparably weak and slower, and their expression is strongly repressed by antagonists. The third class encompasses genes whose expression is ligand-independent, but correlates with PPARβ/δ levels. Surprisingly, PPARβ/δ binding of class III genes occurs in the absence of detectable PPAR response elements (PPREs). Taken together, these analyses led to the definition of different classes of target genes that are distinguished by their mechanism of regulation. PPARβ/δ does not only play a key role in the regulation of metabolic pathways, but also modulates inflammatory processes and has essential functions in tumor stroma, indicating a functional interaction between PPARβ/δ and cytokine signaling. In the second part of this thesis, transcriptional profiling of human myofibroblasts revealed a functional interaction of PPARβ/δ and transforming growth factor β (TGFβ) signaling pathways, and showed that a subset of genes are cooperatively activated by TGFβ and PPARβ/δ. Two different enhancer regions mediating synergistic activation were identified in the angiopoetin-like 4 (ANGPTL4) gene, which was used as a model. A TGFβ responsive enhancer located ∼8.5 kb upstream of the transcriptional start site (TSS) is regulated by a mechanism involving SMAD3, ETS1, RUNX2 and AP-1 transcription factors that interact with multiple contiguous binding sites. A second enhancer (PPAR-E), consisting of three adjacent PPREs, is located in the third intron ∼3.5 kb downstream of the TSS. The PPAR-E is strongly activated by all three PPAR subtypes, with a novel type of PPRE motif playing a central role. 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Transcriptional regulation ETS RUNX 2011-08-10 SMAD doctoralThesis 2010-09-24 Complexity of transcriptional regulation by PPARβ/δ Medizin Life sciences Biowissenschaften, Biologie RUNX SMAD opus:3149 ANGPTL4 urn:nbn:de:hebis:04-z2010-05679 Philipps-Universität Marburg ETS Transkriptionsregulation https://archiv.ub.uni-marburg.de/diss/z2010/0567/cover.png ANGPTL4 monograph